Cao ng Nguyn

(ch bin)

Gio trnh

Cng ngh Protein

Hu 2006

Li ni u
Trong nhng nm gn y cng ngh sinh hc pht trin nh v bo, hng lot cng ngh mi ra i nh genomics, proteomicsv c ng dng vo nhiu lnh vc khc nhau nh nng nghip, cng nghip thc phm v.vc bit l lnh vc y-dc hc. Gio trnh cng ngh protein c bin son trn c s cp nht nhng kin thc hin i, nhng thnh tu mi nht v proteomics trong nghin cu c bn v ng dng thc tin trn th gii v Vit nam, nhm phc v cho vic ging dy v hc tp cho cc ngnh sinh hc v cng l ti liu tham kho ca nhng ngnh hc lin quan khc. Gio trnh bin son c 6 chng vi hai ni dung chnh: - Nhng kin thc c bn v protein nh thnh phn, cu trc, tnh cht ha -l, cc phng php tch, tinh sch v xc nh protein. - Cng ngh sn xut mt s loi protein. Gio trnh bin son c phn cng c th nh sau: Chng 1. M u Chng 2. Amino acid - n v cu to protein Chng 3. Peptide - cu trc v chc nng Chng 4. Cu trc v tnh cht l-ho ca protein Chng 5. Cc phng php chit rt, tinh sch v xc nh protein Chng 6. Cng ngh sn xut mt s protein Cao ng Nguyn Cao ng Nguyn Cao ng Nguyn Cao ng Nguyn Cao ng Nguyn Qu Hai

Gio trnh c xut bn ln u tin chc chn khng trnh khi nhng thiu st. Cc tc gi xin chn thnh cm n v rt mong c s gp ca cc ng nghip v bn c khi ti bn s c hon thin hn

Cc tc gi

Chng 1

M u

I. Khi qut chung v protein

1.1. Nhng c trng chung ca nhm cht protein Protein c pht hin ln u tin th k XVIII (1745 bi Beccari); mi u c gi la allbumin (lng trng trng). Mi n nm 1838 , Mulder ln u tin a ra thut ng protein (xut pht t ch Hy lp proteos ngha l u tin, quan trng nht. Bit c tm quan trng v nhu cu x hi v protein, n nay nhiu cng trnh nghin cu v sn xut hp cht ny c cng b, em li nhiu ngha ht sc to ln phc v cho nhn loi. V vy, nhiu nh khoa hc trn th gii vinh d nhn c gii thng Nobel v cc lnh vc nghin cu lin quan n protein. Nh bit protein l hp cht hu c c ngha quan trng bc nht trong c th sng. V mt s lng, n chim khng di 50% trng lng kh ca t bo. V thnh phn cu trc, protein c to thnh ch yu t cc amino acid qua lin kt peptide. Cho n nay ngi ta thu c nhiu loi protein dng sch cao c th kt tinh c v xc nh c thnh phn cc nguyn t ho hc, thng thng trong cu trc ca chng gm bn nguyn t chnh l C H O N vi t l C 50%, H 7%, O 23% v N 16%. c bit t l N trong protein kh n nh. Nh tnh cht ny nh lng protein theo phng php Kjeldahl, ngi ta tnh lng N ri nhn vi h s 6,25. Ngoi ra trong protein cn gp mt s nguyn t khc nh S 0-3% v P, Fe, Zn, Cu... Khi lng phn t, k hiu l Mr (c tnh bng Dalton)* ca cc loi protein thay i trong nhng gii hn rt rng, thng thng t hng trm cho n hng triu. V d: insulin c khi lng phn t bng 5.733, glutamat-dehydrogengenase trong gan b c khi lng phn t bng 1.000.000 (bng 1.1). 1.2. ngha khoa hc v thc tin ca nhm cht protein T lu, bit rng protein tham gia mi hot ng sng trong c th sinh vt, t vic tham gia xy dng t bo, m, n tham gia hot ng xc tc v nhiu chc nng khc v.v...Ngy nay, khi hiu r vai tr to ln ca protein i vi c th sng, ngi ta cng thy r tnh cht duy vt v ngha ca nh ngha thin ti ca Anghen F. : sng l phng thc tn ti ca nhng th protein. Vi s pht trin ca khoa hc, vai tr v ngha ca protein i vi s sng cng c khng nh. Cng vi acid nucleic, protein l c s vt cht ca s sng. II. Phn loi protein Protein gm hng trm, hng ngn amino acid ni vi nhau bng lin kt peptide to nn mt hay nhiu chui polypeptide c cu trc rt phc tp. Cn c s c mt hay vng mt ca mt s thnh phn c bn cht khng phi protein m ngi ta chia protein thnh hai nhm ln: *1 Dalton = 1/1000 Kilodalton v c k hiu l kDa
Bng 1.1 Khi lng (Mr) v cu trc phn t ca mt s protein

protein

Khi lng s gc s chui (Dalton) amino acid polypeptide

Glucagon Insulin Ribonuclease (ty b) Lysozyme (lng trng trng) Myoglobin (tim nga) Chymotripsin (ty b) Hemoglobin (ngi) Albumin (huyt thanh ngi) Hexokinase (men bia) Tryptophan-synthetase (E.coli) -globulin (nga) Glycogen-phosphorylase (c th) Glutamate-dehydrogengenase (b) Synthetase ca acid bo (men bia) Virus khm thuc l 2.1. Protein n gin

3482 5733 12.640 13.930 16.890 22.600 64.500 68.500 96.000 117.000 149.000 495.000 1.000.000 2.300.000 40.000.000

29 51 124 129 153 241 574 550 800 975 1.250 4.100 8.300 20.000 336.500

1 2 1 1 1 3 4 1 4 4 4 4 40 21 2.130

Protein n gin l nhng phn t m thnh phn cu to ca n gm hon ton amino acid. Th d mt s enzyme ca tu b nh ribonuclease gm hon ton amino acid ni vi nhau thnh mt chui polypeptide duy nht (c 124 gc amino acid, khi lng phn t 12.640), chymotripsin gm ton amino acid ni vi nhau thnh chui polypeptide (c 241 gc amino acid, khi lng phn t 22.600)v.v...Da theo kh nng ho tan trong nc hoc trong dung dch m mui, kim hoc dung mi hu c ngi ta c th chia cc protein n gin ra mt s nhm nh nh: -Albumin: tan trong nc, b kt ta nng mui (NH4)2SO4 kh cao (70-100%). -Globulin: khng tan hoc tan t trong nc, tan trong dung dch mui long ca mt s mui trung tnh nh NaCl, KCl, Na2SO4..., v b kt ta nng mui (NH4)2SO4 bn bo ho. -Prolamin: khng tan trong nc hoc dung dch mui long, tan trong ethanol, isopanol 7080%.
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-Glutein: ch tan trong dung dch kim hoc acid long. -Histon: l protein c tnh kim d tan trong nc, khng tan trong dung dch amoniac long. 2.2. Protein phc tp Protein phc tp l nhng protein m thnh phn phn t ca n ngoi cc - amino acid nh protein n gin cn c thm thnh phn khc c bn cht khng phi l protein cn gi l nhm thm (nhm ngoi). Tu thuc vo bn cht ca nhm ngoi, ngi ta chia cc protein phc tp ra cc nhm nh v thng gi tn cc protein theo bn cht nhm ngoi: -Lipoprotein: nhm ngoi l lipide. -Nucleoprotein: nhm ngoi l acid nucleic. -Glycoprotein: nhm ngoi l carbohydrate v dn xut ca n. -Phosphoprotein: nhm ngoi l acid phosphoric. -Cromoprotein: nhm ngoi l hp cht c mu. Tu theo tnh cht ca tng nhm ngoi m c nhng mu sc khc nhau nh ( hemoglobin), vng ( flavoprotein)... III. Chc nng sinh hc ca protein 3.1. Xc tc v enzyme 3.1.1. Quan im v xc tc enzyme Hu ht tt c cc phn ng xy ra trong c th u do cc protein c bit ng vai tr xc tc, nhng protein c gi l cc enzyme. Mc d gn y ngi ta pht hin c mt loi RNA c kh nng xc tc qu trnh chuyn ho tin RNA thng tin (premRNA) thnh RNA thng tin (mRNA), ngha l enzyme khng nht thit phi l protein. Nhng enzyme xc tc sinh hc c bn cht l acid nucleic c gi l ribozyme. Nhng nh ngha c tnh cht kinh in: enzyme l nhng protein c kh nng xc tc c hiu cho cc phn ng ho hc, l cht xc tc sinh hc vn c ngha c bit quan trng. Hin nay ngi ta bit c khong 3.500 enzyme khc nhau, nhiu enzyme c tinh sch, kt tinh v nghin cu cu trc. 3.1.2. Nhng khc bit v c tnh xc tc, gia xc tc v c v xc tc enzyme. Enzyme l cht xc tc sinh hc, ngoi kh nng xc tc ging nh cht xc tc v c bnh thng n cn th hin mt s tnh cht sau y: a) Hiu sut xc tc rt ln. S chuyn ho c cht khi s dng emzyme xc tc ln hn nhiu so vi cht xc tc v c thng thng. V d: 1mol Fe3+ ch xc tc phn ly c 10-6 mol H2O2/pht. Trong khi mt phn t catalase c mt nguyn t Fe xc tc phn ly 5.10 6 mol H2O2/pht;1 gam pepsin trong 2 gi thu phn c 5 kg protein trng luc nhit bnh thng. Tng t 1gam phn t -amilase sau 1 giy c th phn gii 4.000 lin kt glucoside trong phn t tinh bt, cao hn nhiu so vi xc tc bng cht v c. b) Tnh c hiu cao. Tnh c hiu cao l mt trong nhng khc bit ch yu gia xc tc bng enzyme v xc tc bng cc cht v c khc. Mi enzyme ch xc tc cho s chuyn ho mt hay mt s cht nht nh, theo mt kiu phn ng nht nh. Da vo s tc dng c tnh chn lc, ngi ta c th chia ra mt s kiu c hiu sau: - c hiu kiu phn ng. c hiu ny th hin ch mi enzyme ch xc tc cho mt trong cc kiu phn ng chuyn ho mt cht nht nh. V d: phn ng oxy ho kh, chuyn v, thu phn, v.v...
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- c hiu c cht. L kh nng kt hp ca c cht vo trung tm hot ng ca enzyme v b chuyn ho di tc ng ca chng. Da vo mc c hiu ngi ta li chia ra mt s kiu nh: c hiu tuyt i, l enzyme ch tc dng trn mt c cht duy nht; c hiu tng i, l enzyme c kh nng tc dng ln mt kiu lin kt ho hc nht nh ca phn t c cht m khng ph thuc vo cu to ca cc phn t tham gia to thnh mi lin kt ; c hiu nhm, l enzyme c kh nng tc dng ln mt kiu lin kt ho hc nht nh vi iu kin mt trong hai phn tham gia to thnh lin kt phi c cu to xc nh. Hot tnh ca enzyme ph thuc vo nhiu yu t nh: nng enzyme, nng c cht, nhit v ion kim loi v.v... 3.1.3. C ch xc tc ca mt vi enzyme. Giai on u tin

Giai on cui cng

Hnh 1.1 kinase A

C ch chuyn nhm phosphate ca protein

Hin nay ngi ta bit r c ch hot ng ca nhiu enzyme nh carboxypeptidase, chymotripsin v.v..., mt enzyme cng c nghin cu kh k thuc nhm phosphotransferase xc tc qu trnh chuyn v nhm phosphate l protein kinase A (hnh 1.1) 3.1.4. S phn b enzyme trong t bo. Trong c th enzyme c mt mi m, mi t bo. Nhng ty theo chc nng ca m m cc t bo trong m thng c nhng h thng enzyme ring bit. S khc nhau khng ch gia cc loi t bo m ngay trong mt t bo cng tu theo chc nng ca tng bo quan (organell) ring bit c nhng h thng enzyme c hiu. S sp xp cc enzyme mt cch hp l trong t bo to ra s phi hp nhp nhng trong h thng phn ng dy chuyn lin tc cho hot ng sng ca c th: - Trong nhn t bo c cc enzyme nh ATP-ase, nucleosidase, nicotinicmononucleotide adenylyltransferase, 5-nucleotidase. - Trong ty lp th c cha hu ht cc enzyme lin quan n qu trnh chuyn ho nng lng nh h enzyme ca chui h hp t bo v qu trnh phosphoryl ho to ATP, cc enzyme ca chu trnh Krebs,v.v... -Trong lysosome c cha nhiu enzyme thu phn (hydrolase). -Trong ribosome c cha cc enzyme cho qu trnh tng hp protein. - Trong bo tng cha nhiu loi enzyme, c bit c tt c cc enzyme ca qu trnh ng phn. - Trn mng t bo cng nh mng ca nhiu bo quan c nhng loi enzyme gip cho s vn chuyn mt s cht qua mng v mt s hot ng khc ca mng. 3.2. Isozyme 3.2.1. c tnh phn t Isoyme l cc dng phn t khc nhau ca cng mt enzyme, xc tc cho mt phn ng sinh ha. Mc d cng xc tc cho cng mt loi phn ng sinh ho, nhng do s tn ti cc dng phn t khc nhau nn c mt s tnh cht l, ho v min dch khc nhau. V d: lactatdehydrogengenase (LDH) l enzyme kh hydrogen thun nghch gia lactate v pyruvate c khi lng phn t 130.-140.KDa, cu trc gm 4 tiu n v (subunit), cc tiu n v ny c dch m t 2 gene khc nhau c k hiu l H v M (t ch ting Anh HHeart v M-Muscle). Hai loi chui polypeptide to nn 5 dng phn t khc nhau l: LDH1: HHHH LDH2: HHHM LDH3: HHMM LDH4: HMMM LDH5: MMMM Trong in di theo chiu t m sang dng th th LDH1 chy nhanh nht ri n LDH2, LDH3, LDH4 v LDH5 chy chm nht. 3.2.2. Vai tr chc nng ca cc isozyme. Cc kt qu nghin cu thu c ch ra rng, t l cc dng isozyme c th thay i tu theo tui tc, trng thi sinh l v bnh l. S tn ti ca nhiu dng phn t khc nhau ca cng mt enzyme l mt hin tng sinh hc rt quan trong c v mt l lun v
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thc tin. Chng hn s phn b khc nhau v LDH trong c th ca cc loi c xng sng, loi chui H ch yu tm thy c tim v loi M ch yu tm thy c xng. Bi v loi chui H thng b c ch bi pyruvat vi nng qu tha. Nhng acid pyruvic trong m tim c oxy ho hiu kh mt cch u n trong ty th cung cp nng lng cho m ny, v thng thng khng c s ng ca acid pyruvic. Tri li tuy m c xng c rt nhiu LDH dng M v m ny c nhiu hot ng bt thng gy nn nhng ng pyruvat nht thi, nhng dng M li khng b c ch bi acid pyruvic. 3.3. Cc enzyme di lp th (allosteric enzyme) v phc h enzyme (multienzyme). Trong c th sng cn gp nhng enzyme ngoi trung tm hot ng xc tc cn c mt loi trung tm khc lm nhim v iu chnh hot tnh ca enzyme cn gi l enzyme di lp th. Tuy trung tm iu chnh v trung tm xc tc c tc dng h tr nhau, nhng l hai loi cu trc khc bit nhau. Trong nhiu trng hp c th kho trung tm d lp th m vn gi c hot ng xc tc ca trung tm hot ng. Cc enzyme ny thng c cu to t cc n v nh kt hp vi nhau bng cc lin kt yu , nn rt mm do c th tn ti di nhiu dng khc nhau v c th bin i thun nghch t trng thi ny sang trang thi khc mt cch d dng. Mt trong nhng loi enzyme ny c nghin cu k nht l aspartate-transcarbamylase (ATC-ase) xc tc qa trnh tng hp carbamylaspartate. Enzyme ny gm hai n v nh xc tc v 3-4 n v nh iu ho. Ngoi enzyme di lp th cn gp phc h a enzyme (multienzyme). l nhng phc hp gm nhiu enzyme c lin quan n nhau trong mt qu trnh chuyn ho nht nh. V d phc hp enzyme trong qu trnh to acetyl-CoA t acid puruvic c gi l phc h pyruvatedehydrogenase. Phc hp ny gm ba loi enzyme l pyruvate dehydrogengenase, dihydrolipoyl transacetylase v dihydrolipoyl -dehydrogenase. 3.4. Nhng quan im y hc v protein 3.4.1. Protein l nhng phn chc nng ca c th. Ngoi vai tr l thnh phn chnh trong cu trc ca t bo v m, protein cn c nhiu chc nng phong ph khc quyt nh nhng c im c bn ca s sng nh s truyn t thng tin di truyn, s chuyn ho cc cht l cc enzyme, cc khng th chng li bnh tt, cc hormon dn truyn cc tn hiu trong t bo v.v... u c bn cht l cc protein. 3.4.2. Hnh thnh chc nng mi trn c s cu trc protein. S pht trin ca sinh hc phn t da trn l thuyt trung tm DNA RNA Protein. Nh vy, s bin i DNA s dn n s bin i cu trc ca phn t protein v do chc nng sinh hc ca n s b bin i ko theo nhng thay i c lin quan n ton b c th. Trong qu trnh tin ho ca sinh vt s hnh thnh v thch nghi mt chc nng mi din ra mt giai on lch s lu di. S xut hin mt protein mi bin dng (mt hot tnh hoc t bin cu trc) thng lun i km bnh tt. 3.4.3. S xut hin cc protein bnh l. Y hc l ngnh khoa hc v s sng, ngy nay vi tin b ca khoa hc, nhng hiu bit v bnh l mc phn t vt ra khi gii hn ca gii phu t bo hoc c quan. Sinh hc phn t ra i to ra cuc cch mng trong cc quan nim v bnh. T , s pht trin ca bnh hc phn t lun lun i km vi sinh hc phn t. S bin i cu trc ca mt protein hay s xut hin cc enzyme c cu trc bt thng u do yu t di truyn gy nn. Da theo cc biu hin di truyn ngi ta chia cc protein bnh l ra lm hai loi ln:
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a) Nhng bin i v s lng ca protein: l s thay i do s tng hoc gim protein no , thm ch xut hin nhng protein m t bo bnh thng khng tng hp mt cch thng xuyn. Nhng protein ny vn c cu trc bnh thng v nh vy khng c nhng bin i ca gene cu trc. Nhng lch lc ny do ri lon qu trnh iu ho sinh tng hp protein. Do protein vn c cu trc bnh thng m ch thay i v s lng nn chng vn c chc nng bnh thng v ch thay i v mc hot ng. Trong trng hp l protein enzyme th nhng lch lc v s lng enzyme s dn n nhng ri lon dy chuyn chuyn ho. b) Nhng bin i v cht lng protein: l nhng ri lon v cu trc protein do gene bi bin i, dn n cu trc protein thay i ko theo s thay i chc nng sinh hc ca protein . V d, s bin i cu trc ca hemoglobin (Hb) l protein c chc nng vn chuyn oxygen trong mu dn n bnh thiu mu, hay nh bnh thiu mu do hng cu hnh li lim... 3.4.4. Cu trc v chc nng ca protein min dch. Tham gia vo h thng min dch c nhiu c quan, nhiu loi t bo v c bit nhiu loi protein thc hin cc chc nng ring bit to nn hiu qu min dch c hiu v khng c hiu. Cc protein min c nhc n nhiu hn c l cc khng th, b th v cc cytokine. a) Cc khng th (antibody). Tt c cc phn t khng th c chng minh l cc globulin c chc nng min dch (vit tt l Ig: Immunoglobulin) v c bn cht l glycoprotein. Cc khng th c chia thnh 5 lp. Tu theo cu trc v chc nng min dch l IgG, IgA, IgM, IgD, IgE. Cu trc ca phn t khng th c hnh thnh t hai loi chui polypeptide l chui nng (k hiu: H=Heavy chain) c khi lng phn t t 53-59 kDa v chui nh (k hiu:L=Light chain) c khi lng phn t 22-26 kDa. C bn chui c gn vi nhau bng cu disunfid (S-S). Trung tm hot ng l phn lin kit vi khng nguyn (hnh 1.2) nm vng tn cng N ca chui nng v chui nh c cu trc ch khong 8-10 amino acid. Cc phn t Ig c c tnh hot ng min dch theo hai chc nng: - C kh nng lin kt vi khng nguyn t nht hai v tr tip nhn i vi khng nguyn nh s bin i k diu ca phn tn cng NH2 trn phn t khng th. - Phn tn cng COOH ca phn t khng th c kh nng thc hin mt s ln cc hot ng sinh hc di nh hng ca s lin kt vi th th trn b mt ca t bo. Tt c cc khng th u c cng mt cu trc phn t nhng khc nhau mc ca vng lin kt vi khng nguyn. Nhn chung phn t khng th c chia lm hai phn: phn Fab l phn lin kt vi khng nguyn, phn Fc l phn d kt tinh phn ng vi cc t bo ca h thng min dch qua th th ca cc t bo. Dng enzyme papain hay pepsin c th ct khng th thnh hai mnh Fab v Fc, hoc F(ab)2 tng ng.

Hnh 1.2

Cu trc chung ca phn t khng th (Ig)

b) B th (complement). L nhng protein huyt tng phn ng vi nhau nhm tn cng cc dng tc nhn gy bnh. H thng b th bao gm khong 40 protein c chc nng p ng min dch, chng vi sinh vt v p ng vim. V chc nng, cc khng th tng tc c hiu vi tc nhn truyn nhim bnh, cn h thng b th c c nh ln tt c cc khng th thc hin chc nng min dch. Cc thnh phn ca b th tng tc gia chng vi nhau v cc yu t khc ca h thng min dch. c) Cc cytokine. L ton b cc phn t c tit ra bi cc t bo ca h thng min dch, tham gia vo hot ng tn hiu gia cc t bo trong hot ng p ng min dch. Tt c cc cytokine u c bn cht protein hay glycoprotein v c phn loi nh sau: - Cc interferons (IFN): c cc dng -IFN, -IFN v -IFN, c chc nng ngn nga ca mt s virus gy bnh. - Cc interleukin (IL): c cc dng t IL1 n IL 13, chng c nhiu chc nng, nhng ch yu l kim tra s bit ho v sinh sn t bo. - Cc yu t kch thch qun lc (CSF): c chc nng kim tra s phn chia v sinh sn ca cc t bo ngun v cc t bo mu s khai. - Cc cht dn truyn sinh hc (mediator): l nhng protein ca giai oi p ng min dch cp tnh. 3.4.5. Cu trc v chc nng ca protein vn chuyn. Trong c th c nhng protein lm nhim v vn chuyn nh hemoglobin, mioglobin, hemocianin vn chuyn O2, CO2 v H+ i khp cc m, cc c quan trong c th. Ngoi ra cn c nhiu protein khc nh lipoprotein vn chuyn lipid, ceruloplasmin vn chuyn ng (Cu) trong mu v.v...Mt trong nhng protein lm nhim v vn chuyn c nhc n nhiu nht l hemoglobin. Phn t c cu to t bn tiu n v (subunit), hai tiu n v v hai tiu n v .
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Hnh 1.3 Cu trc ca phn t hemoglobin

Hnh 1.4 So snh cu trc tiu n v ca hemoglobin vi leghemoglobin v myoglobin.

Mi tiu n v ni vi mt heme bng lin kt khng phi cng ho tr. Khi so snh vi protein cng chc nng nh myoglobin c v leghemoglobin thc vt l nhng protein c cu trc ch mt tiu n v (monomer) thy rng cc tiu n v cu trc kh ging nhau (hnh: 1.3, 1.4 ) 3.4.6. Cu trc chc nng v vai tr ca lectin. Lectin l nhng protein hay glycoprotein khng phi ngun gc min dch, lectin c kh nng ngng kt vi nhiu loi t bo, cng nh nhiu loi ng hoc cc hp cht cha ng c tnh cht chn lc. Hu ht lectin c cu trc bc 4, vi khi lng phn t giao ng trong phm vi kh rng t hng ngn cho n hng trm ngn Dalton. V d: lectin t r cy Urtica dioica (h gai Urticaceae) c Mr=8,5 KDa trong khi loi sam bin chu (Tachypleus tridentatus) c Mr=700.KDa. V chc nng, ngi ta thy rng mc d lectin khng phi l khng th chng li tc nhn gy bnh nhng chng c vai tr bo v c th nh tng tc vi mng t bo v gy ngng kt t bo ca chng. H khng nh rng lectin c kh nng gn cc t bo vi khun v khng nguyn l vi cc i thc bo, do vy m vi khun v khng nguyn l b o thi ra khi c th. Ngoi ra c nhng lectin cn c kh nng kch thch s phn chia v bit ho t bo. ng thi ngi ta cng pht hin c nhiu lectin c c hot tnh ca
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enzyme, v d lectin hot tnh kh mnh, c tch ra t ht u mng, c khi lng phn t khong 16.KDa c c hot tnh ca enzyme -galactosidase. 3.4.7. Nhng chc nng khc ca protein. Trong c th ngoi cc protein m nhn chc nng xc tc nh enzyme, chc nng vn chuyn nh hemoglobin, mioglobin, lipoprotein, v chc nng bo v nh cc khng th min dch, cc protein c t nh enzyme nc rn, lectin v.v..., protein cn tham gia nhiu chc nng quan trng khc nh: - Cc protein lm nhim v kch thch iu ho qu trnh trao i cht nh cc hormon - Cc protein lm nhim v cu trc nh v virus, mng t bo, colagen da, fibrolin t - Cc protein lm nhim v co rt nh myosin, actin si c - Cc protein lm nhim v d tr nh casein ca sa, ovalbumin ca trng, v.v...

TI LIU THAM KHO 1.Trn Th n, i Duy Ban, Nguyn Hu Chn, nh H, L c Trnh. 1980. Ho sinh hc. NXB Y hc 2.Phm Th Trn Chu, Trn Th ng. 1999. Ho sinh hc. NXB Gio dc 3.Phm Th Trn Chu, L Minh Chu, Lm Chi, Nguyn Ln Dng, nh H, L Ngc T. 1983. Nhng hiu bit mi v enzim. Tp 8. NXB KH & KT H ni. 4. inh H, i Duy Ban, 1977. Sinh hc phn t v cuc cch mng trong sinh hc, NXB KH& KT. H ni 5. Ngc Lin. 2004. Min dch hc c s. NXB i hc Quc gia H ni (in ln th 2). 6.Fersht A.,1998, Structure and Mechanism in Protein Science, W. H. Freeman, 3rd Rev Edit. 7.Lehringer A.L., 2004. Principle of Biochemistry, 4th Edition. W.H Freeman, 2004 8. Liebler D.C., 2002. Introduction to proteomics. Humana Press Inc. Totuwa, New Jersey. 9. Lodish H., 2003. Molecular Cell Biology. 5th ed.W.H Freeman. 10. Virella G.,1998. Introduction to medical immunology, Marcel Dekker, Inc. New York. Basel. Hong kong. 4th. ed.

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Chng 2

Amino acid - n v cu to Protein

I. Thnh phn tnh cht l- ho ca amino acid

1.1. Thnh phn v cu to ca amino acid Protein l polymer ca cc amio acid ni vi nhau bng cc lin kt cng ho tr l lin kt peptide. Protein c th b thu phn to thnh cc amino acid t do bng nhiu phng php khc nhau. Ngi ta xc nh protein c cu trc t 20 loi amino acid khc nhau. Amino acid l cht hu c m phn t cha t nht mt nhm carboxyl (COOH) v t nht mt nhm amino (NH2), tr prolin ch c nhm NH (thc cht l mt acid imin). Trong phn t amino acid u c cc nhm COOH v NH2 gn vi carbon v tr . Hu ht cc amino acid thu nhn c khi thu phn protein u dng L- amino acid. Nh vy cc protein ch khc nhau mch nhnh, hay cn gi l chui bn (thng c k hiu: R).

Hnh: 2.1

Cng thc cu to chung ca cc amino acid

1.2. Phn loi amino acid 1.2.1. Cc quan im v phn loi amino acid. Hin nay c nhiu ngi phn loi amino acid theo nhiu kiu khc nhau, mi kiu sp xp u c ngha v mc ch ring. Tuy nhin, h u da trn cu to ho hc hoc mt s tnh cht ca gc R. V d: c ngi chia cc amino acid thnh 2 nhm chnh l nhm mch thng v nhm mch vng. Trong nhm mch thng li tu theo s c mt ca s nhm carboxyl hay s nhm amino m chia ra thnh cc nhm nh, nhm amino acid trung tnh (cha mt nhm COOH v mt nhm NH2); nhm amino acid c tnh kim (cha mt nhm COOH v hai nhm NH2); nhm amino acid c tnh acid (cha hai nhm COOH v mt nhm NH2). Trong nhm mch vng li chia ra thnh nhm ng vng hay d vng v.v... C ngi li da vo tnh phn cc ca gc R chia cc amino acid thnh 4 nhm: nhm khng phn cc hoc k nc, nhm phn cc nhng khng tch in, nhm tch in dng v nhm tch in m. y xin c gii thiu cch phn loi cc amino acid mt cch chung nht. Theo cch ny da vo gc R cc amino acid c chia lm 5 nhm: Nhm I. Gm 7 amino acid c R khng phn cc, k nc, l: glycine, alanine, proline, valine, leucine, isoleucine v methionine. 13

Hnh: 2.2 Cng thc cu to cc amino acid nhm I

Nhm II Gm 3 amino acid c gc R cha nhn thm, l phenylalanine, tyrosine v tryptophan.

Hnh: 2.3

Cng thc cu to cc amino acid nhm II

Nhm III. Gm 5 amino acid c gc R phn cc, khng tch in, l serine, threonine, cysteine, aspargine v glutamine.

14

Hnh: 2.4

Cng thc cu to cc amino acid nhm III

Nhm IV. Gm 3 amino acid c R tch in dng, l lysine, histidine v arginine.

Hnh: 2.5

Cng thc cu to cc amino acid nhm IV

Nhm V. Gm 2 amino acid c gc R tch in m, l aspartate v glutamate.

15

Hnh: 2.6

Cng thc cu to cc amino acid nhm V

1.2.2. Cc amino aicd thng gp. Cc amino acid thng gp l nhng amino acid thng c mt trong thnh phn ca cc loi protein. Chng c khong 20 loi v c thu nhn khi thu phn protein. Cc loi amino acid ny c tn gi, khi lng phn t v k hiu c trnh by trn bng 2.1. 1.2.3. Cc aminno acid khng thay th, hay cn thit. Cc amino acid c hnh thnh bng nhiu con ng khc nhau. Nh bit, trong phn t protein c khong 20 loi amino acid, tuy nhin trong c th ngi v ng vt khng tng hp c tt c cc loi m phi a t ngoi vo qua thc n. Nhng amino acid phi a t ngoi vo gi l cc amino acid khng thay th. Ngy nay ngi ta bit c c khong 8-10 loi amino acid khng thay th bao gm: Met, Val, Leu, Ile, Thr, Phe Trp, Lys, Arg v His, ngy nay ngi ta cn xem Cys cng l mt amino acid khng thay th. 1.2.4. Cc amino acid t gp. Ngoi cc amino acid thng gp trn, trong phn t protein i khi cn c mt s amino acid khc, l nhng loi t gp. Cc amino acid ny l dn xut ca nhng amino acid thng gp nh: trong phn t colagen c cha 4-hydrogenxyproline l dn xut ca proline, 5-hydrogenxylysine l dn xut ca lysine v.v... Mt khc, mc d khng c trong cu trc protein, nhng c hng trm loi amino acid khc chng c th tn ti dng t do hoc lin kt vi hp cht khc trong cc m v t bo, chng c th l cht tin thn hay l cc sn phm trung gian ca qu trnh chuyn ho trong c th. Bng 2.1 Cc amino acid thng gp Tn amino acid Glycine Alanine Proline Valine Leucine Isoleucine Tn amino acid gi theo danh php Tn K ho hc vit tt hiu -aminoacetic -aminoprpionic -pirolidincarboxylic -aminoiaovaleric -aminonoisocaproic -amino- -metylvaleric 16 Gly Ala Pro Val Leu Ile G A P V L I Khi lng (Mr) 75 89 115 117 131 131

Methionine Tyrosine Tryptophan Serine Threonine Cysteine Aspargine Glutamine Lysine Histidine Arginine

-amino- -metyltiobutiric -amino- hydrogengenxyphenylpropionic -amino- -idolylpropionic -amino- -hydoxypropionic -amino- -hydrogengenxybutiric -amino- -tiopropionic amid ca aspartate amid ca glutamate , diaminocaproic -amino- -imidazolpropionic -amino- -guanidinvaleric -aminosucinic -aminoglutarate

Met Phe Tyr Trp Ser Thr Cys Asn Gln Lys His Arg Asp Glu

M F Y W S T C B Q K H R D E

149 165 181 204 105 119 121 132 146 146 155 174 133 147

Phenylalanine -amino- -phenylpropionic

Aspartate Glutamate

1.3. Mu sc v mi v ca amino acid Cc amino acid thng khng mu, nhiu loi c v ngt kiu ng nh glycine, alanine, valine, serine histidine, triptophan; mt s loi c v ng nh isoleucine, arginine hoc khng c v nh leucine. Bt ngt hay cn gi l m chnh l mui ca natri vi acid glutamic (monosodium glutamate). 1.4. Tnh tan ca amino acid Cc amino acid thng d tan trong nc, cc amino acid u kh tan trong alcohol v ether (tr proline v hydrogenxyproline), chng cng d ho tan trong acid v kim long (tr tyrosine). 1.5. Biu hin tnh quang hc ca amino acid Cc amino acid trong phn t protein u c t nht mt carbon bt i (tr glycine) v th n u c biu hin hot tnh quang hc, ngha l c th lm quay mt phng ca nh sng phn cc sang phi hoc sang tri. Quay phi c k hiu bng du (+), quay tri c k hiu bng du (-). Gc quay c hiu ca amino acid ph thuc vo pH ca mi trng. Tu theo s sp xp trong cu trc phn t ca cc nhm lin kt vi carbon bt i m cc amino acid c cu trc dng D hay L (hnh 2.7) gi l ng phn lp th. S ng phn lp th c tnh theo 2n (n l s carbon bt i)

17

Hnh 2.7

ng phn lp th ca alanine

Hu ht cc amino acid khc hp th tia cc tm bc sng ( ) khong t 220 280 nm. c bit cng nng 10-3M, trong bc sng khong 280 nm tryptophan hp th nh sng cc tm mnh nht, gp 4 ln kh nng hp th ca tyrosine (hnh 2.8) v phenylalanine l yu nht. Phn ln cc protein u cha tyrosine nn ngi ta s dng tnh cht ny nh lng protein

Hnh 2.8

Ph hp th nh sng cc tm ca triptophan v tyrosine

1.6. Tnh lng tnh ca amino acid Trong phn t amino acid c nhm carboxyl -COOH nn c kh nng nhng proton (H+) th hin tnh acid, mt khc c nhm amin- NH2 nn c kh nng nhn proton nn th hin tnh base. V vy amino acid c tnh cht lng tnh. Trong mi trng acid, amino acid dng cation (tch in dng), nu tng dn pH amino acid ln lt nhng proton th nht chuyn qua dng lng cc (trung ho v 18

in), v tip tc tng pH amino acid s nhng proton th hai chuyn thnh dang anion (tch in m). V vy i khi ngi ta coi n nh mt di-acid.

cation

lng cc

anion

Hnh 2.9

Tnh lng tnh ca amino acid

Tng ng vi phn ly H+ ca cc nhm COOH v NH3+ c cc tr s pK1 v pK2 (biu th phn ly ca cc nhm c 1/2). T ngi ta xc nh c pH i (pI= pH ng in) = pK1 + pK2 / 2. V d: khi ho tan glycine vo mi trng acid mnh th hu nh glycine u dng cation. Nu tng dn lng kim, thu c ng cong chun . Trn ng cong chun thy rng: glycine ln lt nhng 2 proton trc tin chuyn sang dang lng tnh v sau cng chuyn thnh dng anion

Hnh 2.10

ng cong chun ca glycine nng 1 M 25OC

Tng ng phn ly ca nhm COOH c mt na c tr s pK1= 2,34 v phn ly ca NH3+ c mt na c tr s pK2= 9,60. Nh vy ta c 2,34 + 9,60 pHi = ------------- = 5,97 19

2 Mt khc ti pK1 + 2 s phn ly H+ ca nhm COO- glycine l 99%, ch 1% dng COOH v pK2 -2 dng NH3+ l 99%, ch 1% dng NH2. Nh vy trong vng pH t pK1 + 2 n pK2 -2, phn t glycine ch yu dng lng tnh v kt qu ta c mt vng ng in. Ngoi ra cc amino acid trong gc R c thm nhm COOH hay NH 2 s phn ly ca chng s c thm mt tr s phn ly na-pKR (xem bng 2.2) 1.7. Cc phn ng ho hc ca amino acid Cc amino acid u c nhm NH2 v COOH lin kt vi C , v vy chng c nhng tnh cht ho hc chung. Mt khc cc amino acid khc nhau bi gc R, v vy chng c nhng phn ng ring bit. Ngi ta chia cc phn ng ho hc ca amino acid thnh 3 nhm:

Bng: 2.2 Cc tr s pK ca cc amino acid thng gp

Tn cc amino acid Glycine Alanine Proline Valine Leucine Isoleucine Methionine Phenylalanine Tyrosine Tryptophan Serine Threonine Cysteine Aspargine Glutamine Lysine Histidine Arginine pK1(ca COOH) 2,34 2,34 1,99 2,32 2,36 2,36 2,28 1,83 2,20 2,38 2,21 2,11 1,96 2,02 2,17 2,18 1,83 2,17 1,88 2,19 Cc tr s pK pK2(ca NH+3) 9,60 9,60 10,96 9,62 9,60 9,68 9,21 9,13 9,11 9,39 9,15 9,62 10,28 8,80 9,13 8,95 9,17 9,04 9,60 9,67 10,53 6,00 12,48 3,65 4,25 8,18 10,07 pKR(ca R) pI 5,97 6,01 6,48 5,97 5,98 6,02 5,74 5,48 5,66 5,89 5,68 5,87 5,07 5,41 5,65 9,74 7,59 10,76 2,77 3,22

Aspartate
Glutamate

a) Phn ng ca gc R. 20

Do cc amino acid c cu to gc R khc nhau, nn ngi ta c th dng xc nh tng amino acid ring r nh phn ng c trng ca n, v d phn ng oxy ho kh do nhm SH ca cysteine, phn ng to mui do cc nhm COOH hay NH2 ca glutamte hay lysine, phn ng to ester do nhm OH ca tyrosine v.v... b) Phn ng chung. L phn ng c s tham gia ca c hai nhm - COOH v - NH2 . Khi phn ng vi ninhydrin trong iu kin un nng to thnh CO2 , NH3 aldehyde v nihydrin bi kh, cui cng to nn sn phm c mu xanh tm. c) Phn ng ring bit C th chia cc phn ng ring bit theo hai nhm - COOH v - NH2 -Cc phn ng ca nhm - COOH . Ngoi cc phn ng ca nhm COOH thng thng to ester, to amid, to mui ...th n cn c nhng phn ng c trng khc nh c th b kh thnh hp cht ru amino di s xc tc ca NaBH4.

Nhm COOH c th to thnh phc aminoacyl-adenylate trong phn ng hot ho amino acid tng hp protein, hay c th loi CO2 gp rt nhiu trong qu trnh thoi ho amino acid, to cc dn xut amin c hot tnh sinh hc cao nh histamine, sevtnine. - Cc phn ng ca nhm - NH2 . Nhiu phn ng ca nhm amino c dng xc nh cc ch tiu ca amino acid nh: nh lng amino acid ngi ta cho phn ng vi HNO 2 gii phng N2 v nh lng nitrogen

nh lng amino acid ngi ta cho phn ng vi formaldehyde to thnh base schif.

Sau dung NaOH (hoc KOH) chun nhm COOH ca aminoacid xc nh amino acid u N-tn cng ngi ta cho tc dng vi 2-4 dinitrofluobenzen (phn ng sanger) hay phenyliothiocyanate (phn ng Edman). Sau xc nh amino acid N-tn cng tch bit khi chng dng dn xut vi hai loi ho cht trn.

II. Thu nhn amino acid bng thy phn protein

2.1. Thy phn bng acid

21

 thu nhn cc amino acid phng php thng c dng nhiu nht l thu phn bng acid HCL 6N d tha nhit 100-120oC trong khong 24 gi. Sn phm thu c ch yu l cc amino acid t do di dng hydrogenclorate. Mt s amino acid nh serine v threonine b ph hu mt phn, tryptophan b ph hu hon ton, glutamine v asparagine phn ly thnh acid glutamic, acid aspartic v NH4+. 2.2. Thu phn bng kim Ngi ta cng c th thu nhn cc amino acid bng phng php thu phn vi NaOH, bng cch un nng trong nhiu gi. Sn phm thu c hu ht l cc amino acid nhng u b racemic ha, cc amino acid cysteine, serine v treonine b ph hu nhng tryptophan khng b ph hu. V vy, phng php thu phn bng kim thng ch dng xc nh tryptophan. 2.3. Thu phn bng enzyme thu nhn ch phm amino acid ngy nay vic thu phn bng enzyme c ng dng rng ri trong nhiu lnh vc khoa hc khc nhau bi s dng enzyme c nhiu u im nh ni trn. Cc enzyme thu phn protein to thnh cc amino acid hay cc cc peptid c phn t thp c gi chung l peptidhydrogenlase. C nhiu loi peptidhydrogenlase, chng c phn bit nhau bi tnh c hiu khc nhau vi lin kt peptid. Mt s loi ct t lin kt peptid u tn cng ca chui polypeptide c gi l exopeptidase nh carboxypeptidase phn gii lin kt peptide u C tn cng, aminopeptidase phn gii lin kt peptide u N tn cng. Mt s khc ch ct t cc lin kt peptide gia ca chui polypeptide c gi l cc endopeptidase nh pepsin, tripsin, v.v... 2.4. Cc phng php theo di v xc nh tc thu phn bng enzyme Tc thu phn protein ca enzyme ph thuc vo nhiu yu t nh nng enzyme, nng c cht, cc cht km hm, cc cht kch hot, nhit v pH ca mi trng phn ng. xc nh tc thy phn ca enzyme ngi ta khng nh lng enzyme mt cch trc tip m thng xc nh gin tip thng qua hot ca enzyme. Khi thc hin phn ng, enzyme c th lm thay i cc tnh cht vt l, ho hc, v.v... ca hn hn hp phn ng. V vy, theo di nhng bin i thng qua s nh lng c cht b mt i hay sn phm ca phn ng enzyme c to thnh c th bit c chnh xc mc hot ng ca enzyme. Ngi ta chia ra thnh ba nhm phng php: a) Xc nh lng sn phm to thnh hay lng c cht b mt i trong mt thi gian nht nh, ng vi lng enzyme nht nh. b) Xc nh thi gian cn thit thu nhn c mt lng ca c cht hay sn phm ng vi mt lng enzyme nht nh. bin thin nht nh

22

c) Chn nng enzyme nh th no thu c s bin thin nht nh v c cht hay sn phm trong mt thi gian nht nh. Ngi ta thng s dng mt s n v tnh hot nh sau: - n v enzyme Quc t (UI -) l lng enzyme c kh nng xc tc lm chuyn ho c mt micromol c cht sau mt pht iu kin tiu chun. 1 UI = 1 mol c cht (10-6 mol)/pht. - Katal (Kat) l lng enzyme c kh nng xc tc lm chuyn ho c mt mol c cht sau mt giy iu kin tiu chun. 1 Kat = 1 mol c cht/giy 1 1 UI = --- 10-6 Kat = 16,67 nKat (nanokatal) 60 - Hot ring ca mt ch phm enzyme l s n v UI (hoc s n v Katal) ng vi 1 ml dung dch hoc 1mg protein ca ch phm. Nu ch phm enzyme tinh sch hot c th c biu th bng s UI (hoc Kat) trn 1mg enzyme. - Hot ring phn t l s phn t c cht c cht c chuyn ho bi mt phn t enzyme trong mt n v thi gian.

III. Cc phng php phn tch amino acid 3.1. Phng php ho l t lu vic phn tch nh tnh v nh lng amino acid thng l s kt hp ca nhiu phng php ho l v sc k. C th da vo ph hp ph cc amino acid khng ging nhau phn tch. Hoc da v cu trc ca amino acid t do sau khi chui polypeptide b thu phn, c th nh lng amino acid u N-tn cng bng phn ng Sanger hoc Edman. Cng nh vy, c th xc nh amino acid u C tn cng nh phn ng kh nhm carboxyl vi tc nhn kh NaBH4, hoc s dng enzyme carboxypepitdase v.v... 3.2. Sc k Ngi ta c th dng nhiu phng php sc k khc nhau, y ch gii thiu nguyn tc ca mt s phng php thng dng phn tch amino acid. a) Sc k giy. Nguyn tc ca phng php ny l da vo s phn b gia hai pha dung mi: dung mi c nh v dung mi di ng. Dung mi c nh thng l nc gi giy sc k (trong iu kin bo ho hi nc, giy c th gi 15-22% nc tnh theo trng lng 23

giy). Dung mi di ng thng l mt dung mi hu c bo ho nc di chuyn trn t giy theo mao dn ko theo cc cht trong dung dch. Tc di chuyn ca tng cht khng ging nhau v mi cht c c trng bi mt tr s nht nh gi l Rf.

Trong : - a l khong cch chuyn dch ca cht phn tch - b l khong cch chuyn dch ca dung mi C nhiu kiu sc k giy khc nhau l sc k mt chiu i ln, sc k mt chiu i xung, sc k vng nm ngang v sc k hai chiu. Loi giy thng dng l Whatman s 1 v Schleicher-Schull 2044 a v b, dung mi gm cc cht nh 4 Butanol:1 Acetic acid: 5 Nc (dng cho chiu th nht); 3 Phenol: 1 Nc (dng cho chiu th hai). b) Sc k lp mng. Nguyn tc ca phng php ny da trn l thuyt ca sc k giy, ngha l cng da trn s phn b cc cht gia hai pha: cht hp ph c trng rng trn mt phin knh to thnh mt lp mng v pha di ng l dung mi thch hp. Dung mi di chuyn lm dch chuyn cc cht trong mu th. Cc cht hp ph thng dng l silicagel, alumin oxyt, sephadex ,v.v...c kt hp vi thch cao (gypse) dn vo phin knh c) Sc k kh. Nguyn tc ca phng php ny l li dng tnh cht kh bay hi ca cc amino acid nn c th s dng chng trnh nhit chuyn chng thnh cc dn xut (thng l N-acetyl-amin). Cho tc dng amino acid vi cn amylic v HBr khan, sau cho tc dng hn hp ny vi anhydrit acetic. Ct thng dng l loi Chromosorb W (60-80 mesh) c mt lp polyetylenglycol 1% (carbowax 1564 hoc 6000). Chng trnh nhit gia 125o C v 155oC, tc chy 60-240 ml/pht. 3.3. Phn tch bng my t ng a) Xc nh trnh t (sequence) amio acid trong chui polypeptide My phn tch amino acid trong chui polypeptide t ng da trn nguyn tc ca phng php Edman, ngha l tch tch ln lt tng amino acid u N tn cng v xc nh ln lt theo th t tng amino acid c tch ra , v vy c th xc nh chnh xc trnh t sp xp ca cc amino acid trong chui. b) Xc nh thnh phn amio acid trong chui polypeptide bng my sc k lng cao p-HPLC (High Liquid Pressor Chromatography ) Trc ht protein hay peptide phi c thu phn bng HCl 6 N 1100C trong ng hn kn cha nitrogen ( trnh s oxy ho ph hu amino acid) trong thi gian 12-15 gi. Sau trung ho hn hp dch thu phn amino acid v cho vo my phn tch t ng HPLC thnh phn amino acid cng vi mu chun amino acid. My t ng s cho bit hm lng (t l %) ca tng amino acid da theo cc nh (peak) ca amino acid chun. cn nh my HPLC ch cho bit thnh phn (composition) ca tng amion acid ch khng cho bit trnh t cc amino acid 3.4. in di 24

Da vo tnh cht tch in ca cc amino acid trong mi trng c pH nht nh, m c th phn tch amino acid bng k thut in di. Di tc dng ca in trng cc amino acid tch in dng (+) s chy v pha cc (-), cc amino acid tch in m s chay v cc (+). Do kh nng tch in khng ging nhau gia cc amino acid v vy chng di chuyn khng ging nhau trong in trng. Kt qa cc amino acid phn b tri ra trn gi th (giy hoc tm polyamide). 3.5. Phn tch bng quang ph khi Quang ph khi (MS- mass spectrophotometer) l mt cng c phn tch vi chnh xc cao. Nguyn tc ca phng php ny l: Dng mt chm electron bn vo mt lng cht th rt nh, cc phn t cht th

Hnh: 2.11

S hot ng ca quang ph khi

trc ht c ph thnh nhiu mnh ion mang in dng trong iu kin chn khng. Cc mnh ion nh mt b phn pht hin v ghi thnh pic vi cng khc nhau tng ng vi khi lng ca mi ion - l khi ph. C nhiu loi my quang ph khi vi mc phn gii khc nhau, my c phn gii cao l my c kh nng tch c hai mnh ion c khi lng ch chnh nhau phn trm n v khi. 3.6. Phng php ng v Phng php ng v c th s dng xc nh mt hoc vi amino acid trong hn hp c nhiu loi amino acid khc nhau, hay mt amino acid cn c xc nh trong nhiu mu ca mt lot th nghim. Nguyn tc ca phng php ny l da vo hot tnh phng x ca amino acid cng mt loi nh du xc nh v tr v s lng ca amino acid trong chui polypeptide. Ngoi ra ngi ta c th dng phng php ny nghin cu tnh c hiu ca cc amino-acyl-tRNAsynthetase v tRNA 3.7. Phng php enzyme C th xc nh amino acid bng phng php enzyme da trn nguyn tc mi mt loi enzyme c kh nng phn gii c hiu mt loi L-amino acid nht nh. Xc nh sn phm to thnh sau phn ng vi enzyme tng ng c th bit c amino acid trong hn hp. 3.8. Phng php vi sinh vt Mt s vi khun sinh trng trong mt iu kin thch hp, chng rt nhy cm vi pH sn xut ra cc enzyme L-amino acid-decarboxylase c hiu vi tng loi amino acid v hot ng ca chng gii phng CO2 trong mi trng. Xc nh nng CO2 bng p k Warburg suy ra thnh phn ca amino acid. Phn ln trng hp iu kin pH thch hp thng nm trong vng acid, v d: pH 4,5 to ra L-glutamate 1-carboxy-lyase;EC 4.1.1.15 xc tc chuyn ho L-glutamic 25

amino butyric; pH 5,5 to ra L-tyosine-carboxy-lyase EC 4.1.1.25 xc tc chuyn ho Ltyrosine Tyramine v.v... TI LIU THAM KHO 1.Trn Th n, i Duy Ban, Nguyn Hu Chn, nh H, L c Trnh. 1980. Ho sinh hc. NXB Y hc 2.Phm Th Trn chu, Trn Th ng. 1999. Ho sinh hc. NXB Gio dc 4. Nguyn Vit Tu, 1980. Phng php nghin cu ho hc cy thuc. nh xut bn Y hc. TP H Ch Minh. 5. Copeland R. A., 2000. Enzymes; A Practical Introduction To Structure; Mechanism & Data Analysis. Willey-VCH. A John Willey & Sons, INC., Pub. 2nd ed. 6. Daniel L.C., 2002. Introduction to proteomics. Humana Press Inc. Totuwa, New Jersey. 7. Fersht A.,1998, Structure and Mechanism in Protein Science, W. H. Freeman, 3rd Rev Edit. 8. Hans U. B., 1974. Methods of Enzymatic Analysis. Second English Edition Acdemic Press, Inc., New York San Francisco London, Vol., 4. 9. Hollas J. M., 2004. Modern spectroscopy. John Willey & Sons. Ltd. 4th ed. 10. Lehninger A.L., 2004. Principle of Biochemistry, 4th Edition. W.H Freeman, 2004 11. Lodish H., 2003. Molecular Cell Biology. 5th ed.W.H Freeman. 12. Walker J. M., 1996. The Protein Protocols Hand book. 2nd ed. Humana Press Inc. Totuwa, New Jersey.

26

Chng 3
Peptide - cu trc v chc nng I. Tnh cht chung ca peptide
Peptide l nhng protein thng c cu trc on ngn khong t hai n vi chc amino acid ni vi nhau, c khi lng phn t thng di 6.000 Dalton. Chng c th c tng hp trong t nhin hoc c hnh thnh do thoi ho protein. Mc d c cu trc nh nhng nhiu peptide c vai tr kh quan trng trong hot ng trao i cht ca c th. 1.1. Cu to ca peptide.
Nh gii thu trn cc amino acid l nhng n phn t xy dng nn cc chui polypeptide. Trong cc chui cc amino acid c lin kt vi nhau thng qua lin kt peptide (hnh 3.1).

Hnh 3.1

S to thnh lin kt peptide

Dng cng ho tr

Dng ion +

27

Dng lai (hybrid)

Hnh 3.2

S tn ti cc dng ca lin kt peptide

Lin kt peptide (peptide bond) c bn cao bi cu trc ca n c 4 e/ , 2e/ thuc v lin kt C=O cn 2e/ thuc v b i e/ t do ca nguyn t N. Lin kt gia C-N l lin kt phc tp, n c th chuyn t dng n dng lai (trung gian) th b mt phn ghp i ca lin kt (hnh 3.2). Ngi ta cho rng t l ca lin kt kp ny l khong 30% i vi lin kt C-N v 70% vi lin kt gia C v O. Nh vy u ca mt chui peptide l amino acid c nhm -amino ( -NH2) t do c gi l u N-tn cng v u kia c nhm - carboxyl ( -COOH) t do c gi l u C tn cng. Lin kt peptide to nn b khung chnh ca chui polypeptide, cn cc gc R to nn mch bn ca chui (hnh 3.3).

Hnh 3.3

Mch bn v khung ca mt chui polypeptide

Nh vy lin kt peptide gia C-N khng ging nh lin kt gia C-N ca cc cht thng thng, v vy ko theo mt s hu qu sau: - Cc nguyn t C O N H nm trn mt mt phng v khng xy ra s quay t do xung quanh lin kt C-N nu khng c thm nng lng, trong H ca nhm NH lun v tr trans so vi O ca nhm COOH v cu hnh trans ca lin kt peptide l cu hnh n nh.

28

Hnh: 3.4

M hnh cu trc ca lin kt peptide

- Mt khc kh nng quay t do gia C v C gia N v C (hnh 3.4) l rt ln, lm cho mch peptide c khuynh hng hnh thnh cu trc xon. - Ngoi ra lin kt peptide c tnh cht ion mt phn lm tng tnh linh ng ca nguyn t N. Mt khc lin kt n gia C-N trong lin kt peptide c chiu di (1,33 Ao) hi ngn hn so vi a s cc lin kt C-C, C-N ( 1,47A o) v cc lin kt khc nn to cho chui peptide mt cu hnh khng gian c bn cao. 1.2. Cch gi tn v phn loi peptide Cn c vo s amino acid trong peptide gi tn ca peptide, nu c 2 amino acid gi l dipeptide, 3 amino acid gi l tripeptide, 4 amino acid gi tetrapeptide, 5 amino acid gi l pentapeptide v.v...cch gi tn cc peptide theo gc amino acid bng cch tn bt u t amino acid u tin ln lt n amino acid cui cng. Tr amino acid cui cng cn tt c ui ca cc amino acid u b thay bng ui - yl.

Hnh 3.5 Cu to v cch gi tn ca mt pentapeptide Seryl-glycyl-tyrosyl-alnyl-leucine

29

Ngi ta cng c th dng k hiu vit tt 3 ch hoc 1 ch theo th t cc amino acid biu th thnh phn v th t cc amino acid trong chui v d pentapeptide trn (hnh 3.5), c th vit Ser-Gly-Tyr-Ala-Leu hoc SGYAL. Thng thng ngi ta vit amino acid u N tn cng pha bn tri v amino acid u C tn cng pha bn phi v cng c th ghi r u no ca chui peptide l u N-tn cng hay u C- tn cng v nh vy cng pentapeptide trn c th vit H2N-Ser hay Leu-COOH. Trong trng hp c nhng amino acid mt on no trong chui peptide cha xc nh c r ngi ta c th cc amino acid trong ngoc chng hn Ala- Ser-Gly-(Ala,Leu, Val) Glu-Arg-...

1.3. Cc phn ng c trng ca peptide Ngoi phn ng ca nhm NH2 v COOH u tn cng, cc gc R ca peptide cng cho nhng phn ng mu c trng ca cc amino acid t do tng ng. Mt trong nhng phn ng mu c trng nht cho lin kt peptide l phn ng Biure, phn ng ny khng xy ra vi amino acid t do. Trong mi trng kim mnh, lin kt peptide phn ng vi CuSO4 to thnh phc cht mu tm v c kh nng hp th cc i bc sng 540 nm. y l phn ng c s dng rng ri nh lng protein. Phng php xc nh protein theo Lowry cng da trn nguyn tc ca phn ng ny bng cch thm thuc th Folin-Ciocalteau lm tng nhy ca phn ng sau khi thc hin phn ng biure, ng thi da vo cc gc Tyr, Trp nh thuc th to phc mu xanh da tri.

Hnh 3.6

S to phc mu tm trong phn ng Biure

II. Cc phng php tch phn lp v xc nh peptide C mt s phng php tch phn lp v xc nh thnh phn, s lng v trnh t amino acid trong peptide. Nguyn tc chung ca cc phng php tch phn lp v xc nh peptide v c bn cng nh i vi protein. Tuy nhin peptide l nhng on ngn ca chui polypeptide v th c th b qua giai on ct chui polypeptide thnh cc peptide nh m c th tch phn lp ngay bng phng php in di hay sc k tch ring tng peptide. Sau khi tch ring cc peptide ngi ta tin hnh thu phn n hon ton thnh 30

cc amio acid t do. T xc nh cc amino acid u N-tn cng v amino acid u Ctn cng. Cc d liu thu c qua phn tch s c so snh i chiu v tng hp li. V d, Puppy v Bodo phn tch mt peptide ca dch khi thu phn Cytocrom C thu c cc d kin sau y: - Thnh phn amino acid ca peptide sau khi c thu phn hon ton v sc k l 2Cys, 1 Ala, 2 Glu,1His, 1Thr, 1Val,v 1Lys. - Dng phng php Sanger xc nh c amino acid u N-tn cng l Cys v phng php carboxypeptidase xc nh c amino acid u C-tn cng l Lys. - Cu to ca peptide nh (bng cch thu phn tng phn ban u v xc nh cc amino acid , amino acid u N-tn cng v amino acid u C-tn cng ca mi peptide nh): Cys- Ala Cys-(Ala,Glu) Ala- Glu Glu- Cys Cys- His Glu (Cys, His) Thr (Val, Glu, Lys) Tng hp cc d kin trn, h xc nh c trnh t cc amino acid ca peptide nghin cu l: H2N-Cys-Ala-Glu-Cys-His-Thr-Val-Glu-Lys-COOH. y l nguyn tc chung xc nh mt trnh t trong peptide. Tuy nhin i vi nhng peptide di vic xc nh rt phc tp. III. S tn ti t nhin v vai tr chc nng ca peptide 3.1. Khi nim chung Trong t nhin tn ti nhiu dng peptide c chc nng quan trong lin quan n hot ng sng ca c th nh l cc hormon, cc cht khng sinh hay nhng cht tin thn ca t bo vi khun v.v... Bn cnh cng c nhng peptide chc nng cha r rng, c nhng peptide l sn phm thu phn ang cn dang d ca protein. Trong phm vi ca chng trnh ny xin c gii thiu mt s peptide quan trng, c nhiu ngha cho hot ng sng ca sinh vt. 3.2. Glutathion v cc cht tng t Glutation l mt tripeptide -glutamyl-cysteyl-glycine vi cng thc cu to nh sau: (Val- Glu) Thr (Val, Glu) Glu- Lys

31

Trong cu trc nhm SH ca cysteine l nhm hot ng, v vy ngi ta thng vit tt ch glutation l G-SH. Trong mi trng hot ng glutation c th nhng hydrogengen (H) thnh dng oxy ho v ngc li c th nhn H thnh dng kh: Nh phn ng trn, glutathion ng vai tr ca mt h thng oxy ho kh (vn chuyn hydrogen). Glutathion l mt trong nhng peptide ni bo ph bin nht, n phn b nhiu trong cc m v cc c quan nh: gan, thn, lch, tim, phi, hng cu v.v...

Dng kh

Dng oxyha

3.3. Cc hormon c bn cht peptide v protein 3.3.1. Adrenocorticotropic hormone (corticotropin, ACTH). ACTH hay cn gi l kch t v thng thn kch thch s tng hp steroid, adrenocortic v thng thn. L hormon polypeptide, cu trc phn t bao gm 39 amino acid, c khi lng phn t khong 4.500. Nghin cu cu trc v chc nng thy rng, cc on trinh t amino acid trong chui peptide c nhng chc nng hot ng sinh hc khc nhau: - on 24 amino acid u tin l phn hot ng sinh hc trong 13 amino acid u tin tng ng vi chui -MSH (melanin stimulating hormone- kch hc t) v 5 amino acid t 6-10 c hot ng ca MSH, 16 amino acid u tin ca ACTH mang tnh cht hot ng to ra steroid. - on trong chui t amino acid s 25 n 39 c s thany i tu theo loi ng vt. Trong c th ACTH kch thch ch yu t bo v thng thn bi tit ra hormoon chuyn ho ng(corticosteron,cortisol), ACTH xc tc phn ng hydrogenxyl ho v c bit l phn ng ct chui ngang ca cholesterol (tng hp steroid), phn ng 11 hydrogenxyl ho. 3.3.2. Cc hormon kch thch bi tit melanin (MSH). MSH c bi tit thu gia tuyn yn. Ngi ta chia MSH thnh hai loi: MSH gm 13 amino acid, ging vi 13 amino acid u ca ACTH nn c tc dng yu ca ACTH v - MSH. Hin nay ngi ta tng hp c MSH hon ton. S iu ho tng hp MSH c thc hin bi hai yu t: mt yu t c ch gii phng MSH l MIF (melanocyte release inhibiting factor) c cu to l mt trpeptide gm pro-leu-gly-NH2; mt yu t kch thch gii phong MSH l MRH (melnocyte release stimulating hormone), cu to l mt peptide gm 5 amino acid. MSH gy tng sc t ln, nhng MSH khng tn ti nh mt s hormon ring bit ngi. Hin tng sm da ca nhng con bnh nghin (addision) c th do ch yu l s tng tit ca MSH. 3.3.3. Oxytocin, Vasopressin Vasotocin. 32

Oxytocin l hormon thc , gy co d con, l mt peptide c 9 amino acid. ng vt c v, oxytocin ch khc s thay i ca 2 amino acid nh sau: amino acid v tr th ba l isoleucine v amino acid v tr th tm l leucine (Bng 3.1). Vasopressin ca loi ch nhi c cu trc trung gian gia vasopresin v oxytocin ca ng vt c v (amino acid th ba l isoleucin v amino acid th tm l arginin). Vasopressin l hormon gy co mch, l mt peptide c cu trc gm 9 amino acid. Phn ln ng vt c v amino acid th 8 ca vasopressin l lysine(lysVasopressin), tr ln v h m, amino acid th 8 l lysine (lys-vasopressin). Bng 3.1 VaSo snh cu trc ho hc gia oxytocin v vasopressin ca mt s loi ng vt Lysin Vasopressin soArgi-nin vasop- 1 2 3 4 5 6 7 8 9 ressin Cys-Tyr-Phe-Glu-Asn-Cys-Pro-Arg-Gly-NH2 Phn ln ng vt c v 1 2 3 4 5 6 7 8 9 Ln, H m Cys-Tyr-Phe-Glu-Asn-Cys-Pro-Lys-Gly-NH2

presVasotocin 1 2 3 4 5 6 7 8 9 ng vt c xng sng, khng c v

sin

Cys-Tyr-Ile-Glu-Asn-Cys-Pro-Arg-Gly-NH2

Oxytocin

ng vt c 1 2 3 4 5 6 7 8 9 xng Cys-Tyr-Ile-Glu-Asn-Cys-Pro-Leu-Gly-NH2 sng, c ( ch khc vasopressin chui bn amino acid v, chim th ba v th tm)

ng vt c v amino acid th 3 l isoleucine; vasopressin c tn l la argininvassotocin. Oxytocin c tc dng trn c trn ca t cung v tuyn v, gy co khi t cung sinh con v kch thch s tit sa khi cho con b. Vasopressin c tc dng chng li niu, tng cng ti hp thu nc thn, ng thi lm co mch, do c tc dng tng huyt p. 3.3.4. Cc hormon sinh trng (HGH). Hormon sinh trng ca ngi (human growth hormone) cn c tn gi STH (somatotropin hormone) l mt chui polypeptide bao gm 191 amino acidc khi lng phn t 21 kDa (L. Stryer, 1998) . Trong cu trc c hai cu disulfua c to thnh gia amino acid 53-165 v gia amino acid 182-189 (hnh 21). Hot ng sinh hc ca HGH l chui gm 134 amino acid. HGH c cu to rt ging vi hormon lactogen ca rau thai (85% amino acid ging nhau) v gn ging prolactin ca ngi (32% amino acid ging nhau) . Hormon sinh trng c tc dng s tng trng ni chung, kch thch s to sn hn l to xng, n cng l mt hormon chuyn ho. Hormon sinh trng kch thch s 33

tng hp protein t nhng amino acid c vn chuyn d dng vo trong t bo nh HGH, v l hormon gy tng ng huyt, sinh i tho ng ( kch thch s bi tit glucagon), ng thi kch thch s thoi ho lipid m bo nhu cu v nng lng cho c th, gy tng acid bo t do trong huyt tng. S thiu ht HGH nu xy ra trc tui dy th s dn n chng ngi ln, s d tha HGH nu xy ra trc tui dy th s xy n chng ngi khng l, nu xy ra sau tui dy th s dn n chng ngi b to d thng (pht trin chiu dy ca u, xng v mt). 3.3.5. Prolactin (PRL- kch nh t) Prolactin hoc LTH (luteotropic hormon), l mt polypeptide c khi lng phn t 23.000. ngi c cu trc 198 amino acid. cc loi ng vt s lng amino acid trong chui ging nhau khong 70%. Cu trc bc 1 v hot ng ca PRL c tnh cht tng t HGH v c hormon to sa ngun gc rau thai. Prolactin c bi tit lin tc khi c thai, chng kch thch th vng bi tit ra progesteron trc khi progesteron c bi tit bi nhau thai. PRL chun b cho tuyn v bi tit sa. Sau khi , khi t cung rng PRL m bo cho s bi tit sa. 3.3.6. Hormon tit (thyrotropin-TRH). TRH l vit tt ca hormon gii phng thyrotropin (tiotropin releasing hormone). Cu trc ho hc ca TRH d c Schally v Guilemin xc nh (nm 1969 v t gii thng Nobel 1977) l mt peptid ngn ch 3 amino acid l pyroglutamyl-histidyl-prolineNH2. TRH c chc nng tham gia vo qu trnh tng hp v bi tit TSH (kch gip trng t). TRH c chc nng va nh mt hormon gii phng, va nh mt hormon kch thch. 3.3.7. Insulin. T 1953, Sanger (gii thng Nobel 1958) nghin cu, tinh ch v xc nh hon ton cu trc ca phn t insulin. Phn t insulin bao gm 51 amino acid, c cu trc gm 2 chui polypeptide, vi khi lng phn t 5.700: Chui A c 21 amino acid. Chui B c 30 amino acid .

Hai chui c ni vi nhau bng 2 cu disulfua. Trong chui A cung hnh thnh 1 cu disulfua gia amino acid th 6 v amino acid th 11. Phn c hiu (c trng ca mt loi) ch tp trung vo cc amino acid th 8-9-10, 12-14 ca chui A v c bit l amino acid th 30 ca chui B (hnh3.7). Ngi ta cng xc nh c cu trc ba chiu ca insulin v thy rng cu trc phn t insulin c gi vng bi nhiu lin kt mui, lin kt hydro v lin kt cu disulfua gia chui A v chui B. Insulin c tc dng r nht trong tt c cc hormon ca tuyn tu, c bit i vi qu trnh chuyn ho glucid, n c tc dng h ng huyt. Insulin cn kch thch qu trnh tng hp v c ch qu trnh thoi ho glycogen c, gan v m m. c bit insulin tng cng tng glucose hp acid bo, protein v kch thch s ng phn. Tc dng quan trng nht ca insulin l kch thch s xm nhp glucose, mt s ng monose, amino acid trong t bo c v m. Do vy insulin lm gim lng
34

glucose trong mu. Ngoi ra insulin cng lm gim s tn to glucose do lm gim nng enzyme nh pyruvat carboxylase v fructose 1-6 diphosphatase.

3.3.8. Glucagon. Glucagon l mt peptide c tit ra bi t bo alpha ca o langerhans, cu trc phn t glucagon ngi gm 29 amino acid, vi khi lng 3.500. Lc u glucagon c hnh thnh di dng tin hormon, preproglucagon ri n proglucagon c 37 amino acid . Sau khi loi i 8 amino acid tr thnh glucagon. Glucagon lu thng trong mu di dng t do (khng kt hp vi protein), chng c tc dng lm tng nng glucose trong mu bng cch kch thch s phn hu glycogen gan qua trung gian ca AMP vng. Glucagon kch thch s tn to glucose v c ch s phn hu glucose bng cch c ch pyruvate kinase v phospho fructokinase. N cn c tc dng c ch tng hp lipid gan, nhng kch thch s to thnh cht cetonic. i vi protein, glucagon c tc dng thoi ho. 3.3.9. Calcitonin (CT) Calcitonin c pht hin t nm 1962, ngi ta xc nh c cu trc ca n l mt chui peptide gm 32 amino acid c mt cu disulfua gia amino acid th 1 v amino acid th 7 (hnh 3.8).

Hnh 3.8

S cu trc calcitonin ca ln

Calcitonin c tc dng iu ho calci mu. Calci huyt tng c duy tr nng 10 mg/100 ml nh calcitonin (cn c c PTH: parathormon v vitamin D). Cht i trng sinh l chnh ca calci l phosphate. ho tan ca calci phosphate rt thp v s tng ca mt ion ny s gy gim mt ion khc, nu khng phosphate calci s kt ta. Trong mu mt na calci kt hp vi protein, mt na dng t do v c hot ng sinh hc. Xng cha trn 99% calci ca c th v l kho d tr chnh ca calci khi c th cn n. 3.3.10. Angiotensin. Angiotensin c cu trc l nhng peptide c 7 amino acid ( AII-Angiotensin II) hoc 8 amino acid (AIII-Angiotensin III), chng c bi tit bi t bo thn. Chng c tc dng kch thch lp v ca t bo thng thn bi tit aldosteron lm tng lng natri trong mu khi th tch mu hoc natri gim. 3.3.11. Cholecystokinin-pancreozymin(CCK-PZ). L mt peptide c cu trc gm 33 amino acid. Hot ng sinh hc ca n gn lin ch yu C tn cng. Cholecystokinin c bi tit bi nim mc t trng khi c s tiu ho lipid v protein. Trong thc t nhiu t bo bi tit nhiu kiu CCK khc nhau v s 35

lng amino acid (c th l 58; 32; 8 hoc 4 amino acid). CCK c tng hp t mt hormon chung (pre-pro-cholecystokin) c 114 amino acid. Trong c th CCK c tc dng kch thch co bp ti mt, kch thch t bo nang ty bi tit enzyme amylase. 3.3.12. Secretin. c pht hin bi Bayliss v Starling vo nm 1902. l mt peptide gm c 27 amino acid, secretin c bi tit bi t trng nh kch thch ca pH acid. Hin nay ngi ta tng hp c hon ton phn t secrectin Secretin c tc dng kch thch bi tit bicarbonate nhm trung ho acid ca d dy (pH t 1,5-2,5 tng ln 7,0). Ngoi ra secretin cn c nhiu tc dng khc nh kch thch tit mt v bi tit pepsin. 3.3.13. Gastrin. Gastrin c bi tit bi t bo vng hang v ca nim mc d dy. Gastrin c cu trc l mt peptide bao gm 17 amino acid, c bi tit t mt hormon c 34 amino acid. Gastrin tng hp nhn to ch gm 4 amino acid cui cng (t 14 n 17) c hot ng y ca gastrin v c ng dng rng ri trong lm sng (phng php gastrin). Gastrin c tc dng kch thch t bo thnh ca tuyn d dy bi tit HCl v t bo chnh bi tit pepsinogen. Ngoi ra gastrin cn kch thch s vn ng ca d dy, rut, lm gin c vng ca mn v. 3.4. Cc peptide khng sinh l nhng peptide do vi vi khun hoc nm sn sinh ra. Nhiu peptide khng sinh l peptide vng cha cc amino acid dng D v L. 3.4.1. Penicillin. Penicillin c chit ra t dch nui cy nm Penicillium chrysogenum . Penicillin gm nhiu loi, chng c cu to gn ging nhau, bao gm mt vng tiasolidin, mt vng lactam, mt nhm amino c gn vi CO2 v mt mch bn (R), hnh 3.9.

Hnh 3.9

Cu to chung ca phn t penicillin

Ngy nay, ngi ta bit c nhiu loi penicillin chng ch khc nhau bi mch bn.V d: - mch bn ca penicillin F l CH3-CH2-CH=CH2- mch bn ca penicillin K l CH3-(CH2)3-CH2- mch bn ca penicillin O l CH2=CH-CH2-S-CH236

- mch bn ca penicillin G l

- mch bn ca penicillin G l

- mch bn ca penicillin V l

Penicillin ln u tin pht hin c tc dng chng vi khun gram dng Staphylococcus, Diplococcus...nhng hu nh khng c tc dng chng vi khun gram m v nm men. Vi thp nin tr li y pht hin ra nhiu loi penicillin mi trong mt s c hiu qu chng li vi khun gram m v nm men, v d: nng cao (10 mg/ml) pencillin c kh nng chng cc ni nm men Saccharomyces cerevisae n bi v E. coli. 3.4.2. Gramicidin. Gramicidin c pht hin t nm 1942 v bao gm hai loi l gramicidin S c 5 amino acid v gramicidin J c 24 amino acid, chng u l nhng peptide c cu trc vng. Vi nng vi mg/ml gramicidin c tc dng chng c Diplococcus pneumoniae, Streptococcus hemolyticus, Meningococcus, Staphylococcus aureus v Neiseria. Cc cht gramicidin c dng iu tr cc bnh nhim trng do t cu, lin cu qung, nhim trng mu hu sn, vim hng, vim mng no v.v... 3.4.3.Tyrocidin. Tyrocidin c pht hin vo nm 1939 bi Dubos khi ln men vi khun B. brevis. Tyrocidin kt hp vi gramicidin to thnh tyrothricin. Khi thu phn tyrocidin trong mi trng acid thy c cc amino acid ca dang L nh sau: ornitine, valine, leucine, proline, tyrosine, phenylalanine, tryptophan, glutamic, asparaginic. Trocydin lai tip tc phn hu thnh cc tyrocidin A, B v C. Cng ging nh gramicidin nng vi mg/ml tyrocidin u c tc dng chng cc loi vi khn nh Diplococcus pneumoniae, Streptococcus hemolyticus, Meningococcus, Staphylococcus aureus v Neiseria. 3.4.4. Bacitracin. Bacitracin c Johnson v cng s pht hin t nm 1945 t dch chit ca vi khun Baccillus licheniformi (thuc nhm B. subtilis). Bacitracin bao gm 10 loi khc nhau l : bacitracin A, A1, B, C, D, E, F1, F2, F3 v G. Trong bacitracin A chim nhiu nht (37%). Bacitracin A cu trc l mt peptide nh ch gm khong 10 amino acid sau: Lleucine, L-lysine, -L-isoleucine, L-cysteine, L-asparaginic, glutamic, D-asparaginic Dphenylalanin, D-ornitin. Bacitracin tuy khng c hot tnh chng vi khun gram m, nhng li c hot tnh mnh m chng vi khun gram dng, 1n v /ml c th chng c S. pyogenes, S.

37

hemolyticus, S. albus, Clostridium welchii, ...Bacitracin c dng nhiu trong chn nui v cng nghip thc phm. 3.4.5. Polymycin. Polymycin c pht hin cng mt lc vo nm 1947 bi ba nhm nh khoa hc ca M v Anh t dch chit ca vi khun Bac. Polymixa. Polymycin l mt hn hp bao gm cc cht gn ging nhau gi l polymycin A, B, C, E v M. Chng u l nhng peptide c tnh cht kim, d to thnh mui vi acid hu c v v c. Polymycin c hot tnh mnh m chng vi khun gram dng v m, c bit c kh nng chng cc vi khun m xanh (Ps.aeruginosa) khng li cc cht khng sinh khc. 3.5. Cc peptide c ngha sinh l t bo Cc peptide c ngha sinh l t bo thng ch yu l cc peptide hormon c gii thiu trn. Trong phn ny ch mang tnh cht nu ln nhng tnh cht c bn trong hot ng sinh l t bo. l nhng peptide c t 3 n khong 200 amino acid. N gm nhng hormon ca cc tuyn vng di i, tuyn yn, tuyn tu, v.v...S tng hp hormon peptide xy ra li ni cht nguyn sinh di dng mt chui polypeptide di hn, l mt tin hormon (pro-hormon) chng hn nh pro-insulin i vi insulin, proglucagon i vi glucagon...Nhng hormon peptide lu thng trong mu di dng t do, c na i sng ngn (thng di 60 pht), thi gian p ng ngn (vi giy cho tc dng tng ng huyt ca glucagon hoc chng li niu ca vasopressin). Cc hormon peptide khng vo trong t bo ch m tc dng trn b mt ca th th (receptor) c hiu mng t bo. 3.6. Cc peptide c chc nng bo v Trong c th ng, thc vt c nhng peptide lm nhim v bo v cho c th. Loi peptide u tin phi k n ng vt c xng sng l cc peptide khng th trong mu. Chng l nhng yu t nhn bit c lc cc tc nhn vi khun, virus v cc vt l xm nhp vo c th v loi tr chng ra khi c th. Ngoi ra trong mu ca ng vt trn cn c cc interferon vi mt nng nh c kh nng chng li s xm nhim ca virus. Trong mu ca ng vt cn c cc peptide chng chy mu nh fibrin v.v... thc vt c nhiu loi to ra nhng peptide c t, chi vi mt liu lng nh cng c kh nng git cht ngi v ng vt. Ngoi ra trong c th ng vt, thc vt v cc sinh vt khc ni chung u tn ti mt hp cht c bn cht peptide l nhim v bo v l lectin. V c kh nng lin kt ng mt cch c hiu v chn lc, nn lectin c th kt ta cc tc nhn hay t bo l c cu trc ng xm nhp vo c th bo v c th. V th nhiu ngi cn cho lectin l khng th thc vt (xem chng 1). TI LIU THAM KHO 1.Trn Th n, i Duy Ban, Nguyn Hu Chn, nh H, L c Trnh. 1980. Ho sinh hc. NXB Y hc 2.Phm Th Trn Chu, Trn Th ng. 1999. Ho sinh hc. NXB Gio 3. Nguyn Ln Dng, Phm Vn Ty, Nguyn nh Quyn. 1999. Vi sinh vt hc. NXB Gio dc. 4. L c Trnh. 1998. Hormon. NXB Y hc. H ni

38

5. Nguyn Vit Tu,1980. Phng php nghin cu ho hc cy thuc. nh xut bn Y hc TP H Ch Minh. 6.Copeland R. A., 2000. Enzymes; A Practical Introduction To Structure; Mechanism & Data Analysis. Willey-VCH. A John Willey & Sons, INC., Pub. 2nd ed. 7. Daniel C. L., 2002. Introduction to proteomics. Humana Press Inc. Totuwa, New Jersey. 8. Dennison C., 2002. A Guide To Protein Isolation. Kluwer Academic Publishers. New York, Boston, Dordrecht, Lodon, Moscow. 9. Hans U. B. 1974. Methods of Enzymatic Analysis.Second English Edition Acdemic Press, Inc., New York San Francisco London, Vol., 4. 10. Lehninger A.L., 2004. Principle of Biochemistry, 4th Edition. W.H Freeman. 11. Lodish H ., 2003. Molecular Cell Biology. 5th ed, W.H Freeman.

Chng 4

Cu trc v tnh cht l-ho ca protein I. Cc quan im khc nhau v nghin cu cu trc protein
Nh bit, protein l nhng phn chc nng ca c th, s hnh thnh chc nng mi da trn c s thnh phn cu trc ca protein, hay ni cch khc gia chc nng v thnh phn cu trc trong protein c lin h mt thit vi nhau. T s nghin cu cu trc protein c th da trn cc quan im: nghin cu thnh phn cu trc hoc da vo chc nng sinh hc tm hiu cu trc ca chng. Chng hn, vic nghin cu xc nh cu trc bc I ca phn t protein l bc u tin quan trng xc nh phn t hot tnh sinh hc v tnh cht ho l ca protein, l c s xc nh cu trc khng gian ca phn t protein. Da vo cu trc khng gian ca cc phn t protein tng ng, c th d on s nh v cu disunfua, cu trc khng gian ca protein nghin cu. Ngc li s xut hin mt bnh l c trng no lin quan n thay i chc nng ca protein m nguyn nhn ch l thay i 1gc amino acid trong phn t. V d: bnh thiu mu hng cu hnh li lim, khi nghin cu cu trc bc I ca hemoglobin bnh thng v bnh l xc nh c l do gc amino acid glutamic v tr th 6 trong chui ca hemoglobin A (bnh thng) b thay th bng gc amino acid valine II. Cc kiu lin kt trong cu trc protein 2.1. Cc lin kt cng ho tr Trong phn t protein ngoi cc lin kt cng ho tr bnh thng, ngi ta thng nhc n hai kiu lin kt cng ho tr c bit c ngha quan trng i vi cu trc v chc nng ca chng l: - Lin kt peptide (xem chng 3). - Lin kt disunfua (-S-S-). 39

Lin kt disunfua l lin kt ng ho tr to thnh do s kt hp gia hai phn t cysteine vi nhau loi i 2H (hnh 4.1). Lin kt disunfua (cn gi l cu disunfua) c th hnh thnh gia hai phn t cysteine trong cng mt chui polypeptide hoc gia hai cysteine thuc hai chui polypeptide khc nhau. Cu disunfua c vai tr quan trng trong vic duy tr cu trc bc III ca phn t protein. Nhng phn t protein cng cha nhiu cu disunfua th cng cht ch, vng bn. nhng protein khng tan nh protein ca lng, mng, tc, sng rt vng bn vi cc tc nhn ho hc, cha ti 12% l cysteine

Hnh 4.1 S hnh thnh cu disulfua gia hai phn t cysteine 2.2. Cc lin kt yu lm n nh cu trc protein 2.2.1. Lin kt hydro L tng tc yu hnh thnh gia mt nguyn t mang in tch m (gi l nguyn t nhn A-acceptor) v mt nguyn t hydro (H) ang nm

40

Hnh 4.2 sng

Mt s lin kt hydro quan trng trong h thng

Ghi ch: a) gia hydro ca mt ancohol v oxy ca nc; b) gia nhm carbonyl keto v nc; c) gia nhm peptide trong polypeptide; trong mt ni cng ho tr vi mt nguyn t khc (gi l nguyn t cho D- donnor). Ni cng ho tr gia D v H phi l ni phn cc v m my in t ca A phi mang nhng in t khng lin kt c kh nng thu ht in tch + ca H.
Nng lng cn ph v mt lin kt hydro l khong 5 kcal mol -1. Mt c im quan trng ca cc lin kt hydrogengen l H, nguyn t nhn A, nguyn t cho D u xp trn mt ng thng. Nguyn t N trong lin kt N-H cng nh O trong lin kt O-H u l nhng nguyn t cho chnh. Trong h thng sng l cc nhm amine (-NH2) v hydroxyl (-OH), s hin din ca cc nhm ny khin cho cc phn t c mang chng d ho tan trong nc do c s hnh thnh cc lin kt hydrogengen gia chng. c bit, cc phn t nc H2O - nhn t ch yu ca vt cht sng lun lun hnh thnh mt mng li u n nhng hnh t din d th lng hay th rn.

Hnh 4.3 S cu trc mng li hnh thnh bi cc phn t H2O

- Nguyn t oxy biu th bng cc vng trn ln - Nguyn t hydro c biu th bng cc vng trn nh

2.2.2. Lin kt ion L tng tc tnh in gia hai nhm c in tch ngc du. Trong nhiu trng hp cht v c, in t lin kt lun lun b ht v pha nguyn t c m in cao hn gy ra s phn li cation (nguyn t tch in tch m) v anion (nguyn t tch in dng), v d: NaCl Na+ + Cl41

V in t lin kt khng dc phn chia ng u cho hai nguyn t nn lin kt ny khng c xp vo loi cc lin kt cng ho tr. Khc vi cc lin kt cng ho tr hay lin kt hydro, cc lin kt ion khng c nh hng trong khng gian v in trng l khng ng u quanh ion. Trong mi trng nc, cc anion v cation lun lun c vy bc bi cc phn t H2O to thnh mt lp v bc ngoi nn khng th lin kt trc tip vi cc anion v cation khc. Do , ngi ta cho rng tng tc tnh in ny khng ng vai tr quan trong quyt nh cu hnh khng gian ca cc phn t hu c. 2.2.3. Lin kt Van der Waals L cc tng tc khng c hiu xut hin gia hai nguyn t khi chng tin li gn nhau. Tng tc ny khng do s phn phi lch ca cc in t gia hai phn t m do cc bin ng thong qua ca m my in t gy ra s phn cc nht thi trn phn t. Lin kt Van der Waals l kt qu ca lc ht v lc y. Hai lc ny cn bng mt khong cch nht nh, c trng cho tng loi nguyn t. Khong cch ny c gi l bn knh Van der Waals. y l lc lin kt yu nht, vi gi tr ch khoang 1 k cal mol-1. lin kt ny tht s c ngha, n phi tn ti mt s lng ln, ngha l b mt tip xc gia hai phn t phi cc i. in hnh l khi mt phn t c mang mt hc c hnh dng ph hp vi ch li trn phn t kia nh trng hp tng tc gia khng nguyn - khng th, gia enzyme - c cht. 2.2.4. Lin kt k nc (tng tc k nc) Cc phn t khng phn cc, tc l cc phn t khng cha nhm ion ho ln lin kt phn cc, u khng ho tan trong nc, chng l nhng phn t k nc. Lc thc y cc phn t hay cc vng khng phn cc ca cc phn t lin kt vi nhau thay v vi cc phn t H2O (y phn t H2O ra ngoi) c gi l lin kt k nc. y khng phi l mt lc lin kt ng ngha m l khuynh hng loi tr cc nhm khng phn cc ra khi mng li nc. Cn lin kt tht s tn ti gia cc phn t khng phn cc l lin kt Van der Waals. Cc tng tc k nc ng vai tr quan trng trong vic n nh cc protein, cc phc protein vi cc phn t khc cng nh s phn b cc protein trong cc mng sinh hc.

III. Hnh dng kch thc v cu trc ca phn t protein

3.1. Hnh dng kch thc Protein c khi lng phn t (Mr) tng i ln v thay i trong mt di rng t hn 10 nghn n hng trm nghn dalton (bng 4.1). Cc phn t protein c th c dng cu (k c hnh bu dc) hoc dng si. Thuc v dng cu hu ht l nhng protein c hot tnh xc tc hoc vn chuyn nh enzyme, hemoglobin, globulin...t l gia trc di v trc ngn ca phn t b hn hoc bng 20. cc protein hnh si t l ny ln hn nhiu v d tropocolagen (n v cu trc c s ca colagen) c chiu di khong 3000 AO, ng knh khong 15AO. Cc protein hnh si tng i tr v mt ho hc, ch yu gi chc nng cu trc chng c hc v d: colagen ca da, xng, sn, gn, rng; keratin ca tc, lng; fibroin ca t; myosine ca c v.v...

3.2. Cu trc bc nht (cu trc s cp)

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Cu trc bc I biu th thnh phn v trnh t cc amino acid trong phn t protein, cu trc ny c gi vng bng lin kt peptide-lin kt cng ho tr (xem chng 3). Cu trc bc I l bn dch ca m di truyn, vic xc nh cu trc bc I l c s tng hp nhn to protein bng phng php ho hc hoc bng cc bin php cng ngh sinh hc. V d: nm 1953, ln u tin Frederick Sanger (ngi Anh) xc nh trnh t sp xp cc amino acid trong phn t insulin v n nm 1966 protein ny ln u tin c tng hp bng phng php ho hc. Ngy nay ngi ta c th dng vi khun Escherichia coli tng hp insulin (s trnh by k hn chng 6).
3.2.1. Cu trc bc I ca mt s protein bit Bng cc phng php xc trnh t amino acid ca protein, ngoi mt s loi protein bit r cu trc bc I nh insulin c trnh by chng 3, hin nay nhiu loi protein khc bit c trnh t cc amino acid trong chui polypeptide nh: ribonuclease l mt protein c 124 amino acid, ni vi nhau thnh mt chui (hnh 4.4); hemoglobin l protein c 4 chui polypeptide, 2 chui ( mi chui 141 amino acid) v 2 chui (mi chui 146 amino acid); tripsinogen b (229 amino acid); chimotrypsin b (229 amino acid); alcohol dedhyrogenase nga (374 amino acid); glutamate dehdrogenase b (500 amino acid) v.v.. 3.1.2. Tnh quy lut trong cu trc bc nht ca protein Nhng protein ng th ca nhng loi khc nhau c mt s gc amino acid tng i khng i nhng v tr c bit v c nhng gc amino acid thay i, ngha l nhng loi khc nhau, cc amino acid khc c th thay th cho nhau. Th d insulin ca nhiu loi khc nhau c nhng amino acid khc nhau v tr 8, 9, 10 (bng 4.1), tng t nh i vi oxytocin, vasopressin, vasotocin mt s loi ng vt khc.

Hnh 4.4 Cu trc bc nht ca ribonuclesae ca b

Bng 4.1 S thay th amino acid trong chui A ca insulin mt s loi

Loi V tr 8 43 ca amino acid 9 10

B Ln Cu Nga C nh tng Ngi Ch Th

Ala Thr Ala Thr Thr Thr Thr Thr

Ser Ser Gly Gly Ser Ser Ser Ser

Val Ile Val Ile Ile Ile Ile Ile

3.3 Cu trc bc II (cu trc th cp) Biu th ca cu trc bc II l s xon ca chui polypeptide, l tng tc khng gian gia cc gc amino acid gn nhau trong mch polypeptide to thnh hai dng xon v xn . Ni cch khc, l dng khng gian cc b ca tng phn trong mch polypeptide. Cu trc ny c lm bn nh cc lin kt hydro c to thnh gia lin kt peptide k gn nhau, cch nhau nhng khong xc nh.

Hnh 4.5

Cc kiu xon trong cu trc bc II ca protein

3.3.1. Phng php nghin cu cu trc bc II Hin nay ngi ta c th dng nhiu phng php khc nhau phn tch cu trc bc II ca phn t protein nh ph hng ngoi, ph t ngoi- kh kin, ph cng hng t ht nhn, trao i hydro nng, o chit quang v.v..., c s ca mt s phng php thng hay c dng phn tch cu trc bc II ca protein d trn nhng nguyn tc ring sau: - Ph hng ngoi: ph hp th hng ngoi chnh l ph dao ng quay, v khi hp th bc x hng ngoi th c chuyn ng dao ng v chuyn ng quay u b kch thch. 44

Ph quay ca phn t khng nhng l phng php qu nhn dng cc cht m cn cho php xc nh chnh xc khong cch gia cc ht nhn nguyn t v gc gia cc kin kt. - Ph t ngoi- kh kin: Khi phn t hp th bc x t ngoi hoc kh kin th nhng eletron ho tr ca n b kch thch v chuyn t trng thi c bn ln trng thi kch thch. V th ph thu c gi l ph t ngoi kh kin (Ultraviolet and Visible Spectra, vit tt l UVVis) v cng c gi l ph hp th eletron. Mi trng thi eletron ng vi mt ng cong th nng v do ng vi mt gi tr xc nh ca tn s dao ng ring ca phn t. - Ph cng hng t ht nhn: Da trn nguyn l s dng cc ntron v cc proton trong ht nhn nguyn t, s lng spin ca proton v ntron u bng nhau v bng 1/2. Tu thuc vo vic cc spin ca nhng ht nucleon c cp i hay khng m ht nhn ca nguyn t c th c c trng bi mt s lng t spin ht nhn (I) bng khng hay khc khng. Nu ht nhn c mt spin khng cp i th I= 1/2, nu c nhiu spin khng cp i th I 1. Nu chiu vo mu dung dch protein sng v tuyn c tn s xc nh, th cc ht nhn mc nng lng thp s hp th nng nng lng ca sng v tuyn chuyn ln mc cao. Ngi ta ni lc xy ra cng hng t ht nhn (Nuclear Magnetic Resonance, vit tt l NMR). T ngi ta thit k my ph cng hng t ht nhn bao gm ng cha dung dch mu c t gia t trng ca mt nam chm mnh. Mt my pht cung cp sng radio. Mt my thu sng radio theo di s hp th nng lng thng qua cun cm bao quanh mu, tn hiu cng hng t c khuych i, phn tch v truyn sang bt t ghi v ph. My cng hng t ht nhn c pht hin t nm 1946, ng dng vo ho hu c nm 1953, ngy nay cng c pht trin v hon thin. N l cng c c lc cho cc nh ho hc trong vic xc nh cu trc phn t protein. - Trao i hydro nng: Da trn nguyn tc cc hydro nng H 2 (thng c k hiu l D-deuterium) v H3 (thng c k hiu l T tritium) thay th cho cc hydro bnh thng ang nm trong cc lin kt trong cu trc phn t protein. T nh hnh nh ph c trng c pht ra t cc loi hydro nng xc nh cu trc ca phn t. 3.3.2. Cu trc bc II ca mt s protein bit Theo Paulin v Cori (1951) cu trc bc II ca protein bao gm 2 kiu chnh l xon v phin gp Bng 4. 2. S lng xon v phin gp trong chui n mt s protein s gc (%) Protein (s gc) Chymotrypsin (247) Ribonuclease (124) Carboxypeptidase (397) Cytochrom C (104) 45
Xon 14 26 38 39 Phin gp 45 35 17 0

Lysozyme (129) Myoglobin (153)

40 78

12 0

Hnh 4.6

Lt ct ngang si tc vi chui xon keratin

trong tc ngi ta tm thy keratin (hnh 4.6) l loi protein c hai dng cu trc: dng bnh thng v dng dui thng.; cu trc phin gp tm thy trong fibroin ca t.

Cu trc xon hin nay c tm thy trong nhiu loi protein khc nhau Mt khc t l % xon trong cc protein khc nhau cng thay i kh nhiu. V d trong hemoglobin v mioglobin l 75%; lisozym l 35%; ribonuclease l 17% ... - Ngoi ra cn c kiu xon colagen c tm thy trong phn t colagen (hnh 4.7), n v cu trc ca n l tropocolagen bao gm 3 mch polypeptide bn vo nhau thnh mt dy cp siu xon (v mi mch 46

n c cu trc xon chiu cao ca mi gc xon trn trc siu xon ny l 2,9 anstron, mt vng xon l 3,3 gc amino acid . Ba mch polypeptide trong dy cp ni vi nhau bng cc lin kt hydro.

3.4. Cu trc khng gian ca protein 3.4.1. nh ngha v khi nim v cu trc khng gian

Bng phng php nhiu x tia X, ngi ta nghin cu cu trc khng gian ba chiu ca cc phn t protein, l hnh dng do s xon to xon v phin gp to thnh cu trc bc II, l hnh dng do s cun li ca cc chui c cu trc bc II to thnh cu trc bc III v v tr ca s sp xp cc protein c cu trc bc III trong khng gian to thnh cu trc bc IV (hnh 4.8).

Bc I

Bc II

Bc III

Bc IV

Hnh 4.8 S cc bc cu trc ca phn t protein

Trong cu trc khng gian ngoi lin kt hydro th lin kt cu disulfua trong cu trc bc II v c bit trong cu trc bc III c ngha ht sc quan trng gi cu trc cun khc ca chui polypeptide thnh khi. c trng cho potein hnh cu, l tng tc khng gian gia cc gc amino acid xa nhau trong mch polypeptide. Trong nhiu protein cu c cha cc gc Cys to nn lin kt disulfua gia cc gc Cys xa nhau trong mch polypeptide lm cho mch b cun li. Ngoi ra cu trc bc III cn c gi vng bng cc loi lin kt khc nh Van der Waals, lin kt hydro, lin kt tnh in gia cc gc amino acid v.v... Cu trc bc IV ch c trng cho nhng phn t protein c cu trc t hai hay nhiu chui protein hnh cu, tng tc vi nhau sp xp trong khng gian to nn. Mi mt chui polypeptide c gi l mt tiu n v (subunit), chng gn vi nhau nh cc lin kt hydro, tng tc Van der Waals gia cc nhm phn b trn b mt ca cc tiu n v lm bn cu trc bc IV.
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Nh vy ta c th nh ngha mt cch ngn gn cu trc khng gian ca protein l hnh dng ca phn t protein c cu thnh do s sp xp trong chui v gia cc chui polypeptide trong khng gian.
3.4.2. Xc nh khi lng phn t ca protein xc nh khi lng ca phn t protein ngi ta c th dng nhiu phng php khc nhau nh: phng php khuych tn, phng php phn tch rnghen, phng php tn x nh sng v.v... Tuy nhin, cc phng php khc nhau thng cho nhng kt qu khc nhau. Sau y xin gii thiu mt s phng php c tin cy cao v thng c s dng xc nh khi lng phn t protein. - Phng php ly tm siu tc Da trn s xc nh tc lng (hng s lng) ca protein trong dung dch chu s ly tm tc rt ln (hng trm ngn vng trong 1 pht) ri tnh khi lng phn t (Mr) theo cng thc

Trong R l hng s kh tnh theo erg mol-1 -1, T l nhit tuyt i, S l hng s lng tnh theo n v Svedberg (n v Svedberg c k hiu bng ch S v bng 10-13 giy), D l h s khuych tn, V l th tch ring phn ca protein, P l t trng ca dung mi. Bng 4.3 Mi lin quan gia hng s lng (S) v khi lng phn t ca mt s protein Protein Tr s S (n v Svedbrg) Cht c ch pancreatic tripsin Cytochrom C Ribonuclease A Myoglobulin Tripsin Carbonic anhydrase Concanavalin A Malate dehydrogenase Lactate dehydrogenase - Phng php in di Da trn nguyn tc l kh nng di chuyn v phn b trn gi th (thng l gel polyacrylamide hay agarose) ca tng loi protein trong in trng. So snh vi cc protein bit trc khi lng (protein chun) xc nh khi lng protein mu cn tm. Khi lng (hay trng lng phn t Mr) c xc nh tng quan vi s di ng in di ca mt phn t protein trn gel polyacrylamide cha SDS (SDS-PAGE). Ngi ta lp th chun theo log Mr ca cc protein bit khi lng (marker) t l hay tng quan i vi 48 1 1,83 1,78 1,97 2,5 3,23 3,8 5,76 7,54 Khi lng (KDa) 6,520 12,310 13,690 17,800 23,200 28,800 51,260 74,900 146,200

s di ng tng i ca cc protein. Cn c vo th ny s tnh c Mr ca protein cha bit khi lng phn t. Hnh 4.9.

Hnh 4.9

Cch lp th chun tnh Mr ca protein

- Phng php sc k lc gel (lc sng phn t) Ngi ta c th dng phng php sc k lc gel phn tch cc protein c khi lng phn t v kch thc khc nhau. Gel c dng thng l sephadex. l mt polysaccharide c bit, hydrate ho rt mnh thnh nhng ht gm nhng phn t polysaccharide kt hp vi nhau bng nhng lin kt ngang to nn nhng l ry phn t trong khng gian vi cc l c kch thc nht nh (lin kt ngang cng nhiu th l ry cng b v ngc li). Sephadex c nhi vo ct cng vi dung dch m ri cho hn hp protein chy qua, dung dch protein xung ct theo trng lc, cc phn t phn t protein nh c th lt vo l ry nn chy xung ct chm hn cc phn t protein ln khng lt vo l ry (hnh 4.10). Kt qu l hng c ring tng loi protein sau nhng thi gian nht nh. Xc nh khi lng protein bng cch dng phng php ngoi suy vi mt s protein mu bit khi lng phn t.

Hnh 4.10

S minh ho sc k lc gel

A-Hn hp gm c phn t ln v nh B- Cc phn t nh chui vo su trong l gel 49

C- Cc phn t ln ang c rt ra ngoi cn cc phn t nh tm thi c gi li

3.4.3. Cu trc khng gian mt s protein bit Nh s pht trin ngy cng tin b, ngy nay ngi ta bit cu trc bc I cng nh khng gian ca nhiu loi protein c vai tr quan trng trong c th ( xem bng 1.1). Ngoi cu trc khng gian ca mt s protein gii thiu trong phn cu trc bc II trn, t lu ngi ta cng bit cu trc khng gian (bc III v IV) ca nhiu protein c vai tr c bit quan trng khc nh: hemoglobin l protein c nhc n nhiu nht, c khi lng 64.5 kDa, c cu trc t 547 amino acid nm trong 4 chui polypeptide, 2 chui v 2 chui ; myoglobin ch gm mt chui polypeptide kt hp vi nhn hem v chymotripsin l protein c khi lng 22.6 kDa, cu to t 241 amino acid to thnh 3 chui polypeptide (hnh 4.11).

3.4.4. Cc iu kin lm bn vng cu trc khng gian Nh bit cu trc khng gian ca phn t protein c n nh ngoi nh mt s lin kt disulfua bn vng, th phn ln l nh cc lin kt yu nh lin kt hydro, lin kt Van der Waals v.v...V vy cc iu kin lm n nh cu trc ca protein l phi trnh nhng tc ng c th lm ph v nhng lin kt nh: tc ng mnh ca c hc, nhit , pH, cc mui kim loi nng v.v...(s c gii thiu k hn trong mc bin tnh protein phn sau).

Hnh 4.11

Cu trc ca khng gian ca mt s phn t protein 50

3.4.5. Tnh quy lut trong cu trc bc IV ca protein Cc protein c cu trc bc IV, phn t c th c cu to t hai cho ti hng trm tiu n v. Tuy nhin phn ln cc phn t protein c cu trc t cc tiu n v ng nht hoc t cc nhm tiu n v ging nhau, v th phn t protein thng c cu to i xng. V d: cu trc khng gian protein c xc inh u tin l hemoglobin c khi lng phn t 64,5 Kda, gm 4 chui polypeptide, hai chui (141 amino acid mi chui) v hai chui (146 amino acid mi chui). Nh phn tch cu trc bng tia X Max Peutz v John Kendrew (1959) pht hin s sp xp cc tiu n v trong hemoglobin thnh cp i xng, mi mt cp gm mt tiu n v v mt tiu n v . V vy, c th coi hemoglobin c cu trc 4 tiu n v hay 2 tiu n v (mi tiu n v gm c v ). Nhng protein cu trc t cc tiu n v ng nht c mt hay mt s nht nh kiu i xng nh i xng quay trn hay xon c. Nh vy cc tiu n v c th xp chng ln nhau quanh mt hoc mt s trc hay l ng xon c.

Hnh 4.12 Hai kiu i xng vng trn trong cu trc protein

Trong cu trc i xng quay trn cc tiu n v sp xp xung quanh mt trc to thnh dng cu trc ng, cc protein c cu trc i xng ng xon c to thnh cu trc dng m m nhng tiu n v c th xp thm vo theo ng xon c. C nhiu dng i xng quay trn m dng n gin nht l i xng vng trn, cc tiu n v c sp xp bao quanh mt trc duy nht (hnh 4.12). Ngoi ra trong s i xng quay trn c th phn b phc tp hn cn c gi l i xng hai mt, thm ch hai mi mt nh cu trc v ca mt s loi vius. Trong mt s t trng hp phn t protein c th gm nhiu tiu n v khng ng nht th cu trc ca chng c th khng mang tnh i xng v rt phc tp.

IV. Tnh cht l -ho ca protein

4.1. Tnh tan ca protein Cc loi protein khc nhau c kh nng ho tan d dng trong mt s loi dung mi nht nh, chng hn nh albumin d tan trong nc; globulin d tan trong mui long; prolamin tan trong ethanol, glutelin ch tan trong dung dch kim hoc acid long v.v... 4.2. Tnh ngm nc ca protein Trong mi trng nc, protein kt hp vi nc trng ln tr thnh dng keo hay ni cch khc protein trng thi hydrate ho, cc phn t nc bm vo cc nhm a nc trong
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phn t protein nh -NH2, -COOH..., lp o nc bao quanh phn t protein l mt trong cc yu t lm bn vng cu trc, ngn cch cc phn t protein khng cho chng dnh vo nhau thnh ta. 4.3. nht ca dung dch protein Khi protein ho tan trong dung dch, mi loi dung dch ca nhng protein khc nhau c nht khc nhau (bng 4.3). Ngi ta c th li dng tnh cht ny xc nh khi lng phn t ca protein ( nht cng cao th khi lng phn t cng cao).
Bng 4.4 Protein nht ca mt s protein Nng % (trong nc) Gelatin Albumin trng Gelatin Albumin trng 3,0 3,0 8,0 8,0 4,54 1,20 14,2 1,57 nht tng i (ca nc =1)

4.4. Hng s in mi ca dung dch protein Khi thm cc dung mi hu c trung tnh nh ethanol, aceton vo dung dch protein trong nc th tan ca protein gim ti mc kt ta do gim mc hydrate ho ca cc nhm ion ho ca protein, lp o mt nc, cc phn t protein kt hp vi nhau thnh ta. Nh vy, hng s in mi ca dung mi lm ngn cn lc tnh in gia cc nhm tch in ca protein v nc. Mi lin h c c trng bi biu thc:

Trong : D - hng s in mi ca dung dch F- lc tnh in gia cc ion tch in L1 , l2 - in tch cc ion, r - khong cch gia cc ion y lc tnh in gia cc ion t l nhgch vi hng s in mi v khong chc gia cc ion protein. 4.5. Tnh cht in li ca protein Cng nh cc amino acid, protein l cht in li lng tnh v trong phn t protein c nhiu nhm phn cc mnh (gc bn R) ca amino acid v d: nhm COOH th hai ca Asp, Glu; nhm NH2 ca Lys; nhm OH ca Ser, Thr, Tyr v.v...Trng thi tch in ca cc nhm ny
52

ph thuc vo pH ca mi trng. mt pH no m tng in tch (+) v in tch (-) ca phn t protein bng khng, phn t protein khng di chuyn trong in trng th gi tr pH gi l pHi (isoeletric-im ng in) ca protein. Nh vy protein cha nhiu Asp, Glu (amino acid c tnh acid mnh) th pHi trong vng acid, ngc li nhiu amino acid kim nh Lys, Arg, His th pHi trong vng kim. mi trng c pH < pHi , a s protein l mt cation, s in tch dng ln hn s in tch m. pH > pHi phn t protein th hin tnh acid, cho ion H+, do s in tch m ln hn s in tch dng, protein l mt a anion, tch in m.
Bng 4.5 Protein Pepsin Albumin trng Casein Albumin huyt thanh Gelatin 4,9 Prolamin 12,0 pHi 1,0 4,6 4,7 4,9 Gi tr pHi ca mt s protein Protein Globulin sa Hemoglobin Ribonuclease Tripsin Cytochrom C pHi 5,2 6,8 7,8 10,5 10,6

Trong mi trng c pH = pHi , protein d dng kt t li vi nhau v th ngi ta li dng tnh cht ny xc nh pHi ca protein cng nh kt ta protein. Mt khc do s sai khc nhau v pHi gia cc protein khc nhau, c th iu chnh pH ca mi trng tch ring cc protein ra khi hn hp ca chng. 4.6. S kt mui ca dung dch protein Mui trung tnh c nh hng r ti ho tan ca protein hnh cu: vi nng thp chng lm ho tan nhiu protein. Tc dng khng ph thuc vo bn cht ca mui trung tnh, m ph thuc vo nng mui v s in tch ca mi ion trong dung dch, tc l ph thuc vo lc ion ca dung dch ( = 1/2 C1 Z1 2 trong l k hiu ca tng, C1 l nng ca mi ion, Z1 l in tch ca mi ion). Cc mui c ion ho tr 2 (MgCl 2, MgSO4...) lm tng ng k tan ca protein hn cc mui c ion ho tr 1 (NaCl, NH4Cl, KCl...). Khi tng ng k nng mui trung tnh th tan ca protein bt u gim v nng mui rt cao, protein c th b ta hon ton. Cc protein khc nhau ta nhng nng mui trung tnh khc nhau. Ngi ta s dng tnh cht ny chit xut v tch ring tng phn protein khi hn hp. l phng php dim tch (kt ta protein bng mui). Th d dng mui amonium sulfate 50% bo ho kt ta globulin v dung dch amonium sulfate bo ho kt ta albumin t huyt thanh. 4.7. Biu hin quang hc ca protein
53

Cng nh nhiu cht ho hc khc, protein c kh nng hp th v bc x x nh sng di dng lng t h . V vy c th o cng hp th ca protein trong dung dch hay cn gi l mt quang thng k hiu bng ch OD (Optical Density). Da trn tnh cht ngi ta sn xut ra cc loi my quang ph hp th phn tch protein. Nhn chung protein u c kh nng hp th nh sng trong vng kh kin (t 350nm- 800nm) v vng t ngoi (t 320nm xung ti 180nm). Trong vng nh sng kh kin protein kt hp vi thuc th hp th mnh nht vng nh sng 750nm (nh lng protein theo Lowry). i vi vng t ngoi dung dch protein c kh nng hp th nh sng t ngoi hai vng bc sng khc nhau: 180nm-220nm v 250nm - 300nm. bc sng t 180nm-220nm l vng hp th ca lin kt peptide trong protein, cc i hp th 190nm. Do lin kt peptide c nhiu trong phn t protein nn hp th kh cao, cho php nh lng tt c cc loi protein vi nng thp. Tuy nhin vng hp th ny ca cc lin kt peptide trong protein c th b dch v pha c bc sng di hn khi c mt s tp cht ln trong dung dch protein. Mt khc chnh cc tp cht ny cng hp th nh sng t ngoi vng bc sng vng bc sng 180nm-220nm. V th trong thc t thng o hp th ca dung dch protein bc sng 220nm-240nm. bc t 250nm-300nm l vng hp th cc amino acid thm (Phe, Tyr, Trp) c trong phn t protein hp th cc i 280nm (xem chng 2). C th s dng phng php o hp th ca dung dch protein bc sng 280nm nh tnh v nh lng cc protein c cha cc amino acid thm. Hm lng cc amino acid thm trong cc protein khc nhau thay i kh nhiu, do dung dch ca cc protein khc nhau c nng ging nhau c th khc nhau v hp th bc sng 280nm.V c nh gi bng h s tt, v d: h s tt ca albumin huyt thanh b bng 6,7 khi cho nh sng c bc sng 280 nm i qua 1 cm dung dch c nng 10 mg/ml; trong khi h s tt ca khng th IgG bng 13,6. Ngoi ra c nhiu cht khc trong dung dch cng c nh hng n hp th protein. V vy, cc phng php o hp th vng nh sng t ngoi thng c dng nh lng protein c tinh sch hoc xc nh protein trong cc phn on nhn c khi sc k tch cc protein qua ct. 4.8. Kt ta thun nghch v khng thun nghch protein Khi protein b kt ta n thun bng dung dch mui trung tnh c nng khc nhau hoc bng alcohol, aceton nhit thp th protein vn gi nguyn c mi tnh cht ca n k c tnh cht sinh hc v c th ho tan tr li gi l kt ta thun nghch. Cc yu t kt ta thun nghch c dng thu nhn ch phm protein. Trong qu trnh kt ta thun nghch mui trung tnh va lm trung ho in va loi b lp v hydrate ho ca protein, cn dung mi hu c ho nc ph hy lp v hydrate nhanh chng. Trong ch phm protein nhn c cn ln cc cht dng kt ta, cn s dng phng php thch hp loi b cc cht ny. V d c th dng phng php thm tch loi b mui. Ngc li kt ta khng thun nghch l phn t protein sau khi b kt ta khng th phc hi li trng thi ban u. S kt ta ny thng c s dng loi b protein ra khi dung dch, lm ngng phn ng ca enzyme. Mt trong nhng yu t gy kt ta khng thun nghch n gin nht l un si dung dch protein (s ni k hn trong phn bin tnh protein phn sau).
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4.9. Cc phn ng ho hc ca protein Cng nh cc amino acid v peptide protein c cc phn ng ho hc tng t l: phn ng ca cc nhm -COOH, -NH2, gc R v phn ng to mu c trng ca lin kt peptide nh phn ng biure (xem chng 2 v 3. Ngoi ra, cn mt s phn ng mu c trng khc, c ngha quan trng trong pht hin protein v cc gc amio acid trong chui polypeptide: 4.9.1. Phn ng vi thuc th Folin-Ciocalteau Thuc th Folin-Ciocalteau c cha acid phosphomolipdic v acid phos phovolframic. Cc cht ny lm tng nhy ca phn ng biure, mt khc phn ng vi gc Tyr v Trp trong phn t protein. Cc gc amino acid ny tham gia trong qu trnh to phc cht mu xanh da tri. 4.9.2. Phn ng vi ninhydrin Tt c cc amino acid trong phn t protein u phn ng vi hp cht ninhydrin to thnh phc cht mu xanh tm, Phn ng c thc hin qua mt s bc nh sau: Di tc dng ca ninhydrin nhit cao, amino acid to thnh NH3, CO2 v aldehit, mch polypeptide ngn i mt carbon; ng thi ninhydrin chuyn thnh diceto oxy hindrien. Diceto oxy hindrien, NH3 mi to thnh tip tc phn ng vi mt phn t ninhidrin khc to thnh phc cht mu xanh tm (hnh 4.12)

Hnh 4.12

Phn ng ca protein vi ninhydrin

Protein cng c th tham gia nhiu phn ng to mu khc nh: phn ng xanthproteic, cc gc amino acid Tyr, Trp, Phe trong protein tc dng vi HNO3 c to thnh mu vng v sau khi thm kim s chuyn thnh mu nu; phn ng Pauli; cc gc Tyr, His trong protein tc dng vi diasobenzosulfate acid to thnh mu anh o; phn ng Milon gc Tyr tc dng vi thu ngn nitrate trong HNO3 c to thnh kt ta mu nu t v.v...

V. Bin tnh protein

5.1. Khi nim chung Sau khi protein b kt ta, nu loi b cc yu t gy kt ta m protein vn mt kh nng to thnh dung dch keo bn nh trc v mt nhng tnh cht ban u, chng hn ho tan gim, tnh cht sinh hc b mt gi l s bin tnh protein. V vy, i vi vic bo qun protein, ngi ta thng dung dch protein nhit thp thng l 0-4 oC. Song nhit
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 ny dung dch protein dn dn cng b bin tnh, bin tnh cng nhanh khi dung dch protein cng long. S bin tnh nhit thp ca dung dch protein long c gi l s bin tnh b mt: protein b bin tnh to nn mt lp mng trn b mt dung dch, phn di lp mng l nhng nhm a nc nm trong dung dch, phn trn lp mng l nhng gc k nc ca amino acid kt hp vi nhau bi lc Van der Waals. dung dch c cc phn t protein kt hp vi nhau cht ch hn do lm gim bt v hn ch s bin tnh b mt. bo qun tt cc ch phm protein nh enzyme, hormon, -globulin khng c t v.v...ngi ta tin hnh lm ng kh (lm bc hi nc ca dung dch protein p sut v nhit thp), bt thu c c th bo qun c ngay c nhit phng th nghim trong cc ng hn kn. 5.2. Cc yu t gy bin tnh C nhiu yu t tc ng gy ra s bin tnh protein nh: nhit cao, tia t ngoi, sng siu m, acide, kim, kim loi nng. V vy, trong thc t ngi ta rt ch nh hng ca cc yu t c kh nng lm bin tnh protein, v d: khi chit xut v tinh ch protein, c bit l cc protein enzyme, cng nh khi xc nh hot ca chng, phi ch phng bin tnh. Mun vy phi m bo nhng iu kin thch hp nht cho qui trnh k thut, nh tin hnh th nghim trong lnh v m bo pH thch hp ca cc dung dch s dng. 5.3. Tnh cht ca protein bin tnh Nhng thay i d thy nht protein bin tnh l thay i tnh tan, kh nng phn ng ho hc v hot tnh sinh hc nh: hemoglobin b bin tnh khng kt hp vi oxy c, tripsin khi b bin tnh khng thu phn c protein, khng th bin tnh mt kh nng kt hp vi khng nguyn v.v... Nghin cu cu trc khng gian cho thy khi b bin tnh phn t protein khng cn cun cht nh trc m thng dui ra hn, kt qu l ph v cu hnh khng gian cn thit thc hin hot tnh sinh hc. S bin tnh khng lm t lin kt peptide m lm t cc lin kt hydro, lin kt mui v.v...ni cc khc ca chui polypeptide hoc cc chui polypeptide vi nhau, v vy cu trc ca nhm k nc ca protein b o ln, cc nhm k nc quay ra pha ngoi v cc nhm a nc quay vo trong, s hydrate ho ca protein gim (protein mt lp o nc) cc phn t protein d kt hp vi nhau, tan gim v c th kt ta. S bin i cu trc khin protein bin tnh d c tiu ho hn protein nguyn thu, th d tripsin khng thu phn ribonuclease nguyn thu, nhng phn gii rt nhanh ribonuclease bin tnh. Ngi ta phn bit hai dng bin tnh: bin tnh thun nghch (bin tnh tr li dng ban u vi tnh cht v chc nng nguyn thu ca n, l s hon nguyn) v bin tnh khng thun nghch (protein khng tr li dng ban u ca n). Lng trng trng luc l mt v d in hnh v bin tnh khng thun nghch, cn v bin tnh thun nghch ta c th nu trng hp tripsin: un nng tripsin pH 3 ti 90 oC, cu trc ca phn t tripsin b bin i (bin tnh) nhng sau khi lm lnh mt thi gian nht nh, tripsin tr li cu trc ban u v li c hot tnh enzyme.

TI LIU THAM KHO

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1.Trn Th n, i Duy Ban, Nguyn Hu Chn, nh H, L c Trnh. 1980. Ho sinh hc. NXB Y hc 2.Phm Th Trn chu, Trn Th ng. 1999. Ho sinh hc. NXB Gio dc 3. H Hunh Thu Dng. 1998. Sinh hc phn t. NXB Gio dc 4. Nguyn Hu nh-Trn Th 1999, ng dng mt s phng php ph nghin cu cu trc phn t, Nh xut bn gio dc 5.Copyright by The Mc Graw-Hill Companies, 2003. Harpers Illustrated Biochemistry, Twenty-Sixth Edition, Langer Medical Publishing 6. Coreighton T. 1993. proteins, 2nd edition, W.H.Freeman and Company 7. Dennison D., 2002. A Guide To Protein Isolation. Kluwer Academic Publishers. New York, Boston, Dordrecht, Lodon, Moscow. 8. Fersht Alan,1998, Structure and Mechanism in Protein Science, W. H. Freeman, 3rd Rev Edit. 9. Lehninger A.L., 2004. Principle of Biochemistry, 4th Edition. W.H Freeman, 2004 10. Lodish H ., 2003. Molecular Cell Biology. 5th ed, W.H Freeman. 11. Walker John M. . 1996. The Protein Protocols Hand book. 2nd edition. Humana Press Inc. Totuwa, New Jersey.

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Chng 5

Cc phng php chit rt tinh sch v xc nh protein

I. Khi nim
Trong cc t chc ca c th sng, protein c th di dng t do trong cc dch sinh vt hoc di dng kt hp, hoc b cm trong cc t bo. Hn na, trong t bo c cha hng nghn loi protein khc nhau, nu ta cn nghin cu tng loi protein, trc ht phi chit rt v tinh sch chng. Chnh v vy, cc k thut chit rt, tinh sch v nghin cu protein lun v tr trung tm ca cc nghin cu ha sinh v lun c cp nht v hin i ha.

II. Cc bin php cn thit nhn protein nguyn th

Cc phng php chit rt v tinh sch protein u da trn nhng tnh cht ha l ca protein nh tch in, kch thc phn t, ha tan... ca protein cn chit rt. Nhiu protein cn lin kt vi cc phn t sinh hc khc nn vic chit rt cc protein ny cn ph thuc vo bn cht ca cc lin kt. Mun thu nhn c cc protein nguyn th tc l protein c tt c tnh cht t nhin c trng ca n, cn s dng cc bin php khc nhau. 2.1. Nhit tch cc protein khc nhau da trn kt ta ca chng, ngi ta c th s dng phng php bin tnh chn lc nh tc dng ca nhit. Mt iu cn lu l ch nn dng i vi trng hp cc protein enzyme bn vi nhit. Dch protein enzyme c gi 50 700C trong thi gian xc nh, sau protein tp b bin tnh c loi b bng cch lc hoc ly tm. Nh vy, dch chit protein th bn vi nhit c th c thu nhn bng cch cho kt ta khng thun nghch phn ln cc protein tp. thu nhn ch phm protein theo phng php kt ta thun nghch bng cc mui trung tnh, cn tin hnh nhit thp, i vi dung mi hu c cn tin hnh nhit di 00C trnh bin tnh c bit l protein enzyme. 2.2. Nng proton (pH) Protein l cc cht lng tnh, v vy, trong cc dung dch acid v kim chng s b phn ly nh sau:

Do cc acid amin trong chui polypeptide cn tn ti nhiu nhm chc t do di dng cc ion ha l nguyn nhn to ra tnh a in ca protein. Phn t protein rt di nn nhm ion t do tn cng ca chui polypeptide khng ng k, ch yu cc nhm chc t do khc ca chui bn (R) quyt nh tnh cht tch in ca phn t protein (nhm cacboxyl ca
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amino acid, OH ca Tyrosine, - NH2 ca lysine, guamidin ca Arginine, inidazol ca histidine) Mc ion ha ca cc nhm ny ph thuc vo gi tr pH. Cc nhm acid dng anion trong mi trng kim, cc nhm kim tn ti dng cation trong mi trng acid. Nh vy, mt gi tr pH xc nh, mi phn t protein c mt in tch tng s no y m ln ca n ph thuc vo s lng cc nhm tch in dng v tch in m. Kt qu l gi tr nng ion hydro c nh, cc protein khc nhau trong hn hp s c tng in tch khc nhau. Nhiu phng php dng tch cc hn hp protein u da vo c tnh ny. Cc phn t protein mang in tch tng s (dng hoc m) cng du y nhau ra xa nn d tan vo dung dch. Mi mt protein c mt gi tr pH nht nh m tng s in tch m v in tch dng trong phn t bng khng. Gi tr gi l im ng in ca protein. im ng in ca cc acid amin trung tnh c gi tr pH t 5,6 - 7,0; i vi cc acid amin c tnh acid (dicarboxylic) l t 3,0 - 3,2; i vi cc acid amin c tnh kim (diamino) l t 9,7 - 10,8. im ng in, ha tan ca protein l thp nht, protein d b kt ta. Da vo tnh cht ny, ngi ta c th tch tng phn cc protein enzyme trong hn hp. Cng ging nh trng hp tc dng ca nhit trong vic tch chit protein, c th dng phng php bin tnh chn lc nh tc dng ca pH ca mi trng. Dch protein enzyme c gi pH 5 trong thi gian xc nh. Protein tp b bin tnh cng c loi b bng cch lc hoc ly tm. V d citochrom C cng tan trong acid trichloracetic trong khi acid ny lm kt ta phn ln protein. Nh vy cc protein bn vi acid c th c tch chit bng cch ny. 2.3. Tc nhn ha hc C th dng mui trung tnh hoc cc dung mi hu c tch chit cc protein enzyme. Phng php ny c tin hnh da trn c s: ha tan ca protein ph thuc vo s tng tc ca cc nhm tch in trong phn t protein vi cc phn t nc. S tng tc (cn gi l s hydrate ha) s b gim xung khi thm vo dung dch protein enzyme cc dung mi hu c hoc cc mui trung tnh. Dung mi hu c thng dng l etanol, isopropanol, acetone hoc hn hp cc loi ru. Khi s dng cc dung mi hu c, cn ch tin hnh nhit thp (t 50C tr xung). Dng dung mi hu c c th tin hnh tch phn on di 00C v c th n -200C, nh vy c tc dng tt n n nh ca protein enzyme. Khi c kt ta, ch ly nhanh kt ta ra khi dung mi bng cch dng my ly tm lnh. Cc mui trung tnh c th dng l (NH 4)2SO4, Na2SO4, MgSO4... Tuy nhin, ngi ta nhn thy mui (NH4)2SO4 l tt nht v n khng lm hi m lm n nh (lm bn) hu ht cc loi protein enzyme. Loi mui ny li r tin v ph bin. ha tan ca n li rt ln (bo ha 767g/l 250C) Ngoi ra nng (NH4)2SO4 cn thit kt ta protein enzyme khc nhau th khc nhau nhiu. V d: Protease ca nm mc d b kt ta 70% ca (NH4)2SO4 bo ha hon ton, cn amilase ca mm la b kt ta 50% bo ha ca dung dch mui ny. iu ni ln tnh kt ta la chn ca (NH4)2SO4 cao hn cc mui khc.
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C th dng (NH4)2SO4 c 2 dng: dng bt v dng dung dch bo ha. Khi dng bt, ngi ta cho tng t mt vo dch chit protein enzyme. Cch cho cng nh hng ln n lng kt ta ban u ca protein enzyme. Khi cho mui vo dch chit cn phi c my khuy t m bo s ha tan ca mui. Khi dng dung dch bo ha, trong nhiu sch v phng php nghin cu, ngi ta a ra bng tnh s lng mui cn thit pha cc dung dch c bo ha khc nhau nhng nhit nht nh. Khi nim v s phn trm ca bo ha hon ton c cp n. Nh v d trn th protein enzyme c th b kt ta 50% (0,5) hoc 70% (0,7) ca bo ha hon ton ca (NH4)2SO4. Khi cho dung dch (NH4)2SO4 bo ha vo dch chit protein enzyme th nng (NH4)2SO4 khng tng t ngt. Sau khi kt ta xong ngi ta thng lng khong 2 gi hoc qua m, mc ch l to kt ta hon ton ( phng php dng dung mi hu c th khng cn lu). Kt ta c ly ra bng cch ly tm hoc lc qua phu Buchner. Khi ha tan kt ta li ngi ta thng thm ion Ca++ lm bn protein enzyme (dng CaCl2 hoc Ca(CH3 COO)2 tin li, ngi ta a ra cng thc cch tnh lng (NH4)2SO4 cho vo dch c bo ha cho trc (S1) t n mt bo ha cn thit (S2) Ty theo trng thi (NH4)2SO4 cho thm vo dch chit protein enzyme m c cng thc tnh ton khc nhau. - i vi (NH4)2SO4 dng bt

x(g) = Trong V l th tch dung dch, S1, S2 l bo ha cho trc v bo ha cn t (v d: S1 = 0,5; S2 = 0,7 chng hn). Ngi ta cng c th dng bn ton (nomogram) chiu v xc nh c lng (NH4)2SO4 thm vo dch chit protein enzyme t c mt bo ha nht nh. Hoc c th i chiu bng c sn. Lng (NH4)2SO4 a vo dung dch c bo ha nht nh c khc nhau ty theo nhit th nghim. - i vi (NH4)2SO4 dng dung dch bo ha. Th tch (tnh theo ml) ca dung dch bo ha cn cho vo 100ml dung dch c bo ha ban u S1 t n mt bo ha S2 cn thit c tnh theo cng thc sau:

V(ml) = Nh vy, nu thm tng phn cc dung mi hu c hoc tng phn mui (NH4)2SO4 (c nng bo ha khc nhau) th ta c th tch tng phn hn hp protein enzyme. Tuy nhin, phn chia hon ton nhng hn hp phc tp, phng php ny khng cho kt qu tt v ha tan ca mt s protein b tng ln. Mc du vy, cch kt ta tng phn ny cng rt c li, c bit l i vi giai on u ca vic tch chit v lm sch protein emzyme, v phng php kh n gin. Ngoi ra, cc tc ng khc nh c hc, sng siu m... c nh hng n qu trnh tch chit protein emzyme.

III. Ph v t bo v chit rt protein

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3.1. Ph v t bo Protein enzyme c trong tt c cc c th ng vt, thc vt v vi sinh vt. Sau khi c tng hp n c th c tit ra ngoi t bo tn ti trong cc dch c th, dch mi trng (gi l protein enzyme ngoi bo) hoc c gi li bn trong t bo (protein enzyme ni bo). Cc protein enzyme ni bo c th tn ti dng ha tan trong t bo cht v cc bo quan (nhn, microsome, mitochondria v.v...) ca t bo. T bo c bao bc bng mt lp mng.Lp mng ny vi khun i khi rt bn v dy. Ngi ta cn thy nhiu protein enzyme lin kt rt cht ch vi cc bo quan ca t bo. Cc phn t protein enzyme khng c kh nng i qua mng ca t bo v mng ca cc bo quan ca t bo. Do c th chit rt cc protein enzyme ni bo, bc u tin l phi ph v cu trc ca cc t bo c cha protein enzyme v chuyn chng vo dung dch. C th ph v cu trc ca t bo bng cc bin php c hc nh nghin vi bt thy tinh hoc ct thch anh, lm ng ha bng thit b nghin ng th (homogenizator). Thit b ny c chy thy tinh gn vi mt mt quay v c th iu chnh c tc quay theo yu cu. Cc t bo gia chy thy tinh v thnh ci s b ph hy. vic ph v c hiu qu, m thc vt, trc khi nghin, ngi ta thng thi nh mu vo ngn hoc cho trng nc. (v d nh i vi mu ht kh). Cn cc m ca ng vt nh gan hoc thn, khi chit protein enzyme ngi ta cn ct b cc m lin kt. Mun tch c cc protein trong cc cu t ca t bo, ngi ta cn phi dng cc yu t vt l v ha hc khc nh sng siu m, dng cc dung mi hu c nh butanol, acetone, glycerin, ethylacetat... v cht ty (detergent). Cc ha cht tt cho vic ph v cc bo quan ca t bo v trong cc c quan ny thng cha m. 3.2. Chit rt protein Sau khi ph v cu trc ca cc t bo tin hnh chit xut cc protein enzyme bng cc dng dch m thch hp, dung dch mui trung tnh hoc bng nc i vi protein enzyme ni bo hoc bng ly tm tch t bo i vi cc protein enzyme ngoi bo: vic chn phng php tch chit protein enzyme ty thuc vo tnh cht ca protein enzyme cn nghin cu.

IV. Tinh sch protein

4.1. Loi cc tp cht Trong dch chit th thu c ngoi protein enzyme cn c cc protein tp, cc cht cao phn t khc nh polysaccharid, acid nucleic v cc cht phn t nh nh ng monose, cc cht lipid, mui khong v.v... loi b chng phi s dng phi hp nhiu bin php khc nhau. loi b mui khong v cc loi ng... l cc tp cht c phn t lng thp ngi ta thng dng phng php thm tch (dialysis) i nc hay i cc dung dch m long hoc bng cch lc qua gel sephadex. loi b cc protein tp v cc tp cht c phn t lng cao khc, ngi ta hay dng kt hp nhiu bin php khc nhau: phng php bin tnh chn lc nh tc dng ca nhit hoc pH ca mi trng, phng php kt ta phn on bng mui trung tnh hoc cc dung mi hu c (xem 5.2.1; 5.2.2. v 5.2.3), cc phng php sc k trao i ion, in di, phng php lc gel. 4.2. Cc k thut thng thng trong tinh sch protein
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Nh trn trnh by, sau khi nhn c dch chit protein th, ngi ta thng s dng cc phng php khc nhau tch chit v ng thi lm tinh sch protein. Ngoi cc k thut c gii thiu c th cc phn trn, c th s dng cc phng php di y phc v cho vic tinh sch cc protein. 4.2.1. Ly tm Mt trong nhng k thut khng th thiu c trong vic tch chit v tinh ch protein l ly tm. My ly tm c s dng tch cc phn khc nhau khi dung dch. Ngi ta gi pha lng l cht lng bn trn kt ta, pha rn m thng lng kt xung y ng ly tm c gi l kt ta. Thc cht, s ly tm tng tc kt ta ca nhng tiu phn rn nh lc ly tm. S sai khc v t trng ca nguyn liu l lng so vi cht lng cng ln th tc kt ta s cng cao. Mc du ngi ta coi s vng quay trong mt pht l n v thng thng ca lc ly tm, nhng quy c y khng tha mn. Chnh v vy, mc tng lc ly tm c o mt cch chnh xc hn bng nhng bi s ca tr s gia tc ca lc ht mt bin, c ngha l c o mt lc ly tm tng i (relative centrifugal force, RCF) hoc l c o bng cc gi tr l bi s ca lc ht trng trng (xg). Cng thc tnh lc ly tm tng i l:

Trong r l bn knh n y ca ng ly tm tnh ra "pht" (foot, 1foot = 30,48cm), n l s vng quay trong 1 giy. C th tnh gi tr ca lc ly tm tng i mt cch trc tip theo cng thc: Lc ly tm tng i (g) = 1,1118 x 10-5 x R x N2. Trong R l bn knh (cm) np ly tm tnh t tm ca trc n y ng ly tm, N l s vng quay trong mt pht. Cng c th dng bn ton (nomogram) tnh lc ly tm tng i Thng thng c th lm kt ta nhng tiu phn ln vi lc ly tm tng i b bng cc my ly tm bn. lm kt ta nhng tiu phn b hn k c nhng thnh phn ca t bo cn c nhng my ly tm c hiu bo m c cc gi tr lc ly tm tng i cao hn 15000 x g i vi bt k nh lng no. C nhng nguyn tc lm vic an ton hn vi my ly tm i vi ngi thao tc cng nh i vi nguyn liu c x l m lc th nghim cn ch tun th. l nguyn tc cn bng i xng khi ly tm v thng bng my ly tm khi t my lm vic. Do tnh cht khng bn vi nhit ca phn ln cc protein enzyme, khi tch chit tinh ch chng, cn s dng my ly tm lnh. Trong trng hp iu kin th nghim c hn ch, c th s dng mt s phng cch nhm m bo duy tr mu nhit thp. Cn lm lnh tt mu v ng ly tm trc khi ly tm. Nu trong my c cc m thay th c th lm lnh chng trong t lnh trc khi ly tm. Cn phi tin hnh ly tm vi thi gian ti thiu trnh nng my. C th t trc tip my ly tm b vo t lnh, a dy dn ra ngoi qua lp m ca cnh ca t lnh. 4.2.2. Thm tch Thm tch l s khuch tn vi phn qua mng vn khng thm i vi nhng cht keo ha tan (protein, mt s cc polysaccharid) nhng thm i vi cc dng dch cc tinh th.Cc tinh th (cc mui, cc hp cht hu c c trng lng phn t thp...) c th khuch
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tn qua mng theo nh lut Fick. Nc s khuch tn t dung dch c nng thp hn (thng l dung dch ra) vo dung dch keo, trong khi cc ion (cation v anion) v cc cht phn t nh s chuyn vo dung dch c nng thp hn (thng chuyn vo dung dch ra). Trong qu trnh tch chit v tinh sch protein, loi mui ammonium sulphate ra khi dung dch protein th cho dung dch protein vo ci ti c hiu lm bng nguyn liu bn thm. Thng thng ngi ta hay dng ti colodion hoc cellophane (loi sau hay c dng hn). Sau t c ti vo bnh cha lng ln nc hoc lng ln dung dch m c pha long (v d m phosphate c pH = 7, nng 0,01M). V mng cellophane l mng bn thm, c kch thc l ch cho cc cht c phn t i qua vo cc dung dch m long theo nh lut khuch tn. Nh vy, mui s khuyn tn vo nc hoc dung dch m long (di chuyn theo hng gim nng ), cn nc hoc m long s di chuyn t dung dch ra vo ti cha protein. Protein l nhng i phn t khng th vt qua ti thm tch v c gi li trong ti. Bng cch thay i thng xuyn dung dch ra c th ty sch mui ra khi protein , mc du trong qu trnh thm tch, n l dung dch c pha long hn. C th lm gim bt hoc loi tr s pha long nh th khi tin hnh thm tch di p sut, c ngha l khi dung dch c x l nm di mt p sut thy tnh y , dng thy ng hc ca nc t dung dch s cn bng s khuch tn ca cc phn t vo dung dch. Phng php ny thng i hi c thit b c bit.

Hnh 5.1 Thm tch loi mui (NH4)2SO4 trong kt ta protein.

Phng php thm tch thng thng l cho dung dch c kt ta protein vo ti thm tch (khng qu 2/3 th tch ti). Cho thuc st trng hoc toluen bo qun protein (v thi gian thm tch lu, protein c th b thi hng). Buc ti vo mt que thy tinh, gc que thy tinh ln ming chu nc gi ti gia chu. Cho vi nc chy nh lin tc vo y chu thay i nc thng xuyn (hnh 5.1). Mui (NH 2)SO4 ha tan trong nc v b loi dn. Thi gian thm tch c th t 24 - 48 gi, lm thm tch ln cui cng bng nc ct. C th tng tc thm tch khi khuy dung dch ra bng my trn c hc hay my trn t hoc l quay chm ti nh ng c khng ln. Khi thm tch cc protein thng ngi ta tin hnh tt c cc thao tc mi trng lnh. 4.2.3. Sc k lc gel Sc k lc gel l mt trong nhng k thut sc k ct c dng ph bin trong tch chit v tinh sch protein. Dch chit protein enzyme c loi b phn ln cc protein tp nhng vn cha m bo ng nht cn thit c tip tc lm sch bng phng php sc k ct. Phng php sc k (chromatography) l do hai ch "chroma" l mu sc v "grapho" l vit, ngha l vit bng mu. Thu ban u, ngi ta s dng phng php sc k tch cc cht mu v ch sau ny ngi ta p dng cho vic tch cc cht khng mu.
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Sc k lc gel cn c gi l phng php dng cht ry phn t, lc gel (gel filtration) C s ca phng php lc gel l da vo s khc nhau nhau v kch thc, hnh dng v phn t lng ca protein enzyme c trong hn hp tch chng ra. m bo cho vic tch protein enzyme c tt, cht ry phn t phi l cht tr, khng phn ng vi protein enzyme. Cht ny cng khng ha tan v tng i bn vi cc yu t v c hc cng nh sinh hc. Ngoi ra cht c s dng cho mc ch lc phn t phi l cht khng c tnh n hi (khng co) v phi l cht a nc (hidrofil). Gel sephadex l cht tha mn cc yu t trn. Sephadex l ch phm dextran do cc loi vi sinh vt khc nhau l Leuconostoc to ra khi chng c nui cy trn mi trng cha saccharose. Trng lng phn t ca dextran c th t ti hng triu v ln hn. Phn t dextran bao gm cc chui do cc gc glucose to thnh cc lin kt 1,6. Sephadex Nhn t dextran bng cch x l ha hc (do tc dng ca epichlohidrin) to ra cc li phn nhnh c lin kt ngang gi l "sng phn t" v cht ny tr thnh khng tan trong nc. S lin kt ngang to ra cng nhiu, kch thc ca l sng phn t cng nh. Phng php lc phn t trn Sephadex c tin hnh nh sau: cho sephadex vo ct thy tinh di v cn bng bng dung dch m c pH nht nh. Sau cho dung dch protein enzyme ln ct. Khi lc v chit bng dung mi thch hp, cc phn t c trng lng phn t nh ( y l cc mui) s khuch tn chm chp qua cc l nh ca cc ht Sephadex b trng phng, cn cht c trng lng phn t ln hn ( trng hp ny l protein enzyme) khng c kh nng i vo m lch nhanh qua cc ht sephadex v s c chit nhanh ra khi ct (hnh 5.2 v hnh 5.3). V vy ta c th tch c cht c trng lng phn t cao hn thot ra khi ct gel trc so vi cht c phn t lng nh. Hng Sephadex (Pharmacia) ca Thy in tung ra th trng cc loi sephadex c kch thc khc nhau c k hiu t G10 n G200. S k hiu ch ra mc nhn (ht) nc ca chng. V d G10 ch khi trng phng th 1g gel kh nhn 1ml nc (1ml/g)

Hnh 5.2 Hot ng ca lc phn t sephadex.

Cc sephadex c k hiu khc nhau t G10 n G200 phc v trong vic lc phn t cho php cc cht c trng lng phn t khc nhau lt vo cc ngng khc nhau.

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Ngi ta cn s dng sephadex loi mui thay cho qu trnh thm tch. Cng nhm cht ry phn t c ngun gc polisacharid, l ch phm dextran nh sephadex (pharmacia) cn c molselect (Reanal) - l sn phm ca Hungary c ng dng nhiu trong nghin cu. C th dng lm c c cc cht c trng lng phn t ln nh protein, peptid, loi mui khi protein enzyme (dng nhanh hn so vi thm tch), lc gel tch theo trng lng phn t (nh protein huyt thanh)

Hnh 5.3 Tch cc phn t theo kch thc bng sc k lc gel.

hoc tch cc sn phm protein c hnh thnh di tc dng ca enzyme phn ct (nh - G - globulin b ct bi papain). Ngoi nhm cht ry phn t l ch phm dextran cn c nhm cht ry phn t l ch phm gel acrilamid bao gm Biogel (Bio - Rad) v Acrilex (Reanal). Acrilex gel l loi copolimer, sn phm ca Hungary c to ra t acrilamid v N, N' - metilen bisacrilamid. Cc acrilex gel c k hiu t P - 300 dng tch cc cht c trng lng phn t trong ngng t 100 - n 300.000. C th s dng vng pH t 2 - 11. Cht th ba l agarose gel, loi sulphate. Hay ph bin l loi sepharose (Pharmacia). Ngi ta thng dng cht ny tch cc phn t c trng lng ln hn 106. Tm li bng phng php lc ry phn t ngi ta th tch cc cht c trng lng phn t khc nhau c trong hn hp (nh polimer, polisaccharid, acid nucleic, protein). Ngi ta c th dng k thut ny loi mui thay cho qu trnh thm tch. V hn th na, trong qu trnh tinh ch protein enzyme, chng cn c s dng c c dung dch protein enzyme. 4.2.4. Phng php sc k trao i ion. Phng php sc k trao i ion da vo s khc nhau v in tch tng s ca cc protein enzyme. Hay ni cch khc, phng php ny c da trn c s ca phn ng trao i ion gia protein c tan trong nc hoc dung dch m long v cc tc nhn trao i
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ion. Tc nhn (hay nguyn liu) trao i ion c th l cht nha c tch nhm sinh ion hoc l cht ionit. y l nhng cht gi tr, khng tan trong nc, c bn cht l cellulose hoc cht gel dextran c li phn nhnh (Sephadex, Molselect) hoc l cht nha polistirol. Cht gi th ny thng kt hp vi cc nhm ion ha. Cc cht trao i ion c cht gi l celluose, sephadex, molselect thng thng c dng tch protein enzyme, cn cc cht trao i ion c cht gi l polistirol (v d nh Dowex, Amberlite) ch dng tch cc peptid c trng lng phn t nh hn. * Cc cht trao i ion c cht gi cellulose - Cationit CM - cellulose (carboxylmetyl - cellulose)- l mt dn xut este ca cellulose. Cellelose - O - CH2 - COOH Khi phn li cho ra COO-. y l cht trao i cation. Trn nhng cationit, th cc protein kim c tha nhng nhm amin v nhng nhm kim khc c hp ph (lin kt ion). S hp ph trn cc cationit c tin hnh vi nhng dung dch long pH 1,5 - 6,5. (Cc protein kim c cha cc acid amin diamino - mono carboxylic nh lys, Arg, His) - Anionit DEAE - cellulose (diethylamino - ethyl - cellulose) l dn xut este ca cellulose)

Trong H2O n c phn ly:

y l cht trao i anion, bn thn tch in dng. Cc anionit c p dng phn tch cc protein acid c tha nhng nhm carboxyl t do. S hp ph protein trn nhng ionit nh vy c tin hnh vi nhng dung dch m c lc ion thp (0,005 - 0,1M) pH 7,5 - 8,5. (cc protein acid c cha cc amino acid monoamin dicarboxylic nh glu, Asp) Trong hn hp cht ionit vi dung dch m c pH tng ng, cc cht ionit ni trn tr nn tch in. V vy trn b mt lp cht gi s hnh thnh mt lp in tch c du ph thuc vo kiu nhm chc ha hc ca n. Nu thm protein enzyme vo dung dch m th cc phn t protein enzyme mang in tch s b cc nhm tch in tri du ca cht trao i ion ko li. Khi dng mt dung dch m phn hp ph c pH khc hoc khi thm mt loi ion khc c lc ion ln hn th phn t protein enzyme s b y ra khi cht trao i
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ion. Khi tin hnh phn hp ph, thng ngi ta thm vo cc ion Na + v Cl- trong NaCl c nng tng dn theo bc thang hay theo gradient. Cc phn t protein enzyme no c in tch tng s nh th s b y ra trc do lc lin kt vi cht trao i ion yu. Cn nhng protein enzyme no c lin kt vi ionit ln hn th s b y ra bng mt lc ion ca mui ln hn. Nh vy, bng cch ny, chng ta c th tch c tng phn cc loi protein enzyme. Vic tch tng phn c la chn tt nht l khi tng dn nng cc ion thay th. Nh nng ion ca mui tng dn (gradient) ngi ta c th rt ra t ct cc loi protein enzyme khc nhau. C th thu nhn dch chit enzyme protein sau khi qua ct bng my thu phn on t ng. Theo th t tng phn dch thu c, ngi ta tin hnh nh lng protein theo cc phng php Lowry hay phng php o quang ph v xc nh ot hot ca enzyme. * Nu cht gi l sephadex th chng ta c cht trao i ion sephadex. l cc loi DEAE - sephadex v CM - sephadex. u im ca loi ny l va tch c protein enzyme v kch thc v v in tch tng s ca cc protein enzyme. Trng hp CM - sephadex trn cht gi sephadex c gn nhm COO-

COO-: mang in tch m l cht trao i cation 4.2.5. Phng php dng cht hp ph c hiu sinh hc hay l phng php sc k i lc (affinity Chromatography). C s ca phng php ny l ngi ta gn nhng phn t gn (ligand) vo cht mang (cht gi) rn bng lin kt cng ha tr m protein enzyme cn tch s tng tc c hiu vi n. Nhng cht c th l c cht (Substrate) hoc cht c ch (inhibitor) cnh tranh. Trong trng hp hai cht khng nguyn v khng th c i lc lin kt c hiu vi nhau, ngi ta thay th v tr cht gn vo gi th gia khng nguyn v khng th nghin cu tng loi ging nh c cht, cht c ch v enzyme c hiu ca chng. Hay ni cch khc dng cht ch c kh nng lin kt c hiu vi mt enzyme hoc protein ta nghin cu. Cht mang th rn c th l bt k mt loi no phc v cho lc gel nh sephadex, nhng ngi ta hay s dng nht l gel Sepharose trn ct gi th hay cht mang cha c cht c nh pH v lc ion ph hp, ch c protein enzyme no c kh nng chuyn ha c cht mi gn vo, cc protein khc th chy xung ct. Bng cch thay i pH v lc ion ph hp hoc c th bng cch thm cht c ch cnh tranh c ha tan vo th c th tch c protein enzyme khi ct trng thi sch. 4.2.6. Kt tinh protein. y l phng php c hiu tt nht tch tng phn protein enzyme giai on tinh ch cui cng. Khi protein enzyme c lm tinh khit hon ton, trong nhng trng hp ring bit, ngi ta c th tin hnh kt tinh chng. Mt iu cn ch l protein enzyme trng thi tinh th khng th c coi l bng chng v s tinh khit. Cc tinh th protein enzyme kt tinh ln u i khi c sch khng vt qu 50% v c th cha cc protein enzyme
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khc. Ngi ta thng tin hnh kt tinh protein enzyme trong dung dch (NH4)2SO4. Qu trnh kt tinh c th tin hnh t t ko di vi ngy thm ch hng tun nu mun nhn c cc tinh th tt. Thng thng l thm mui (NH4)2SO4 vo dung dch protein enzyme kh m c cho n khi lm vn c nh nhng dung dch. Sau t dung dch vo mt ni, ng thi tng rt t t nng mui trong dung dch. C th tin hnh tng nng mui theo nhiu cch, thm dung dch mui m c hn vo dung dch protein enzyme theo tng git, thm mui qua mng bn thm hoc c th cho bay hi chm chp dung dch protein enzyme. Trong qu trnh kt tinh c th thay i ch s pH hoc nhit . kt tinh protein enzyme c d dng, nhng giai on trc , ngi ta thng tch tng phn cc protein enzyme bng cc dung mi hu c. iu ny c l lin quan n vic cc cht c bn, cht lipid b loi ra khi dung dch protein enzyme to iu kin tt cho qu trnh kt tinh. 4.2.7. Lm kh v bo qun ch phm protein. Tnh c nh cu trc ca protein c bo m nh kh nng lin kt nc ca chng. Nu dung dch protein enzyme b kh nhit trong phng th a s protein enzyme b bin tnh. Protein enzyme ch c th c gi dang kh trong trng hp nu vic sy kh c tin hnh v cng nhanh hoc nhit thp. Nhiu protein nh chng ta bit, nhit thp c th c kt ta bng ru hoc acetone. Nu kt ta thu c bng cch em x l bng ru tuyt i, bng acetone hoc bng ether v sau lm kh tht nhanh th protein s khng b bin tnh. dng kh, bt protein c th gi mt thi gian lu, khi ha tan n trong nc n th hin nhng tnh cht ban u ca mnh. Tuy nhin, nhiu protein khng chu c cch x l nh vy. V vy, ngi ta s dng phng php lm ng kh l phng php lm kh protein thn trng nht, phng php ny c th s dng c cho hu ht protein. Dung dch protein c thm tch c lm ng bng v lm thng hoa bng p sut 0,01-0,001 mm thu ngn (c to ra nh my ht chn khng mnh). Ni nc c to ra u c ng bng trong bu d tr nhit thp hn (-70, -80 oC). Sau mt vi gi ch cn li bt protein. Sau bt ny (trong chn khng) c th c gi nhit cao hn. Protein c lm ng kh bng cch nh th khi ho tan trong nc ct s cho dung dch protein (nguyn th) ngay sau mt vi nm. Ngi ta bo qun huyt thanh mu bng phng php nh th, huyt thanh kh ny c th c s dng trong mc ch truyn mu. V. nh lng v nh gi tinh sch ca ch phm protein 5.1. Cc phng php xc nh hm lng protein Protein sau khi c tch chit v lm sch c th nh lng c. Nu protein nghin cu l enzyme th vic nh lng thng qua xc nh hot ca enzyme (theo quy c quc t: 1 n v hot enzyme l lng enzyme lm chuyn ho 1 mol c cht trong 1 pht cc iu kin tiu chun). Nh vy c sau cc bc tch chit v lm sch protein ngi ta thy tng lng protein gim nhng hot enzyme tng ngha l hot ring tng rt cao. Protein c coi l sch nu nhng bc tch chit v lm sch sau khng lm tng hot ring ca chng na, v ch khi ta mi hon thnh vic tch chit v lm sch protein. Nu protein khng phi l enzyme th ta c th nh lng chng bng cc phng php khc. Nu protein ta nghin cu l hormon hoc cc c t th chng c xc nh qua hiu ng sinh hc m chng gy ra; v d nh hormon sinh trng (growth hormone) s kch thch s sinh trng ca cc t bo ch nui cy. Nu l protein vn chuyn th c th xc nh chng thng qua nng cht m chng vn chuyn. Sau y l mt s phng php thng c s dng xc nh hm lng protein. - nh lng nitrogen theo phng php kjeldahl:
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Phn ln cc phng php gin tip xc nh protein u da trn c s xc nh lng nitrogen. Lng nitrogen c trong cc protein l gn ging nhau khng ph thuc vo cht lng v ngun protein. ng nhin trn c s lng nitrogen c th xc nh ch cc protein c tinh sch hoc lng protein ca cc mu nghin cu m ngoi protein ra khng cha nhng cht cha nitrogen khc. Trong nng nghip v cng nghip thc phm, protein th c nh lng bng cch xc nh lng nitrogen ton phn v kt qu nhn vi 6,25, ngha l coi protein lun lun cha 16% nitrgen. Thc t, trong thc phm, bn cnh protein cn c nhng cht hu c khc c cha nitrogen nh amid, alcaloid, ammonia (v d nh trong thc phm ln men) acid nitric... do hm lng nitrogen ton phn chnh thc cao hn 16% (16-17%) nhng protein thc vt th hm lng ny li thp hn 16%. H s 6,25 l h s trung bnh th. Trong mt vi trng hp, mun c kt qu chnh xc, nn dng nhng h s c bit. Nguyn l ca phng php ny l v c ho mu bng H2SO4 m c v cht xc tc. Dng mt kim mnh (NaOH hoc KOH) y NH3 t mui (NH4)2SO4. Sau hng NH3 vo dung dch acid c nng xc nh. Sau dng kim c nng xc nh chun acid d. T tnh c lng nitrogen c trong nguyn liu. - nh lng protein theo phng php Lowry: Phng php ny da trn c s dng my o mu xc nh mu sn phm kh ca phosphomolipden - phosphowolframate (thuc th Folin - Ciocalteau) vi phc hp ng protein. Phc mu xanh to thnh c th o bc sng 675nm. Cng mu ca hn hp phn ng t l thun vi nng protein. Da vo th chun protein (thng thng dng tinh th albumin huyt thanh b) ta c th tnh c hm lng protein trong mu nghin cu. Phng php Lowry c dng rng ri xc nh nhiu loi protein khc nhau. Phng php ny c nhy cao, cho php pht hin c protein trong dung dch nng 1 g/ml. Tuy nhin cng mu cn tu thuc nhiu vo loi protein. V d, cng mt nng , dung dch trypsin cho cng mu cao gp 3 ln gelatin, hemoglobin cho cng mu thp hn trypsin nhng cao hn gelatin. Ngoi ra, nhiu cht khc c th lm tng hay gim cng mu phn ng, v vy phng php ny cho kt qu chnh xc khi xc nh protein c tinh sch. - nh lng protein bng phng php quang ph: Phng php n gin nht o nng protein trong dung dch l hp th tia cc tm ca n. Nu protein tinh sch th nng tuyt i ca n c tnh theo gi tr o c. Nu protein khng tinh sch (v d, dch chit t mt sc k) th nng ca protein tng c tnh tng i t hp th. Nguyn tc ca phng php ny l cc protein hp th tia cc tm cc i bc sng 280nm do cc acid amin thm nh tryptophan, tyrosine v phenylalanine. hp th 280nm thay i tu loi protein nhng h s tt o c (ngha l hp th ca dung dch protein 1% vi ng sng truyn qua 1cm) cho mi protein cho php tnh nng ca protein tinh sch. Vi mt hn hp cc protein hoc vi bt c mt loai protein no m khng bit h s tt th nng protein c tnh nh sau: Nng protein (mg/ml)=1,55xA280-0,77xA260 Trong : A280: hp th bc sng 280nm A260: hp th bc sng 260nm
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Phng php ny khng dng c cho cc dung dch c nng protein thp hn 0,1mg/ml hoc khi c mt nhiu cht khc m hp th cng mt vng cc tm (v d, m, acid nucleic v mt s cht bo), hoc khi protein trong dch truyn ph ch khng phi trong dung dch. (V d, trong mng hoc cc phc hp c trng lng phn t ln). Cng cn ch l nu t l A280/A260 thp hn 0,6 ngha l dung dch protein cha sch, b ln cc cht khc, c bit vi acid nucleic th nn s dng phng php Lowry o nng protein. V vy, phng php o hp th tia cc tm thng c dng nh lng protein tinh sch hoc xc nh protein trong cc phn on nhn c khi sc k tch cc protein qua ct. 5.2. nh gi tnh ng th ca protein: Khi nhn c mt protein enzyme trng thi kt tinh, ngi ta phi th li mc tinh khit hay tnh ng th ca n. ng th ca ch phm protein enzyme phi c kim tra bng mt s phng php da trn nhng nguyn l khc nhau. Trong mt s trng hp protein enzyme c coi l ng th khi ly tm, nhng li c th phn chia thnh mt s isoenzyme bng phng php in di trn gel. Chnh v vy, nu dng nhiu loi phng php khc nhau kim tra sch ca protein m kt qu u cho l ng th th protein c th c cng nhn l tinh khit. Nhng phng php kim tra tnh ng th hay dng l xy dng th v ho tan, in di v siu ly tm. - Phng php kim tra tnh ng th (hoc cn gi l tnh ng nht) ca protein n gin v nhy nht l xy dng ng biu din v ho tan. Cch lm nh sau: Trong hng lot mu dng mt th tch khng i mt loi dung mi (nc hoc dung dch mui) lc vi nhng s lng enzyme khc nhau. Sau lc v xc nh s protein trong dch lc. Cui cng xy dng ng th. Trong nhng loi mu u, tt c cc protein thm vo b ho tan v s lng protein thm vo bng s lng protein c trong dch lc hay dch ly tm. Kt qu nhn c biu din l mt ng thng. Sau dung dch t c bo ho. Nu protein em ho tan l tinh khit ngha l ng nht th khi thm protein trong dch lc s khng tng ln v ng biu din c mt im un. Nu trong mu c mt protein th hai th sau khi t c bo ho i vi protein t ho tan hn, loi protein th hai cn c th ho tan c na. Kt qu l c mt im bo ho th hai v ng biu din c hai im un. Nu dch chit (hn hp) c nhiu protein enzyme th s c nhiu im un. Phng php ny c Northrop v Kunitz s dng rt c kt qu. Nay vn cn ng dng nhiu. - Phng php th hai xc nh ng th ca protein enzyme l phng php in di. Phng php in di l ng dng tnh cht lng tnh ca protein, da trn c s dch chuyn ca cc tiu phn ch phm protein enzyme mang in trong in trng. em ch phm protein enzyme in di pH v lc ion nht nh. Nu trn in di c mt bng protein th chng t protein enzyme l n th. Nu c hai bng chng t c hai protein enzyme trong ch phm . Bn in di ny c ua vo my detector pht hin khng ch nng ca bng in di m cn pht hin c s lng cc bng vt.

70

Hnh 5.4: ng biu din ho tan protein

- Phng php siu ly tm: y cng l phng php rt quan trng xc nh tnh ng th ca protein enzyme. Phng php c thc hin nh sau: Dng lc ly tm rt ln bng cch tng s vng quay ly tm ln hng nghn, hng vn vng trong mt pht. Vi tc ly tm rt ln, ngi ta c th tch ra c cc phn t enzyme c trng lng phn t khc nhau. Tc kt ta ca protein enzyme trong my siu ly tm c xc nh bng trng lng phn t ca n. Khi dng quay th cc phn t protein li khuych tn vo dung dch. Bi vy, cn phi quan st tc lng trong qu trnh siu ly tm. Chnh v vy, trong cc siu ly tm, ngi ta gn mt thit b quang hc c bit, v cc ng nh sng ca kt qu phn tch ln mt mn. Nu trong dung dch c mt loai protein enzyme (c mt trng lng phn t) th trn mn sng s cho ng th c mt nh. Nu lm vic vi hai protein enzyme th s c hai nh v.v... TI LIU THAM KHO 1. Nguyn Hu Chn, Nguyn Th H, Nguyn Nghim Lut, Hong Bch Ngc, V Th Phng. 2001. Ho sinh. Nxb Y hc - H Ni. 2. Phm Th Trn Chu, Trn Th ng. 1999. Ho sinh hc.Nxb Gio dc - H Ni. 3. Nguyn Tin Thng, Nguyn nh Huyn. 1998. Gio trnh sinh ho hin i. Nxb Gio dc - H Ni. 4. L Ngc T, L Vn Ch, ng Th Thu, Phm Quc Thng, Nguyn Th Thnh, Bi c Hi, Lu Dun, L Don Din. 2002. Ho sinh cng nghip. Nxb Khoa hc & K thut - H Ni.
71

5. Ajtai K., Szilagyi L..1985. Biokmiai gyakorlatok.Tankonyv. Jankonyv Kiado, Budapest. 6. Kerese I. 1984 Methods of Protein analysis. Akademiai Kiado, Budapest 7. Lehninger A.L. 2004. Principles of Biochemistry, 4th Edition. W.H Freeman, 2004 8. Stryer l., 1981. Biochmistry. W. H. Freeman and company, San francisco.

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Chng 6
Cng ngh sn xut mt s protein

I. Mc ch yu cu
T nhng hiu bit v kin thc rt c bn v bn cht, ngha ca protein, cng vi cc phng php chit rt, tch tinh sch protein thng thng qua cc chng c gii thiu trn, trong chng ny chng ti mun gii thiu cng ngh sn xut mt s protein t cc ngun nguyn liu khc nhau. Trong u tin cp n sn xut mt s loi protein ti t hp. Qua chng ny yu cu sinh vin nm c nguyn tc chung v cc bc chnh ca mt quy trnh sn xut, trc khi i vo nhng vn c th chng ta cn tm hiu thm mt s vn sau y: 1.1. ngha ca protein i vi qu trnh sinh hc Nh chng ta bit, mi sinh vt tn ti c nh lun gn vi iu kin ca mi trng thng qua qu trnh trao i cht. Qu trnh trao i cht bao gm nhiu khu chuyn ho trung gian, mi chuyn ho trung gian l mt mt xch ca cc qu trnh ng ho v d ho. Trong cc qu trnh sinh hc , ngoi cc cht glucid v lipid th protein v cc sn phm th cp ca n ng vai tr ht sc quan trng, to nn mt chu trnh trao i cht v nng lng cho c th. Ngy nay ngi ta bit c 1010- 1012 loi protein khc nhau cc dng c th sng trn tri t. Mc d trong c th, v c bn protein ng vai tr l cc thnh phn cu to ca cht nguyn sinh v l thnh phn cu to chc nng ca cc enzyme v mt s hormon, nhng cc sn phm phn gii ca chng vn c s dng lm cht liu tng hp nhiu cht khc nhau v c bit l vic cung cp ngun nng lng cho c th. V kt qu ca vic phn gii amino acid to thnh CO2, NH3, amin, cetoacid v hng lot trng hp cn to ra nhng cht kh phc tp thuc cc nhm hp cht hu c khc nhau. c bit trong cc sn phm phi k n cc qu trnh kh amin ho oxy ho, cc amino acid c chuyn thnh acid carboxylic v cung cp hydrogen theo phn ng chung sau y:

V d: Khi oxy ho proline to thnh pyroline-carboxylic, cht ny c th phn gii theo con ng to thnh aldehytglutamic, sau chuyn thnh acid glutamic hoc ornithine cung cp hydrogen. Bn cnh proline cng c th oxy ho dn dn to thnh hydrogenxy-proline, sau mt s bin i to thnh sn phm tham gia trong chu trnh Kreb cung cp ATP. Tm li, s phn gii protein tip n l cc amino acid l cc phn ng cung cp nng lng cho cc qu trnh sinh hc. T ch cung cp hydrogen xut pht t nhiu loi c cht to thnh mt phc h trong trao i cht, tu trung cc c cht, cui cng du nhng hydrogen cho chui h hp t bo to thnh cc phn t ATPngun d tr nng lng cho mi hot ng sng ca sinh vt. 73

1.2. Nhu cu khoa hc v x hi i vi protein Protein l hp phn ch yu quyt nh ton b cc c trng ca khu phn thc n, ch trn nn tng protein cao th tnh cht sinh hc ca cc cu t khc mi th hin y . Khi thiu protein trong ch n hng ngy s dn n nhiu biu hin xu cho sc kho nh suy dinh dng, st cn mau, chm ln tr em, gim kh nng min dch chng ca c th vi mt s bnh. Thiu protein s gy nh hng xu n hot ng bnh thng ca nhiu c quan chc nng nh gan, tuyn ni tit v h thn kinh v.v... Thiu protein s lm thay i thnh phn ho hc v cu to hnh thi ca xng nh lng calci gim, lng magie tng cao. Do , mc protein cao cht lng tt (protein cha amino acid khng thay th) l cn thit trong thc n ca mi la tui. Theo cc bo co ca UNO v FAO t nm1972, ngi ta i n kt lun rng vic sn xut protein ca th gii hon ton p ng nhu cu ca dn s hin ti trong thi gian . Tuy nhin 2/3 protein sn xut ra li ch c tiu th bi 1/3 dn s th gii. Tc sinh cc nc ang pht trin ln gp i so vi cc nc pht trin v theo tnh ton nm 2000 dn s th gii t khong 7000 triu ngi, nh vy nhu cu ca protein cng phi tng gp i so vi nm 1972. Ngoi ra nhng nc pht trin c mc sng cao, cc thc n hn hp dnh cho ng vt cng i hi cha cc ngun protein c cht lng cao sn xut ra trng, gia cm, b, ln... tiu chun. Cc thc n ny phi tho mn hon ton cc nhu cu dinh dng ca ng vt v thng l 10- 30% protein tnh theo trng lng. Ngoi nhu cu protein lm thc n cho ngi v chn nui th trong lnh vc y dc, s hiu bit v bnh hc mc phn t ang pht trin nhanh chng, nhu cu s dng cc ch phm t protein nghin cu cha bnh cho ngi v ng vt ngy cng tng. Ngy nay ngi ta c th sn xut cc thuc men qu him t protein nh cc hormon sinh trng, hormon chng tiu ng l insulin, cc globulin min dch, cc yu t ng mu, cc kch thch t sinh trng hng cu, cc khng th n dng, cc loi vaccine v.v...bng con ng cng ngh gene c th va to ra sn phm t nhin, va h gi thnh, li c th to ra mt lng ln nhanh chng m trc y bng con ng tch chit hay tng hp ho hc khng bao gi p ng kp nhu cu trong chn on v iu tr cc bnh. Hin nay nhiu cng ty tranh nhau sn xut cc sn phm ny theo phng php k thut DNA ti t hp (recombinant DNA technology) mang li nhng li nhun khng l hng nghn triu la mi nm. Chng hn loi protein albumin huyt thanh ngi (HSA) c c tch ra t mu ngi cho v c dng iu tr trong bnh mt huyt tng, thay th cht lng iu tr bng, sc chn thng hay mt s trng hp bnh nhn b phu thut. Loi ny trn th gii mi nm tiu th khong 300 tn v thu ring khon ny ti 1000 triu la mi nm. 1.3. Tnh hnh nghin cu v sn xut protein c hot tnh sinh hc Insulin l protein hormone kt tinh u tin (Abel, 1925); enzyme u tin c kt tinh l urease (Summer, 1926). Ngi ta tip tc i su vo nghin cu cu trc v chc phn ca protein cng nh mi lin quan gia chng. Vi cng trnh nghin cu ca nh sinh ho hc ngi M J.H. Northrop kt tinh c pepsin (1930), tripsin (1932) v chymotripsin (1935) v mt s cng trnh khc ngi ta cng hiu r hot tnh sinh hc ca cc loi enzyme ny. n nm 1953, Sanger xc nh c cu trc bc1 ca insulin gm c 51 amino acid; tip Hirs, Stein More v cng s (1960) xc nh c cu trc bc 1 ca enzyme ribonuclease gm 121 amino acid; cng nm Braunitzer xc nh c hemoglobin ngi gm 4 chui polypeptide, hai chui - mi chui141 amino acid v hai chui - mi chui 146 amino acid v hng chc cc protein khc nh cytocrom, papain v.v...Mc d t nm 1953 ln u tin Du Vigneaud tng hp c cc 74

hormon peptide oxytocin v vasopressin bng phng php ho hc v ch 10 nm sau (1963) Trung quc, M v Ty c tng hp c insulin theo mu cu trc bc 1 ca Sanger a ra; 1969 tng hp c ribonuclease v 1970 tng hp c protein sinh trng. T con ngi bc vo giai on tng hp nhn to cc protein mt cht c tm quan trong bc nht i vi s sng. Trong nhng thp nin 70 vic tch chit v nghin cu chc nng cc protein c hot ng sinh l, c bit trong lnh vc v tnh khng khun ca peptide din ra mnh m nh: vai tr bo v ca cc cationic protein trong cc ht bo tng (Zeya, 1971; 1975); protein BPI (bacterial permeability inducing factor) tch t bo tng bch cu trung tnh ca bnh nhn b bnh ung th bch cu tu mn tnh. Tnh cht cng nh trnh t amino acid ca protein ny c xc nh bi cc cng trnh ca Pohl (1990) v Morgan (1991). Vo nm 1993-1996 Shafer cng b nghin cu kh nng khng khun ca mt loi peptide tng hp ho hc da trn cu trc bc nht ca cathepsin G, ng cn cho bit thm rng peptide c tng hp t cc dng D-amino acid v dng L-amino acid c hot tnh khng khun tng ng. Bn cnh vic tch chit v nghin cu cc protein c hot tnh sinh hc trong t nhin v bng tng hp ho hc, ngy nay vi k thut di truyn hin i cc nh nghin cu xy dng c phng php mi thu nhn cc peptide c hot tnh sinh hc cao nhm chm sc v bo v sc kho cng ng. Da trn tnh t cc amino acid c xc nh t cc peptide tch chit ngoi t nhin hoc cc peptide c tng hp theo phng php ho hc, cc nh nghin cu tng hp cc on oligonucleotide hoc tch v nhn cc gene m ho cc peptide t nhin bng k thut PCR (Polymerase Chain Reaction), sau to cc vector ti t hp v bin np vo cc t bo thu nhn cc sn phm ti t hp c hot tnh mong mun. Mc ch sau to cc chng sn xut thu nhn sn phm mc pilot. y l phng php mi, song cng c nhng kt qu ng khch l v ang c tin hnh nhiu phng th nghim trn th gii. Thnh tu ni bt nht trong lnh vc sn xut protein ti t hp l cc loi vaccine nh phng chng vim gan B, st xut huyt, bnh u ma v.v...cho con ngi v nhiu loi vaccine cho ng, thc vt m chng ti s gii thiu lng ghp trong cc mc v sn xut vaccine phn di. Vaccine c coi l phng thc hu nghim nht phng chng v lm gim phn ln t l mc v cht do cc i dch ri ro a n. V nhng l do khc nhau, khng c mt quc gia no c c s cung ng vaccine ngay t u i dch m thng l nhiu thng sau . Nn sn xut vaccine ln mang tnh cht thng mi mun c vaccine cng phi c ti 3-6 thng sau i dch virus bng n. Gn y, ngi ta thng ni nhiu n cm gia cm, kh nng sn xut cc vaccine cm c tp trung nhiu chu u v Bc M. Nng lc sn xut hin nay c tnh 300 triu liu vaccine tam lin (ba thnh phn) trong mt nm, kh nng ny cn km xa vi nhu cu tng ln trong i dch. T chc y t th gii (WHO), qua mng li c bit cc phng th nghim cm quan st lin tc s din bin ca virus H5N1 t khi ca nhim u tin trn ngi Hong Kong nm 1997. Cc phng th nghim ny chun b chng vaccine nguyn bn (u tin), n ang c chng minh trong mt k ngh nh mt gc ging pht trin vaccine. Cc phng th nghim ny hng dn vic phn tch cu trc phn t ca virus, bo m gip cng vic pht trin vaccine tin trin. Mt phn vic quan trng l khm ph ra s t bin ca virus trong nm 2005.

75

Vi trong s 10 nc c cc c s sn xut vaccine ni a, hin ang tin hnh pht trin mt vaccine cho i dch. Mt vi ni trong s cc d n pht trin vaccine i dch tin n giai on th nghim lm sng, mong c thi gian ngn nht ca th nghin lm sng i vi cc vaccine d tuyn khc nhau. Mt cng ty cho bit s bo co kt qu th nghim lm sng vi WHO vo u thng 12 nm 2005. Hin ti cha a ra c mt cng thc vaccine cm c hiu qu v kinh t v vic s dng hm lng khng nguyn-thnh phn khng nguyn ca vaccine kch thch p ng min dch. Bi vy nhng cuc th lm sng din ra vi cch kim chng cc cng thc vaccine khc nhau, k c cht hp ph. Cht ny tng cng p ng min dch, v v l thuyt th n c th cho php bo v thch hp mc hm lng khng nguyn thp. Xu hng ny cng ang tin hnh. Nh chng ta bit, vaccine th cn cho mt i dch thc s ang ti gn, m vic sn xut thng mi th khng th bt u khi mt i dch virus bng n. Bi vy, WHO khuyn khch v mt k ngh v nhng c quan c thm quyn a ra nhng quy trnh nhanh chng cho vic cp giy php v s dng vaccine i dch. Vic ny WHO thc hin. Ngoi ra, WHO ang s dng din n Quc t c v cng ng quc t tm kim nhiu cch tng kh nng sn xut v bo m rng cc nc ang pht trin c c mt vaccine hiu qu trong mt n lc c th c v gi c. Tuy nhin, theo xu hng hin ti hu ht cc nc ang pht trin s cha c vaccine dng trong thi im bt u i dch m ch c c trong thi gian xy ra i dch Vit nam ta, trong nhng thp nin va qua tnh hnh nghin cu v sn xut cc cht protein c hot tnh sinh hc cng thu c nhng kt qu ng k. Nhiu cng trnh nghin cu phn lp v tch chit cc protein c hot tnh sinh hc c cng b. Tuy nhin, hu ht cc cng trnh ch cp n vic iu tra trong t nhin, nghin cu cu trc, chc nng v ng dng, m cha cp nhiu n sn xut cc loi protein . Mc d khng nhiu nhng cng c nhng ghi nhn bc u nh vic sn xut thnh cng mt s loi vaccine phng bnh ca Vin v sinh dch t Trung ng nh vaccine virus vin gan B(khng nguyn b mt- HbsAg), vaccine phng vim no Nht bn, vaccine virus di (khng nguyn G)v.v...Gn y nht (2003), bng k thut di truyn nhm tc gi L Trn Bnh (Vin cng ngh sinh hc-TTKH & CNQG) thu nhn c mt loi peptide ti t hp c hot tnh khng khun v mt loi protein khc c tn gi l melittin (loi protein c trong nc ong dng iu tr bnh nhim khun v lm tan cc khi u). II. Sn xut protein bng con ng vi sinh vt Ngay t thi c xa vi sinh vt vt c con ngi s dng ch bin v sn xut thc phm, n l mt b phn trong khu phn n ca con ngi v cc loi vt nui. Ngy nay vic sn xut protein t ngun vi sinh vt khng ch cn l mc ch ch s dng lm thc n n thun, m cn c s dng cho nhiu mc ch khc nhau na nh sn xut cc loi hormon, cc peptide khng sinh v cc cht min dch v.v... V nguyn tc chung: sn xut protein t vi sinh vt, tu theo mc ch ring s dng loi vi sinh vt (ging) v mi trng nui cy (ln men) thch hp, nhm to iu kin thun li cho vi sinh vt tng hp ti a lng cc loi protein mong mun. thu nhn ch phm protein t vi sinh vt, vic tch chit c tin hnh nh vic chit xut cc protein khc. Di y xin c gii thiu cng ngh sn xut mt s protein bng con ng vi sinh vt: 2.1. Sn xut protein n bo 2.1.1. Khi qut chung v sn xut protein n bo. 76

Protein n bo (SCP- Single Cell Protein), l tn gi theo quy c ch vt cht t bo vi sinh vt c s dng lm thc n cho ngi v ng vt. Thut ng ny tht ra khng chnh xc hon ton, v lng sn phm tao ra khng phi l mt protein thun khit m l cc t bo c x l thuc nhiu loi vi sinh vt khc nhau, c a bo ln n bo v bao gm vi khun, nm men nm si v to. So vi sn xut cc ngun protein truyn thng sn xut SCP c nhiu u th sau y: - Tc sn xut cao. - Hm lng protein cao(30- 80% tnh theo trng lng kh). - C th dng cc ngun carbon khc nhau (m mt s vn c coi l cht ph thi). - Cc chng c nng sut cao v thnh phn tt: d kim, d chn. - Din tch sn xut khng ln, cho sn lng cao (tr to). - Khng ph thuc vo ma v, kh hu. 2.1.2. Quy trnh sn xut SCP. Quy trnh sn xut SCP bao gm cc bc chnh sau y: - Chun b ngun C, thng l phi qua mt t hp x l vt l v ho hc. - Chun b mi trng thch hp cha ngun C v cc ngun N, P v cc cht dinh dng khc. - Ngn nga s nhim tp ca mi trng hoc thit b sn xut. - Cy vi sinh vt mong mun. - Tch sinh khi t bo vi sinh vt khi mi trng tiu dng. - Hu x l sinh khi tinh khit hay l khng. 2.1.3. Nhng vn cn lu trong sn xut SCP. 2.1.3.1. Nui cy: D l ln men din ra di iu kin v trng hoc iu kin sch u cn phi c bin php trnh tp nhim nh: un nng hoc lc cc thnh phn mi trng v kh trng thit b ln men; khc vi to, cc qu trnh sn xut SCP u cn thng kh mnh; nhit to ra phi c h thng lm lnh. 2.1.3.2. Thu hi sinh khi: Nm men v vi khun thng c thu hi bng ly tm (vi khun cn nng lng ly tm cao hn), trong nhiu trng hp cn c nhn t vn cc (kt bng/ kt cm). Cc vi sinh vt dng si c th thu hi nh ly tm vt (lc vt), gi thnh s r hn. Phi loi bt cng nhiu nc trc khi lm kh trnh tn km tr nhng ni c th phi nng v s dng lao ng gi r, tuy nhin sn phm s c cht lng thp. Tu thuc vo loi c cht v loi sn phm, sinh khi cn c hu x l loi cc thnh phn c cht hay thng gp hn l lm gim hm lng cc cht khng mong mun (v d acid nucleic) hoc thm ch tch ring phn protein. Mt nhc im quan trng ca SCP l thng cha hm lng acid nucleic cao, c bit l vi khun, nm men v nm si. Nu cc loi SCP dng lm thc n cho ngi th l c vn , bi v con ngi thiu enzyme uricase xc tc cho s oxy ho acid uric thnh allantoin ho tan hn. n nhiu cc dn xut purine s lm tng hm lng acid uric trong mu, acid ny kt ta s to thnh tinh th trong cc khp v ng gp vo vic to nn cc vin si trong ng tit niu. Theo Edozien(1970) hng ngy mt ngi ch nn n 20 gam nm men (tnh theo trng lng kh). 77

 c nhng phng php ra nhm lm gim hm lng acid nucleic trong SCP. Nhng thng th cc phng php x l y s dn n lm gim gi tr sinh hc ca protein n bo. Cc phng php chnh l: - Thu phn bng kim - Chit bng ho cht. - iu khin v sinh trng v sinh l t bo. - Hot ho RNA ase (bng x l nhit ngn). phng khng loi ra khi mi trng ngoi nhng s lng ln vi sinh vt k c dng cht ln dng sng. Khi dch thi c hm lng BOD (Biologycal Organic Demand) cao th cn phi x l trnh nhim mi trng. Sau khi x l mi trng c th ti s dng, vic ny nhm ng thi hai mc ch l gim lng nc mi cn thit v gim gi thnh. Theo tnh ton, nhu cu nc cn s dng l 18- 45 x 10 6 lt cho mt nh my sn xut 100.000 tn SCP/ nm. 2.1.3.3. Tuyn chn vi sinh vt. Mt vi sinh vt c s dng cho mc ch sn xut SCP lm ngun thc n protein cho ngi hoc ng vt cn phi c mt s c im l: - Khng gy bnh ng thc vt v ngi. - C gi tr dinh dng cao. - c chp nhn nh l mt loi thc phm hoc thc n gia sc. - Khng cha cc c t. - Gi thnh sn xut thp (ngha l ph thuc vo tc sinh trng; sn lng; hm lng protein; c i hi b sung cht dinh dng hay khng; c u th pht trin chn lc trn mi trng sn xut; d tch v d lm kh) v c th vi tng loi vi sinh vt nh sau: a) i vi to. to ngi ta thng dng ba chi sn xut SCP l: Chlorella, Spirulina v Scenedesmus chng c th c phng thc sinh dng l quang hp, ho tng hp hoc l d dng. Phng php hay dng l phng php quang hp. Trong trng hp ny nhn t gii hn l nh sng, v vy kinh t nht l dng cc h h di nh sng mt tri. Tuy nhin khi tin hnh nui to cc h thng c quy m ln th kh c th gi c cc iu kin v trng vi gi thnh thp v trong trng hp ny nguy c nhim tp l nghim trng. Mt khc, mt t bo khi nui to thng thp ( cc h thng nui ln ch thu c 1-2 gam/lt tnh theo trng lng kh) c th t c iu kin chiu sng tt, song iu ny s dn n chi ph tn km hn khi thu hoch. Ring to Spirulina c th thu hoch kinh t hn nh bin php lc hoc vt. Thnh phn cc cao phn t ca to thay i rt nhiu ph thuc vo cc iu kin sinh trng. Hm lng protein th (N x 6,25) c th chim ti 60%, thnh phn cc amino acid cn i, ring amino acid cha lu hunh thp. To cha nhiu sc t, mt u im khi c dng lm thc n gia sc, song li khng tt cho dinh dng ca ngi. Hin nay SCP ca ba chi to ni trn c th dng lm thc phm cho ngi tuy nhin vi mt t l thp. Ring Spirulina t lu vn c coi l ngun dinh dng protein l tng v r tin Mehico v vng b bc h Tchad ca chu Phi, to ny ngoi hm lng protein cao cn cha nhiu nguyn t vi lng v rt giu vitamin B12 ( gp 4 ln so vi gan b). h c th vt spirulina t cc thu vc t nhin. b) i vi vi khun. 78

Vi khun c th c dng sn xut SCP, tuy nhin cn ch mt s u v nhc im sau y: - Tc sinh trng nhanh. - Dng c nhiu loi c cht. - pH cn gi 5-7, nu khng s c nguy c b nhim vi khun gy bnh. - Thu hi bng li tm kh. - Hm lng protein th c th rt cao ( ti 80%) song hm lng bnh thng ca cc acid nucleic c bit l RNA cng cao (ti 20%) v cn phi c loi b. - Thnh phn amino acid cn i nhng hm lng amino acid cha S hi thp. - Khi dng cc vi khun gram m sn xut SCP cn lu kh nng sn sinh c t ca chng. c) i vi nm men Nm men c s dng sn xut SCP vi quy m ln t lu, c bit l vic s dng cc loi trong nhng chi nh Saccharomyces, Torulopsis v Cadida. Tuy nhin cng nh vi khun vic sn xut SCP t nm men cn lu cc c im l: - Thng tc sinh trng ca nm men tng i cao nhng khng bng cc chng vi khun sinh trng nhanh nht. - pH trong ln men thng gi 3-5, iu ny to iu kin thun li cho vic lm gim nguy c nhim trng. - C th thu hoch bng bin php ly tm lin tc. - Hm lng protein nm vo khong 50- 60%, tuy nhin hm lng acid nucleic c th ln ti 15%, v vy cn phi tin hnh cc bin php lm gim lng acid nucleic. - Thnh phn amino acid cn i nhng so vi vi khun th hm lng cc amino acid cha lu hunh cn thp hn. s dng cn phi b sung thm methionine. - Giu cc vitamin nhm B. d) i vi nm si. Khi chn ging nm si sn xut SCP trc ht cn lu c nhiu loi mang c t, chng thng xuyn e da tnh mng m c bit gy ung th cho con ngi. Ngy nay ngi ta tm c trn 20 loi hot tnh khc nhau cc c t nm si So vi vi khun v nm men, nm si thng c tc sinh trng chm hn. Gn y ngi ta phn lp c cc chng c tc sinh trng gn bng nm men v nm si c mt s c im sau: - Gii hn thch hp cho sinh trng kh rng (t 3- 8), tuy nhin trong sn xut cn gi pH < 5 trnh nhim khun. Vic nhim nm men thng hay xy ra nu mi trng khng c kh trng tt. - Khi nui cy chm nm si thng to thnh cc m si, iu ny c u im l lm cho vic thu hoch d dng, nhng li c nhc im l hn ch s phn b ng u khng kh trong ton b h si. - Hm lng protein th nm trong khong 50- 55%, song khi sinh trng nhanh hm lng acid nucleic s cao (RNA ti 15%). - Thnh phn amino acid ca nm si cn i, tuy nhin cc amino acid cha lu hunh cng c mt nng thp. 2.2. Tng hp cc hormon bng vi sinh vt 79

Cch y khong 60 nm v trc, cc nh khoa hc mi bit cch s dng nm ch to thuc khng sinh penicillin v ch to cc loi thuc khng sinh khc c s dng rng ri hin nay. Hn 20 nm trc y din ra mt cuc t ph thc s mang li li ch to ln cho con ngi c bit trong lnh vc y dc. Lc , cc nh khoa hc bit cch a gen ngi vo phn t ADN ca vi khun v t bo ng vt, ri buc chng phi sn sinh ra mt loi protein c tc dng cha bnh. Sn phm u tin ca phng php l insulin (mt loi hormon chng bnh i tho ng) c Hng Eli Lilly&Co pht minh nm 1982 thng qua vi khun bin tnh gen E. coli. Ngy nay c hn 130 dc cht sinh hc c chnh thc cng nhn bi Tng cc kim sot tn dc ca M v c s dng trong y hc. y a s khi lng hng bn i thuc cc loi dc phm nh erythropoetin, interferon, hormon tng trng v insulin con ngi ca mt s hng sn xut ng u th gii . 2.2.1. Sinh tng hp insulin bng E. coli. Trc y insulin c tch t tuyn tu ca b, ln i khi gy d ng cho ngi. Trong c th insulin c tng hp di dng proinsulin gm c ba chui polypeptide l A, B v C. Khi chuyn thnh insulin th chui C c loi b. Bng k thut ti t hp DNA, ngi ta chuyn gene m ho insulin vo vi khun, v khi c nui cy trong mi trng thch hp, vi khun E. coli s sinh tng hp to ra loi peptide ny. Quy trnh sn xut theo phng php ny c th tm tt gm cc bc c bn sau y: - Chun b on oligonucleotide m ho cho insulin: da vo trnh t v cu trc ca cc amino acid ca insulin gm c 51 amino acid v 2 chui polypeptide A v B ni vi nhau bng hai cu disulfua. Ngi ta to dng gene ring bit m ho cho hai chui A v B - Chun b vector (c th dng plasmide ca vi khun hoc nm men). - Dng enzyme hn ch ct plasmide v ni on gene m ho cho insulin to vector ti t hp (loi thng dng l pBR 322 ). - Chuyn vector ti t hp pBR 322 vo vi khun E. coli. - Nui cy (ln men) vi khun E. coli trn mi trng thch hp. - Tch chit v thu nhn sn phm l hai loi polypeptide A v B ring. -Trn hai loi peptide li vi nhau v x l bng phng php ho hc hay enzyme to cu disulfua. - Kim tra cht lng ca sn phm Vic tch chit v tinh sch insulin c th tin hnh theo cc cc phng php sinh ho thng thng nh kt ta, ly tm v sc k. Th hot tnh ca insulin (hin nay c dng ph bin l k thut ELISA). S dng vi sinh vt mang gene ti t hp sn xut cc loi hormon ni chung, c mt s vn cn lu l: a) Cc peptide c to ra khng phi l sn phm ca gene t nhin m c dn ra t mt gene tin thn (precursor gene). b) Mt khc vi sinh vt th khng c cng c ch hot ng nh l c th bc cao nn khng c s bin i sau dch m (post-translation modifications) nh glycosyl ho, methyl ho hay amin ho v.v... c) Hn na peptide khng bn trong mi trng vi khun, d b thoi ho bi s tiu ho ca nhng enzyme to thnh nhng peptide ngn. 2.2.2. Sn xut yu t gii phng hormon sinh trng.
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Yu t gii phng hormon sinh trng (GRF- Growth hormon Releasing Factor) l mt peptide c 44 amino acid vi u tn cng c nhm NH2 c bi tit vng di i trong c th. Bng con ng cng ngh gene c th thu c GRF 1-44 NH2 ti a v c gi thnh r hn so vi phng php tng hp ho hc.
Quy trnh sn xut GRF bng k thut DNA tai t hp cng bao gm cc bc chnh tng t nh i vi insulin. Tuy nhin, i su vo chi tit k thut bit, hin nay c ba phng php sinh tng hp GRF theo con ng cng ngh gene l: a) Phng php 1. Trc ht cn thit k gene tng hp nhn to cho GRF 1-44 OH. Song cn ch rng amino acid tn cng khng th thu c bng amin ho trc tip vi khun v gene nhn to cn c b m ho cho Met-GRF 1-44 OH codon m u ca gene. Khi thu c GRF 1- 44 OH th Met cn phi c tch ra ngoi. Hn na protein ny ngn d dng b phn hu bi protease ca E. coli v nh vy GRF c th b thay i cht t trc khi tch t mt lng nht nh. b) Phng php 2. Mt hng khc l lin kt mt gene peptide GRF vi mt gene protein ca vi khun, trn c s protein ca vi khun bo v peptide GRF khi b thu phn. Sau protein ny s c tch ra bng con ng ho hc hoc enzyme. c) Phng php 3. Hng th ba l dng s chn ca enzyme t -pheromone trong nm men. Gene GRF c dung hp vi pheromone v nh vy protein lai cha nhng tn hiu Lys- Arg(Ala)- 2 hoc 3 trong u ca GRF 1- 44 OH. Sn phm GRF c bi tit vo mi trng nui cy. Cui cng GRF 1-44 OH c tch, lm sch v amin ho bng phng tin enzyme. 2.3. Sn xut cc cytokin Cytokin ngy nay ang tr thnh mt hng kinh doanh ln cnh tranh gia cc cng ty Tonen ( Nht bn), Genetech (M), Delta (Anh), Rhone Poulene (Php). Gn y ngi ta pht hin hng lot cc cytokin c bi tit ra t hai nhm khc nhau ca t bo lymphocyte T. Nhm Th1 bi tit interleukin 2 (IL-2) v -interferon (IFN- ); nhm Th2 bi tit IL-4, IL-5, IL-6 v IL-10. sn xut mt s lng ln cytokin p ng cho nhu cu cha bnh cc cng ty trn th gii ngy cng u t mnh m vo con ng cng ngh gen v cng ngh sinh hc. sn xut cytokin ti t hp ngi ta s dng cc k thut ti t hp DNA tiu chun v nhng cht bin d nh hng v oligonucleotide c tin hnh khi dng h thng bin d in vitro ca hng Amesham. Cc oligonucleotide c tng hp trn my tng hp DNA 391. Dng t bo E. coli IB 392 c bin hnh biu hin vector (vt ch) cha on DNA (m ho IL-2, IL-1 v INF- ) ca cu c bin i sn xut cc protein ti t hp. Vi khun c nui cy ln ln qua m trong mi trng Mq 30oC ri ho long 20 ln bng mi trng ti, gi 34oC cho n khi c DD 600 =1, s trng phng plasmide c gy ra bng t bo 42oC qua 20 pht. Sau tip 5 gi 37oC to ra cc protein ti t hp. Cc t bo c ly tm 5000 vng /pht trong thi gian 30 pht v lng cn gi nhit 20oC cho n khi s dng. 2.4. Sn xut cc cht min dch v vaccine 81

Vi sinh vt t lu c xem l cng c c lc phng nga v iu tr bnh tt. Ngoi vic s dng vi sinh vt phc hi li khu h ng rut b hu hoi bi iu tr khng sinh lu ngy, t lu con ngi cng bit s dng cc vi sinh vt cn sng hoc b git cht, c bit l cc vi khun, lm khng nguyn to khng th hay lm c t to khng c t trong min dch. Trong sn xut vaccine cc vi khun gy bnh thng c nui i tr . 2.4.1. Sn xut cht c t min dch (Immunotoxin-ITs). Cht c t min dch l cc c t gy c cho t bo, ITs c cu trc gm hai phn: cht gy c (toxin) c bn cht protein v tc nhn hng ch. c t min dch hin ang c pht trin mnh m nhiu nc trn th gii iu tr cc bnh v ung th v bnh t min. Trong iu tr HIV, tr ngi ln nht l kh nng thm nhp ca virus vo mt loi t bo lympho T c vai tr nh b nh ca h thng min dch. Kh nng ngy trang ca virus HIV tinh xo n mc cha c loi thuc chng ti pht no c kh nng tm dit c. gii quyt vn ny, nhm nghin cu trng i hc Texas thnh cng trong vic to ra mt loi c t min dch, bao gm mt khng th chuyn nhn dng t bo T ct giu virus v mt phn t c ngun gc t cht c ricin thn kinh. H tin hnh th nghim 24 bnh nhn HIV v nhn thy loi c t trn lm gim mt lng ln cc t bo T nhim virus trong mt thi gian ngn. Tuy nhin, theo gio s Frances Gotch thuc trng Imperial London (Anh), nu loi c t trn nu khng c lp trnh cn thn, n c th hu dit bt k t bo min dch no trong mu v khin h min dch s yu hn do cc t bo b nh b tiu dit. Mt liu php ang iu tr dng ung th mu him gp cho nhiu kt qu ha hn trong th nghim ban u v cng gip bnh nhn iu tr cc bnh bch cu khc. Trong bnh bch cu t bo c lng l ung th t bo lymphocyte B, h dng c t min dch gm mt phn khng th v mt phn c t. c t ny c tn l BL22, c phn khng th nhn dng gn vi t bo ung th v phn c t tiu dit t bo. Ngoi ra, BL22 cung c hiu qu trong iu tr bnh bch cu lymphocyte mn tnh (CLL), mt dng bnh bch cu ph bin hn bnh bch cu c lng. Cc nh khoa hc c d nh dng BL22 iu tr bnh bch cu nguyn bo lymphocyte cp tnh (ALL) tr em chng li c t ngi ta c th to ra cc khng c t, thng l trong c th th ng vt. sn xut cc cht ny ngi ta thng dng phng php nui chm cc vi khun, chng hn Clostridium tetani hay C. butilinum. Sau tim vo cc ng vt thch hp nh nga chng hn. T huyt thanh nga ngi ta tch khng c t, tinh sch mc cao ri em iu tr. Cc c t hay c dng sn xut ITs l cc protein t vi khun nh: Pseudomonas exotoxin (PE) hay diphtheria toxin (DT), hoc t thc vt nhm protein bt hot ribosome (RIPs) nh ricin, pokweed anti-viral protein (PAP), saporin (SAP)... Ngay nay ngi ta c th to c t min dch bng cng ngh DNA ti t hp trn c s gene m ho cho protein v hot ribosome (RIPs). 2.4.2. Sn xut vaccine v hot (inactivated). Vaccin v hot hay cn gi l vaccine cht, chnh l tc nhn gy bnh nhng b git cht. Khi mt thc th vi sinh b v hot th protein b mt vn c gi nguyn cu trc. V gc vaccine m ni, n vn gi c tnh khng nguyn, ngha l chng vn c kh nng kch thch min dch, tuy khng hon ton v bn lu. 82

 sn xut loi vaccine ny, cc vi khun gy bnh thng c nui i tr. Trc kia thng dng phng php b mt, ngay nay hay dng phng php chm. Sau git cht bng nhit, acid, formalin, phenol hoc hoc cc cch khc sao cho khng nguyn b mt ca chng vn c gi li, ri c ch thnh thuc tim. Loi vaccine ny t vi khun c sn xut vi mt s lng ln, tuy nhin ngy nay ngha ca chng phn no b gim st, mt phn v s khc bit v tnh khng nguyn ca nhiu vi khun, mt phn v kh nng iu tr v khng sinh hin nay rt ln. Ngay c trong vic nui cy vi khun sn xut vaccine cng phi thay i phng php nui lin tc c s dng. 2.4.3. Sn xut vaccine nhc c (attenuated vaccine). L loi vaccine cha tc nhn gy bnh cn sng, nhng b lm gim c lc, khng cn sc gy bnh. sn xut loi vaccine nhc c ngi ta dng phng php tip truyn nui cy trong iu kin bt li gim bi c lc. Trong cc vt ch khng thch hp qua nhiu th h (c th hng trm th h), lm cho vi sinh vt c nhng bin i v gen thch nghi iu kin mi. Nhng bin i dn n vic mt i hon ton hay gim n mc khng cn kh nng gy bnh, nhng vn cn kh nng kch thch min dch v chng vn cn sng. Mt trong cc loi vaccine nhc c th loi vaccine BCG (Bacillus-Calmette- Gurin) c ngha rt ln. vaccine ny c sn xut t mt chng vi sinh vt l Mycrobacterium tuberculosis v. bovis c Calmette, mt hc tr ca Paster v Gurin nui qua 230 ln cy chuyn trn mi trng mt b lm mt hot tnh. sn xut loi vaccine ny ngi ta dng c phng php b mt ln phng php chm. Trong hu ht cc trng hp nui cy chm hin nay ngi ta dng glycerin (2-10%)lm ngun carbon, Tween 80 (0,1-2%), L-asparagine v dung dch allbumin (5%) lm ngun nitrogen v cc mui dinh dng thng thng khc. Sau iu chnh t nng t bo 5x 10-7/ml v lm ng kh vi mt dung dch cha 2% Natri glutamte; 8% dextran; 7,5 saccharose v 0,1% hydroxylamine. Khi tim phng sn phm s c pha long ra. Loi vaccine ny thng c kh nng kch thch cho min dch lu bn (1-2 nm), hnh thnh y cc loi hnh min dch v tin li khi s dng. Tuy nhin, v l cn sng, nn chng c kh nng li c (pathogenicity inversion), c bit thng gy phn ng vaccine vi c th yu. 2.4.4. Sn xut vaccine ti t hp ( Recombinant Vaccines). Vaccine ti t hp gene hay cn gi l vaccine di n v, y l loi vaccine th h mi c to ra nh k thut di truyn. Vaccine ti t hp gene c sn xut da trn nguyn tc chuyn gene khng nguyn ca i tng gy bnh vo mt vi sinh vt no . Vi sinh vt ny sinh si ny n nhanh chng v sn xut ra cc phn t khng nguyn. Tch chit , tinh sch cc khng nguyn lm vaccine di n v. Quy trnh sn xut vaccine ti t hp gene nhn chung phc tp, nhng nguyn tc chung u tri qua cc bc sau y: - Tuyn chn ngun gene c biu hin khng nguyn chnh ca virus (khng nguyn b mt hoc khng nguyn li), thao tc ct gene. - Chn vector thch hp (plasmide), ct plasmide v gn gene khng nguyn vo to vector ti t hp. - Chn vt ch thch hp a vector vo. Vt ch c nhiu loi nh vi khun, nm men, virus (chng hn E. coli, S. cerevisae, adenovirus, virus u, baculovirus v.v...). - Nui cy, ln men. 83

+ Nu vi khun, nm men th nui cy trong mi trng nhn ln lng ln khng nguyn. + Nu l virus th phi nhn ln trong t bo ng vt hay thc vt thch hp. - Tch chit v lm sch cc protein khng nguyn bng phng php ho sinh thng thng. - To vaccine phn t dng chng, dng tim. - Tin hnh th hiu lc trn ng vt v trn lm sng. Hiu lc ca vaccine ph thuc vo nhiu yu t. Bn cnh bn cht khng nguyn mnh, yu th con ng a vaccine vo c th cng rt quan trng: da hay nim mc, tim hay chng, thi gian s dng vaccine, ln s tim chng v khong cch gia cc ln, liu lng dng, tui dng v theo di di min dch. Nu l min dch dch th th vaccine bng phn ng trung ho khng nguyn khng th, ELISA, min dch hunh quang, min dch phng x. Nu l min dich t bo th pht hin o lng cc t bo T, B v pht hin cc yu t T v B.

2.4.5. Mt s m hnh ti t hp gene trong sn xut vaccine ng dng trong nng nghip. Hin nay c mt s m hnh ti t hp gene trong sn xut vaccine cho cy trng, chng hn: ngi ta bit vi khun Bacillus thuringensis (Bt) sn xut ra protein chng cn trng, ngy nay ngi ta tch rin ra c cc gene gn vo Agrobacterium tumefaciens ri dng sng bn gene chuyn vo khoai ty, bng.., phng bnh cho cy; Hay Roundup l mt trong nhng thuc dit c di tt. Roundup dit c c di chnh l nh n kh nng c ch hot ng ca mt enzyme tham gia tao amino acid thm cn cho s tng trng ca c di . Ngi ta nghin cu cu trc gene ny v lp vo vi khun no ri tm cch a vo cy ng ni chung t sn xut ra Roundup tr c di. Hn na Roundup li thoi ho nhanh chng trong mi trng tr thnh thnh phn t nhin v hi.
i vi ng vt, hin nay nhiu loi bnh do nguyn nhn t virus cng c nhiu vaccine c sn xut bng k thut ti t hp gene nh: bnh Newcastle c vaccine virus u ti t hp, bnh Influenza c vaccine virus pox ti t hp, bnh virus bch cu b c virus u ti t hp v.v...

III. Sn xut protein ngun thc vt

3.1. C s ho sinh v ngha ca nui cy m v t bo 3.1.1. C s ho sinh v di truyn u th k XIX cc nh khoa hc chnh thc xy dng hc thuyt t bo, h cho rng trong c th a bo cc t bo c th tn ti c lp v ring bit. n nm 1962, Haberlandt l ngi u tin xng ra phng php nui cy t bo thc vt chng minh cho tnh ton th ca t bo. Theo ng mi mt t bo bt k ca c th sinh vt a bo u c kh nng tim tng pht trin thnh mt c th hon chnh, ngha l: Mi t bo ring r ca mt c th a bo u cha y ton b lng thng tin di truyn cn 84

thit ca c sinh vt v gp iu kin thch hp th mi t bo c th pht trin thnh mt c th hon chnh. iu kin thch hp chnh l cc yu t trong mi trng gm: - Cc loi mui khong. C nhiu loi mui khong khc nhau cn thit cho i sng thc vt c cung cp di dng a lng nh P, Ca, K, Mg, N, S v dng vi lng nh Fe, Cu, Zn, Mn, Mo v.v...Chng u l nhng tc nhn quan trng tham gia v cc phn ng ho hc trong qu trnh trao i cht - Ngun carbon v ngun nng lng. M v t t thc vt ch yu sng theo phng thc d dng (mc d trong mt s trng hp chng c th sng bn d dng nh iu kin nh sng nhn to lc lp quang hp to ra ngun carbon), v vy vic a vo mi trng nui cy ngun carbon l iu kin bt buc. Ngoi vic cung cp nng lng, ngun carbon l khung chnh cho s tng hp cc cht cn thit trong qu hot ng sng. - Cc ph gia hu c. y l cc loi nh vitamin c s dng xc tc cho cc phn ng ho hc, chng c th c tng hp trong t bo nui cy nhng lng thng khng do phi b sung t ngoi vo. Cc amino acid c th c tng hp trong t bo thc vt, nhng vic b sung thm cc amino acid cn c tc dng kch thch sinh trng trong nui cy protoblast v hnh thnh cc dng t bo. Ngoi ra cn c mt s ph gia hu c khc u quan trng cho qu trnh trao i cht. - Cc cht iu khin sinh trng. Trong nui cy m b t bo thc vt thnh phn quan trong nht l cc cht iu khin sinh trng. Ngi ta thy rng c tt c 4 nhm cht iu khin sinh trng chung l auxin, cytokinin, gibberellin v acid abcisic. S sinh trng v pht trin ca m nui cy khi ch b sung mt hoc mt s cht thuc 4 nhm trn vo mi trng nui cy. T l ca cc loi hormon cn cho s to mt c quan no rt khc nhau. chnh l c s c ngha rt quan trng cho mc ch thu nhn ch phm mong mun trong nui cy m v t bo thc vt bng vic thay i hp l cc iu kin mi trng. tch chit protein t thc vt cn ch rng: t bo thc vt, c thnh t bo c cu to bi hydratcarbon, lignin bao quanh mng sinh cht. Cc si nh cellulose bao quanh mng nguyn sinh cht theo dng li lm cho t bo c dai bn nhng c kch thc ln (i vi t bo nui cy m c kch thc khong 100 m). V vy vic nghin t bo trong nitrogen lng s d dng thu c cc phn t peptide cha trong khng bo. Sau khi thu nhn bng ly tm 10.000 vng /pht, 4oC trong 10 pht, dich chit th c chit vi th tch tng ng dung dich m Tris-HCl 0,1M; pH= 7,2- 7,5. Sau peptide c tinh ch theo cc phng php tinh ch protein hin ang s dng trong cc phng th nghim (chng 5). 3.1.2. ngha ca nui cy m v t bo thc vt. Vic tch chit ngun protein trong t nhin bng phng php ho, l (da trn s pht trin nhanh chng ca k thut ho hc hin i) phc v cho nhu cu i sng con ngi ngy cng pht trin. Thc vt bc cao vn l ngun cung cp c cc hp cht c gi tr cho nhiu mc ch khc nhau, c bit trong lnh vc dc phm. Tuy nhin trong nhng nm gn y ngun thc vt t nhin ngy cng b cn kit do s khai thc t ca con ngi. Sn lng khai thc khng n nh do hu qu ca mt s yu t nh: iu kin 85

t nhin khng thun li, chi ph lao ng ngy cng cao, kh khn v k thut v kinh t trong trng trt v.v... Phng php nui cy m v t bo s gip gii quyt nhng vn . Mt khc s chn dng t bo c th to ra dng c kh nng tch lu cao hp cht mong mun. Chng hn nh cng trnh ca Zenk v cng s (1978) nghin cu hm lng anthraquinon (mt loi sc t) trong t bo ca cy nhu (Morinda citrifolia) cho bit rng nng c hm lng ca cht ny ln gp 20 ln so vi r l c quan tch tr chnh ca hp cht ni trn. Cng nh vy, thng bo ca tp on cng nghip ho du Mitsui (Nht bn) nm 1983 v vic sn xut dc cht shikonin l v thuc dn tc c kh nng chng vim. Bng phng php nui cy t bo trong thng ln men loi nh 750 lt, h chn c dng t bo c kh nng tch lu shikonin nhiu hn gp 10 ln vi ngun nguyn liu t nhin cy shikokon ( Lithospermum erythozhion). Ngi ta khng nh rng khi so snh hm lng cc cht trong nhng t bo chuyn sn sinh ra cht , th hm lng cc cht trong t bo nui cy vn ln hn t 2-4 ln. (Zenk, 1978) K thut nui cy m v t bo thc vt cn cho php iu khin t bo to ra cht quan tm. Chng hn c th iu chnh cht kch thch sinh trng hoc nh k thut ti t hp DNA, chuyn gene m ho cht mong mun vo thc vt to ra cy bin i gen. Gn y c rt nhiu cng trnh cng b kt qu s iu khin t bo thc vt sn xut protein mong mun, trong ch yu hu ht cc cng trnh u tp trung vo vic sn xut vaccine v nhng protein ng dng trong lnh vc y hc. Ngoi ra nui cy m v t bo thc vt cn loi b c s nhim virus gy bnh, ngn chn s nh hng cht lng ca protein quan tm sau khi c tch chit. 3.2. Cc thnh tu sn xut protein t thc vt trn th gii

T lu, trn th gii thng thu nhn protein thc vt bng con ng chit xut t cc ngun t nhin. Xc nh c hng i s khng tn ti lu di, cc nh khoa hc tm ra ngun nguyn liu mi da trn c s nui cy m v t bo thc vt. Ngy nay k thut nui cy m v t vo c trin khai ngy cng tin b cc nc cng nghip tin tin. Mt lot cc cng ty trn th gii nh: cng ty Escagenetics (California, M); hng Phyton Catalitic (New York, M) v rt nhiu cng ty khc cng b thnh tu sn xut cc dc cht da trn k thut nui cy m v t bo. Theo mt cng b gn y nht loi thuc chng HIV v nhng bnh khc nh bnh di c th c bo ch t hoa mu bin i gene, hay ni cch khc ngi ta ang bin i mt loi cy n c th tr thnh mt loi thuc tr bnh. Cc cng ty dc phm ln ang nghin cu kh nng , v chi hng triu la nghin cu nhng tn dc ny. Theo thng bo ca phng th nghim Arizona (l ni ang i u v nghin cu nng dc), gim c phng th nghim cho bit iu n gin trong s dng thc vt bin i gene l khi chuyn cho cy mt gene, cy tr thnh mt h thng sn xut v chng ta s c c mt h thng sn xut chi ph thp. Ti y ngi ta ang sy lnh nhng ming c chua - mt loi cy c trng th nghim, mng mt loi gen chng loi ung th c t cung - mt phn trong cng ngh ch bin thc phm tng i n gin chuyn t thc vt sang dc phm. Khi ra khi my sy lnh, chng nh nh polystyrene, nhng vn mang y mi c chua v vn gi c thnh phn hot cht. Sau , chng c nghin thnh bt v ng vin. Mi loi thuc vin c ngun gc thc vt khc nhau c th tr nhng bnh khc nhau. Nhng tri c chua thm mang vaccine tr tiu chy, cy thuc l c th chng li virus AIDS. Tt c s c gi thnh r hn cc loi dc phm hin i hin nay.
IV. Sn xut protein t ngun ng vt 86

4.1. Nguyn tc v iu kin sn xut protein Vic tch chit sn xut protein t ngun ng vt tu theo mc ch la chn phn m c s tch lu hp cht ln nht, chng hn sn xut insulin thng tch chit t tuyn ty, cc enzyme tiu ho nh pepsin, tripsin, c t nim mc d dy v.v... vic tch chit cc hp cht ny tt nht ton b qu trnh tin hnh trong phng lnh v u thng da trn nguyn tc v cc bc chung sau y: - Nghin v chit rt bng m c pH thch hp. - Ly tm thu dch lng v loi b phn b. - Kt ta dch lng (c th dng mui hay dung mi hu c). - Ly tm thu phn dch ni (phn ta), ta c ch phm th. - Tinh sch bng sc k, thu ch phm c tinh khit cao. Tuy nhin tu theo tng loi hp cht mong mun c nhng thay i iu kin thch hp khi sn xut. V d: nhng peptide tch t t bo bch cu trong sn xut khng th n dng, thng dng cc dung dch c tnh acid v gm cc bc nh sau: - Cc t bo bch cu c thu nhn bng ly tm trong iu kin lnh cng vi cht chng ng mu. - Chit bng m pH=7,5-8,0 (thng l m Tris-HCl, nng 20-50 mM, cha 50 nM NaCl). - Ly tm thu phn lng. - Nghin vi dung dch c tnh acid (thng 15- 20mm HCl). - Ly tm thu dch ni ta c dung dch cha peptide. 4.2. Sn xut khng th n dng 4.2.1. Khi nim v khng th n dng. Khi kt hp dng t bo bch cu lymphocyte sn sinh khng th vi mt dng t bo ung th s to thnh mt dng t bo lai c tn gi l hybridoma. Tnh cht c bit nht ca loi t bo lai ny l c kh nng tng sinh vnh vin, chng c bt t ho. trong mi trng nui cy v sn sinh ra mt loi khng th c gi l khng th n dng. Cc phn t khng th ny c cu trc hon ton ging nhau, chng c s dng rt a dng trong phn tch sinh hc v cha bnh. Ngy nay cc khng th n dng c p dng nhiu trong chn on v iu tr y hc v mang v nhng li nhun ln cho cc cng ty dc trn th gii. 4.2.2. Mt s lu khi sn xut khng th n dng. - Cc khng th n dng ca chut (lai t bo ung th vi t bo lymphocyte ca chut), tuy c dng nhiu trong iu tr nhng c mt s nhc im. l trong nhiu trng hp khng th chng li Ig ca chut biu hin trong tun hon mu bnh nhn, nh vy ngn cn s iu tr lu di. - Khng th chut c th lm yu hot ho h thng b th ngi in vivo dn ti hu qua gy c trc tip t bo. khc phuc nhng nhc im trn ngy nay ngi ta pht trin sn xut khng th n dng c ngun gc t ngi. Bi v cc khng th n dng ngi c dung np tt trong cc bnh nhn. Tuy nhin, sn xut khng th n dng c ngun gc t ngi cng c mt s bt cp nh: sn xut khng th n dng ca ngi (lai t bo ung th vi t bo lymphocyte ca ngi), phi tm c ngun t bo lymphocyte min dch ph hp nhm tng t l bi tit khng th. Khng th n dng ca loi gm nhm c sinh 87

ra bi s lai to vi t bo lymphocyte ca lch hay hch lympho t nhng ng vt c min dch nhc li. Nhng nhng ngun lymphocyte khng c th ly c mt cch bnh thng ngi. V th nhng cch n gin nht l dng t bo lymphocyte c tch ra t mu ca nhng ngi tnh nguyn c gy min dch vi cht c un vn, hay t nhng ngi pht trin khng th tun hon sau bnh nhim trng, bnh t min hoc ca ung th. Nhng khng th c hiu c th c tch ra t mu nh nhng khng th c mt trong tun hon. Mc d lymphocyte c th kim c ch yu l t mu, nhng cng c th thu c nhng t bo min dch t cc c quan khc nh nhng hch lymphocyte, amidan, ung th tu xng v lch. C mt s nghin cu cng ch ra rng, lymphocyte c ly t nhng c quan rn nh hch lymphocyte, amidan c th d hp nht li hn so vi lymphocyte t mu. Ngoi ra h cn cho bit thm cc lymphocyte t mu chut thi khng hp nht vi t bo lch nh l vi t bo u tuy xng (myeloma) chut. 4.2.3. Lm giu i vi cc lymphocyte c hiu Hiu qu ca qu trnh bt t ca t bo ph thuc vo t l bi tit khng th c hiu, v vy trc ht cn phi lm giu cc t bo lymphocyte c hiu khng nguyn. C ba phng php lm giu lymphocyte c hiu khng nguyn sau y: a) Hng cu ph khng nguyn phn ng vi khng th c hiu to thnh th hoa hng v th ny c tch ra bng ly tm theo gradien nng . b) Cc khng nguyn c nh du bng cht hunh quang phn ng vi khng th trn t bo B. Ri cc t bo lin kt khng nguyn c th tch ra bng my FACS (fluorescence Activated Cell Sorter). c) Cc t bo B sn xut khng th dnh vo nhng a plastic ph khng nguyn v ri c tch ra d dng. 4.2.4. Cc phng php lm bt t t bo. C ba phng php lm cho cc t bo lymphocyte c hiu khng nguyn tr thnh bt t: a) Ti np (transfection) vi t bo ung th thnh dng t bo bin hnh. b) Lai (hybridization) lymphocyte vi t bo ung th thch hp (myeloma) thnh dng lai (hybridoma). c) S bin hnh ko di theo nhim virus Epstein Barr (EBV) thnh dng t bo nguyn bo lympho (lymphoblastoid). Ri cc t bo c lai tr thnh hybridoma. S lai to l hp nht cc t bo lymphocyte min dch vi cc t bo ung th lympho to ra cc t bo ny tip tc bi tit cc khng th, ng thi li bt t v l t bo cha gene ung th. T l hp nht t bo ngi th thp, mc t 0,08 x 10-7 ti 60 x 10-7. Trong khi t l lai to ca lymphocyte chut l t 30x 10 -7 ti 60 x 10-7. T l lai to thp do tnh khng bn ca cc t bo lai, m cc t bo lai cha mt lng ln nhim sc th gp 2 hoc 3 ln bnh thng. Thng tin di truyn cho tnh c hiu khng th nm t nht hai nhim sc th tng ng, c hai ca chng u cn c mt trong nhn v c biu hin bi t bo sn xut khng th v bi tit lin tc. S mt nhim sc th tng ng hay mt s biu hin ca chng c th dn n t bo bi tit s tr thnh khng bi tit. Mt trong nhng giai on u tin v l ch yu l chn lc thun ho t bo giai on rt sm. S thun ho c tin hnh t nhng t bo n gin, nhng nhiu t bo bi tit khng th th khng ln ln t t bo n gin, do nhng t bo nui khc nhau v nhng yu t tng trng thng c a thm vo nui cy thun ho trong giai 88

on sm ca s ln ln ca t bo. S thun ho thng c nhc li cho n khi chng ln ln v bi tit c khng th c hiu. V giai on ny n thng c coi l to c dng t bo bn. Song s thun ho vn cn c nhc li. Ngc li vi hiu qu thp ca s hp nht t bo ngi, s nhim v bin dng ca cc lymphocyte bi virus Epstein Barr (EBV) to thnh t bo lymphoblast xy ra tn s cao 1 x 10-1. EBV l mt virus herpes lm nhim trng cc t bo lymphocyte B gy nn bnh vim st hch (mononucleosis). Hu ht nhng ngi trng thnh th gii phng Ty biu hin EBV v chng c mt qun th lymphocyte tun hon c th ngn cn s nhim EBV tip tc. K thut hybridoma-EBV c kt cu bi mt dng nguyn bo lymphoblast (KR4) c pht trin vi s nhy cm HAT (mi trng la chn c cha Hypoxanthin, Aminopterin v Thymidin) v khng ouabain lai vi dng sn xut khng th. Mt hng khc l dng nhng on DNA hoc l ca t bo ung th hoc l ca virus gy ra s hnh thnh nhng dng t bo b bin hnh t nhng t bo lympho bi tit khng th. Tuy nhin k thut ny c coi nh l mt s truyn nhim nn khng chc chn v t bo c truyn nhim th ln ln chm hn. 4.2.5. S dng hp cc thnh vin lai vi nhau v phng php la chn t bo lai. S lai gia t bo ca ngi vi nhau gp tr ngi ln do thiu mt dng t bo thnh vin ph hp. Trong h thng gm nhm, s nui cy cc t bo myeloma tc l nhng t bo ng th c dn ra t cc lymphocyte bi tit khng th (t bo plasma) th c th hot ng nh l mt thnh vin trong qu trnh hp nht ny. Ngc li, myeloma ca ngi kh c xc nh trong nui cy v nhng dng t bo c nhng du hiu khng thuc th thch hp cho la chn d dng cc t bo lai. Cc nguyn bo lympho (lymphoblast) cng c dng nh l thnh vin trong qu trnh hp nht. c nhng cng b nghin cu so snh v tn sut lai to gia lymphocyte ca ngi vi cc dng t bo khc v theo di tnh bn ca chng cho bit rng: myeloma ca chut (NS-1) hp nht vi tn sut cao nht v hng km bn hn l cc t bo lai gia ngi vi ngi. H cng to ra cc t bo lai gia ngi v chut thnh cng v thu c cc khng th c hiu. Vn l tm kim t bo thnh vin thch hp hp nht vi lymphocyte ngi l mt hng mi. Cc t bo lai c gi l heteromyeloma c xy dng gia myeloma ca chut v ngi. Cc t bo khng bi tit Ig ca chut hoc ngi v chng sn sng hp nht vi t bo lymphocyte ca ngi hnh thnh t bo lai sn xut khng th bn. Song cho n nay, heteromyeloma vn cha c a n cc phng th nghim khc th nghim. Phng php la chn t bo lai sau khi hp nht nh sau: Sau cc bc k thut lai to, mi trng nui cy cha hn hp cc t bo lympho cha hp nht v cc t bo lai. Cc t bo lai gia myeloma v lymphocyte cn phi c chn ra nui cy trn mi trng HAT. Ni chung s nui cy da trn nguyn tc thiu enzyme dn n lm cho t bo nhy cm vi cht i khng ca aminopterin v acid folic. Ch c nhng t bo lai gia myeloma v lymphocyte c kh nng s dng hypoxanthin cung cp trong mi trng HAT. Tm li mc d cn nhiu hn ch nhng hin ti c nhiu loi khng th n dng hin ang c s dng chn on v iu tr nhiu loi bnh tt ( bng 6.1). Trong mt tng lai khng xa na vi nhiu nghin cu ci tin trong lnh vc cng ngh sinh hc, chng ta hy vng rng: sn xut khng th n dng ngi hay gm nhm c gi 89

thnh r cung cp cho iu tr cc bnh him ngho nh ung th v khng ch c s tr li min dch khng mong mun thng xy ra trong vic ghp c quan hay trong bnh t min thp khp. 4.3. Sn xut cc hormon, enzyme v protein khc 4.3.1. Sn xut bng tch chit trong t nhin. Cc horrmon, enzyme v nhng protein c hot tnh sinh hc khc trong t nhin c bi tit v tch ly trong cc m, t bo rt khc nhau Bng: 6.1 Mt s khng th n dng ang c s dng trong y hc Ngun xut x Lai gia t bo ngi v chut Loi khng th - c t un vn. - Virus si - Ung th phi - Ung th v Lai gia t bo ngi v ngi - Virus si - Dinitrochloronbenzene - c t un vn - Glioma Cc bin dng EBV (EBVtransfections) - Rhesus D - c t un vn - Khng nguyn b mt vin gam B - Cytomegalovirus - Chlamydia trachomatis -Virus cm -Melanom Lai ti EBV(EBV hybrids) - c t un vn

ph thuc vo chc nng ring bit ca n. V vy, thu nhn cc loi hormon, enzyme hay protein khc nhau, ngi ta s dung cc m khc nhau tch chit v tinh sch chng. V d: sn xut hormon tng trng ngi (HGH- Human Growth Hormone) hay hormon kch t v thng thn (ACTH- Adrenocorticotropic Hormone) v kch non t(FSHFollicle Stimulating Hormo), ngi ta tch chit t tuyn yn ca ngi cht; insulin, glucagon v stomatostatin, c trch ra t tuyn ty ca b, heo; hormon erythropoetin, angiotensin-25-hydrogenxy-cholecalciferon, c trch ra t thn, cc enzyme tiu ho nh protease, lipase, amilase v ribonuclease, c trch ra t tuyn tu, mng nhy d dy v.v... Vic tch chit v tinh sch hormon, enzyme v cc loi protein c hot tnh sinh hc gm cc cc bc v phng php tch chit v tinh sch cc cht trong t bo ng vt bnh thng c trnh by trn. Tuy nhin, cc hormon v cc protein c hot tnh sinh hc khc vic tch chit trong t nhin thng ch thu nhn c mt lng nh, khng p ng c nhu cu i hi trong cha bnh. Mt khc cc hormon v cc protein tch chit ngi v ng vt thng gy d ng hoc nhim cc bnh ca virus 90

v.v... khc phc mt s nhc im ngy nay ngi ta pht trin mnh theo hng sn xut hormon v cc protein ti t hp DNA. 4.3.2. Sn xut hormon, enzyme v protein ng vt bng cng ngh gene. Hin nay trn th gii c nhiu loi hormon, enzyme v cc loi protein quan trng khc, c bit cc loi protein ng dng trong y hc chn on v iu tr tung ra th trng (bng 6.2), nhiu nh sn xut thu c hng chc triu la mi nm nh con ng sn xut hormon bng DNA ti t hp. Ngoi khng th n dng, cc vaccine, cc t bo ng vt cn c ng dng rng ri trong vic sn xut cc hormon trong iu tr cho ngi v ng vt. Bng: 6.2 Cc hormon v protein iu tr c sn xut t t bo ng vt S 1 2 3 4 5 6 7 8 9 Cc horrmon v protein Hormon sinh trng ca ngi Hormon sinh sn Yu t VIII Erythropoetin Cc yu t ng mu Cht hot ho plasminogen-m (tPA) Khng th n dng Vaccine vim gan B Alpha-interferon

a) Cc bc sn xut hormon ti t hp DNA trong t bo ng vt. Nhn chung cc bc sn xut hormon ti t hp trong t bo ng vt u gm nhng bc chnh tng t nh sn xut cc loi protein ti t hp trong t bo vi khun. S khc bit ln nht trong sn xut protein t bo ng vt so vi t bo vi khun l: thay v t bo vi khun l ni sn xut protein th y l protein c sn xut trong t bo ng vt, v chi tit hn gm nhng bc chnh nh sau. - Tuyn chn h biu hin thch hp biu hin gene. - Da vo trnh t gene m ho protein thit k cc on mi nhn gene. Tng hp mi. - Chun b DNA khun nhn gene. - Nhn gene bng PCR t DNA h gene ca vi khun hoc t ngn hng cDNA, nhng tt nht l l nhn gene trc tip t chnh plasmid mang gene tng dng dng c trnh t gene . - a gene vo vector tch dng v bin np vo E. coli khuych i dng gene. - Tch plasmid t cc th bin np E. coli kim tra gene trong vector tch dng bng enzyme hn ch. - a gene vo vector biu hin v bin np vo E. coli. - Tch plasmid t cc th bin np trong E. coli v kim tra gene, c bit l chiu di ca gene trong vector biu hin. Gen cn phi nm ng chiu promotor. - Bin np vector biu hin c mang gene biu hin vo t bo ng vt. - Kim tra s biu hin. 91

- Tch, tinh sch protein. b) Mt s c im cn ch khi sn xut hormon v cc loi protein bng DNA ti t hp trn t bo ng vt. Vic sn xut protein ni chung v hormon ni ring trong t bo ng vt c mt s u im hn so vi h thng t bo vi khun hay nm men. Cc t bo ng vt c h thng ci bin sau dch m nh: glycosyl ho, phosphoryl ho, amin ho v s ct b t cc preprotein, tp hp cc tiu n v v.v...Hn na, mi trng kh ca vi khun E.coli khng dn n hnh thnh cu disulfua m ch tm thy t bo ng vt. S thiu nhng bin i thch hp l nh hng su sc n cu hnh v tnh ho tan ca protein. Trong nhiu bin i mt s trng hp biu hin E.coli tch t nhng dng khng ho tan v mt hot tnh sinh hc (cng c mt s trng hp s bin i ch xy ra hot tnh sinh hc), chng c th lm nh hng t nhiu n bn cht khng nguyn v l tnh ca protein. Trong khi mt s protein, nhng bin i th cp chnh xc th i hi mt cch tuyt i cho chc phn ca phn t.Tm li, s gp khc protein, s tp hp cc tiu n v v s bi tit chng l nhng l do cng thm chng ta hiu ti sao phi bt buc dng nhng t bo ng vt trong sn xut cc hormon v nhng protein c hot tnh sinh hc. Mt vector t bo ng vt in hnh gm c ba thnh phn chnh: - Thnh phn 1: S gii m trnh t DNA ca vi khun cho php truyn b v sn xut DNA vector trong vi khun trc khi gy nhim vo t bo ng vt. - Thnh phn 2: Vng vector t bo ng vt cha nhng on trnh t, m nhng on cho php la chn nhng t bo c gy nhim dy ln thng xuyn t 103106 . Thm vo , vng ny c th cha nhng on trnh t c i hi duy tr v phng i mt lng ln s bn copy ca vector. - Thnh phn 3: Vng th ba gm nhng on trnh t m ho hormon hay protein cn sn xut cng vi nhng on iu ho (promotor), tn hiu thm nh poly A, introns v.v... Nhiu vector dng trong t bo ng vt i hi duy tr v phng i c mang n t cc ngun virus ng vt loi DNA nh SV 40, polyoma, papiloma bovin (PBV) v Epstein-Barr. Tt c chng u c bn cht lm bin tnh di truyn. Thng nhng on trnh t lm iu ho gene mong mun cng c dn ra t cc virus. Song vn cng c th tp hp cc vector khng c yu t virus, bi v bao gm nhng yu t kim tra t bo nh h thng phng i c da trn emzyme reductase dihydrofolate v cc h thng khc. Tt c cc vector t bo ng vt c thit k n nay u dng nhng dng t bo xc nh lm vt ch. c) Nhng phng php chuyn gene. DNA c th a vo t bo hoc cng lng ta vi calci phosphate hay lin kt vi DEAE-dextran. Vo nhn, DNA c gi vo mt cch ngu nhin trong mt hay nhiu chromosome ca t bo ch. Dng t bo bn cha DNA c st nhp th biu hin nhng tn hiu chng t c s st nhp. Quan trng hn c l s chuyn gene dng vector retrovirus. Nhng virus RNA st nhp vo vo trong nhim sc th ca t bo vi nhng bn sao DNA ca chng. Tt c nhng on m ho protein ca b gene virus c th b mt v gene c chuyn ti c t gia i ca nhng on trnh t nhc li nhng u cui ca virus (LTRs-long terminal repeat). Nu nh DNA virus ti t hp c bao gi bi herpes virus (virus ny cung cp on m ho protein cn thit dch m), th on ti t hp ny c th gy nhim 100% cc t bo nhn. Hn na v c ch st nhp bao gm s 92

ti t hp c hiu LTRs nn DNA chim ch gia chng lun lun st nhp nguyn vn vo b gene. d) Nhng k thut lm tng cng biu hin ca gene. Mun ti u ho s biu hin ca gene chuyn ngi ta cho rng nn ghp thm mt on biu hin promotor mnh v thng ci t mt hay nhiu ch trong gene ca virus. Da vo phng php ny, vector ca adenovirus c dng biu hin nhiu gene c hiu qu trong c gene thymidine kinase ca virus herpes v gene ny cu thnh ti 10% ca sn phm c tng hp mi. Retrovirus c dng mt cch tng t sn xut mt lng ln hormon v protein hu dng c th dng trong thng mi, bi da vo nhng tn hiu biu hin tt cha trong LTRs ca chng (bng 6.3). Dng thay i ca cu trc vector bao gm phi hp nhng yu t c chc phn t nhng ngun khc nhau. Nh trong s phi hp vi gene khi ng tng cng (enhancerpromotor) cn cho s phin m c hiu qu hay trong s phi hp nhng on thch hp c u 5 tn cng v 3 ca vng m ho protein lm tng s dch m cng nh tng tnh bn truyn tin sau khi sao chp xong. Trong trng hp ny, nhng n v sao chp hon ton c hnh thnh bi cung cp nhng on m ho. Ngoi ra cn c hai yu t na c th lm tng cng biu hin ca gene st nhp vo trong b gene l: - V tr st nhp ca gene. Gene c gi vo chromosome mt cch ngu nhin, hoc vng tr hoc vng hot ng. Chng hn cc on ca -globin a vo dng t bo erythroleukemia, ch c xc nh c mt trong s 1000 t bo b nhim l c cha on vector st nhp locus -globin. Do phi c s sng lc v tch dng c s ti t hp v tr c bit. Mun lm c iu ny phi c bin php la chn nhng v tr st nhp cho php biu hin mc cao. Tuy nhin hin nay vn ny ang cn c nghin cu. - S lng bn sao (copy) Lng sn phm thu nhn c t nhng gene c chuyn thng t l thun vi s lng bn copy ca gene c mt, do lm tng s lng copy ca nhng gene c st nhp l rt cn thit. Ngi ta thy rng, s khuych i gene trong cc t bo nui cy chu s tnglng thuc c th dng bin i ca n c chn lc khng thuc hn so vi dng t bo hoang di. Trong phn ln trng hp l do s sn xut qu nhiu nhng enzyme c kh nng c ch tc dng ca thuc. S sn xut qu nhiu enzyme li thng do tng nhiu s lng Bng 6.3 Mt s sn phm ti t hp biu hin trong t bo ng vt

93

Vector

Promotor

S copy Hormon

Vt ch Sinh trng C127(chut) Fibroblast

lng S phn t (x106) 6-18 15 200-600 250

BPV Retrovirus

MMT-1 RSV-LTR

10-100 100

Cht Plasmin m DNA genomic TPA Khng xcL. chut nh Khng xcCHO nh cng) CHO cng) (chut (chut 0,07 50 60 gan B 0,125 3,8 0,03 4,5 1,6-2,0 0,18 0,6 0,6 1-2 0,45 25 90 0,7 100 34-42 3,5 13 13 22-24 10 0,6 440 520

Amplifiable SV 10E (DHFR)

Amplifiable 10E 100 (DHFR) Adenovirus Khng nguyn SV 40 SV 40 SV 40 SVORI SVORI BPV BPV BPV BPV Retrovirus (MSV) SV40L/ HBV SV 40L SV 40L HBVb HBV HBVb HBV HBV MMT-1 MSV-LTR

b mt vim

Khng xcCV-1 (kh) nh Khng xcCV-1 (kh) nh Khng xcCV-1 (kh) nh 5 x 105 COS Khng xcCOS nh 50-100 20-200 10-100 150-160 5-10 C127(chut) C127(chut) NIH3T3 (chut) C127(chut) NIH3T3 (chut)

Ghi ch: lng = pg / t bo / ngy bn copy, ngha l c s khuych i gene cu trc m ho cho enzyme. Trn thc t ngi ta thy c trn 10 trng hp khuych i gene ni bo c bit trn t bo ng vt c v, chng hn gene m ho enzyme dihydrofolate reductase, asparagine synthetase v.v...chng biu hin ch l m ho cho nhng protein c ch thuc. Nh vy c th gi rng nhng gen chn lc mong mun a vo s st nhp mt cch ngu nhin vo b gene s c th khuych i. Hn na vng DNA khuych i rt ln so vi vng gene cu trc la chn (thng l hn 1000Kb c khuych i nguyn vn). Hin nay sn xut cc protein iu tr mang tnh cht thng mi, ngi ta cung khuych i vi gene DHFR (xem bng 6.3). 94

Cc vector c s lng cao nht l cc vector da trn virus SV 40 ca kh. Mt h thng thun li bt ngun t dng t bo thn kh CV-1, m dng ny b bin tnh bi virus SV 40 lm khim khuyt phn gc sao chp. Thiu chc phn ny, DNA hp nht vo nhim sc th ch n ch biu hin chc phn gene c m ho ca cc virus l khng nguyn T m thi. Cc t bo COS (CV-1 khuych khuych phn gc SV 40) s cho php sao chp cc plasmid vi khun, chng cha t nht 228 bp ca DNA SV 40 bao gm c phn gc sao chp. Vector SVORI c th sao chp vi 4x 10 5 copy /t bo/ 2 ngy v biu hin mc cao, nhng phn ln vector ca virus ny d cht v t bo b nhim ch sng st c 2 tun trong nui cy. Virus ng vt dng cu trc vector biu hin l virus papiloma ca b (BPV), sao chp theo cch th b sung (episome) trong cc t bo nguyn bo si (fibroblast) ca loi gm nhm. S lng copy ca n thp hn SV 40, nhng u im nht ca n l khng git cht t bo ch nn cc dng t bo bn v c th dng sn xut. Trong mt s trng hp vector BPV c ch ra l duy tr epiosome ging con ng ca virus nguyn vn. Trong nhiu trng hp khc vector BPV hp nht vo t bo ch thng nh sp t hng i t u n cui. S phi hp gia promotor v enhancer mnh (nh promotor I ca metallothionein chut v enhancer ca BPV) ng thi vi s lng copy cao tng i s cho s biu hin cao trong cc t bo fibroblast chut ca nhiu cc gene. V. Sn xut protein t ngun ph thi Sn xut protein t ngun ph thi hin ang l vn thi s, bi v s dng ngun ph thi ngoi ngha to ra cc sn phm mong mun dng trong cc lnh vc nh dc phm, chn nui , m phm v.v...s dng ngun ph thi cn c ngha to ln trong vic x l nhim mi trng. nc ta cng nh trn th gii hng ngy c hng trm tn rc thi cn c x l. Vi ngha chng ti xin gii thiu cng ngh sn xut mt s protein v amino acid t ngun ph thi. 5.1. Sn xut cystine v cc amino acid 5.1.1. Vi nt v ng dng ca cystine. Cystine c cu trc t hai phn t cysteine lin kt vi nhau qua cu disulfua. Gn y ngi ta pht hin thy cystine c nhiu ng dng trong i sng: Trong dc phm nh thuc chng vim gan, bo v gan trong nhim c kim loi nng, chng lon dng da, chng rng tc, chng bnh ngho m v chng nhim c thai nghn. Ngoi ra cystine cn c dng lm thuc chng bng da v vim lot gic mc, thuc phng v iu tr ung th, thuc b min dch, thuc chng x ho v thp khp, thuc chng phng x, phng cht c ho hc v hn gn nhanh nhng vt thng, vt m v.v... i vi m phm, cystine c lm thuc tr ha, thuc sy tc bn, thuc lm mt tc v.v... i vi thc phm cystine tr thnh loi thc phm cao cp, c dng vo sa kh, bnh m kh v sp cao cp. Vi ngha nh vy, cystine hin nay tr thnh thng phm c gi tr trn th trng Quc t, c bit l cc nc Ty u v Nht bn. Ring Nht mi nm tiu th ln ti 500 tn cystine. 5.1.2. Cng ngh sn xut cystine v amino acid t ngun ph liu. Trong cc cng ngh ph bin sn xut amino acid bng con ng vi khun, nm men, con ng tng hp enzyme, con ng tng hp ho hc, th cystine vn ang 95

c tch chit t ngun nguyn liu giu cystine v trong nm 1980, th gii sn xut c 700 tn dng L-cystine. y chng ti ch trnh by vic sn xut cystine v cc amino acid t ngun ph liu l lng g, lng cnh vt lng ln v tc vn, Qu trnh tch chit phi tri qua 11 cng on sau y: Cng on 1: Thu phn bng HCl 100oC. Cng on 2: Trung ho dch thu phn bng Na2CO3. Cng on 3: Lng , lc v thu ta. Cng on 4: Ho tan ta bng HCl 5%, thu ly dch trong. Cng on 5: X l than hot tnh. Cng on 6: Trung ho NaOH, thu cystine th. Cng on 7: n cng on 11 lp li cc bc trn thu c cystine sch v cui cng phi xc nh sn phm bng mt trong nhng phng php khc nhau thng s dng trong phng th nghim. Bng cng ngh trn h thu c hm lng cystine t tc l 6%; t lng cnh vt 2,3%; lng g 3,5% v lng ln 2,13% (tnh theo hm lng amino acid tng s). 5.2. Sn xut protein n bo t b ma Vic gii quyt ngun thc n cn bng v mt dinh dng cho ngi v ng vt ang c ngha thi s. Mt trong nhng hng c nhiu trin vng v c c bit ch l lm giu protein n bo t cc ph liu c ngun gc thc vt dng lm thc n thc vt giu protein. Sn xut protein n bo t b ma nhm nng cao cht lng ca thc n gia sc, ng thi gp phn lm gim mc gy nhim mi trng v nh nng cao hiu qu kinh t ca ngnh cng nghip sn xut ng ma. Nhn chung quy trnh sn xut protein n bo t b ma gm cc bc tng t nh sn xut protein n bo bng vi sinh vt ch khc ngun carbon y l b ma.Tuy nhin cn ch mt s c im sau y: - Thu phn nguyn liu thch hp pH 1,5-3,0 nhit 121 oC thi gian 30- 50 pht. - Ln men chm vi chng nm men thch hp (ch yu l S. tropicalis SK-4). - Mi trng ln men cn b sung (NH4)2HPO4 vi hm lng 1,0 gam/ lt v MgSO4 hm lng 0,7 gam/lt.

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TI LIU THAM KHO 1. Kiu Hu nh, 1999. Gio trnh vi sinh vt hc cng nghip. Nh XB KH& KT H ni. 2. i Duy Ban, L Th Cm Vn, 1994. Cng ngh gen v cng ngh sinh hc ng dng trong Y-Dc hc hin i. Nh XB Y hc. 3. i Duy Ban, L Th Cm Vn, 1994. Chuyn Cng ngh gen trong sn xut vaccine th h mi ng dng trong Y hc v nng nghip hin i. TT T liu, TT KHTN& CN QG, H ni. 4. i Duy Ban, L Thanh Ho, 199 .Cng ngh sinh hc i vi vt nui v cy trng. Nh XB Nng nghip 5. L Trn Bnh, Phan Vn Chi, Nng Vn hi, Trng Nam Hi, L Quang Hun, 2003. p dng cc k thut phn t trong nghin cu ti nguyn sinh vt Vit nam. Nh XB KH&KT H ni. 6. Phm Anh Cng, Panfilov V.I, 1999, Nghin cu quy trnh cng ngh sn xut thc n gia sc giu protein n bo t b ma. Bo co Khoa hc Hi ngh cng ngh sinh hc ton quc. Nh XB KH&KT. H ni 7. Nguyn Quc Khang. 2002. nng lng sinh hc. NXB KH& KT 8. Nguyn Hong Lc. 1998. Gio trnh nui cy m v t bo thc vt. i hc Khoa hoc Hu 9. L Ngc T, 2000. Ho sinh cng nghip. Nh XB KH& KT H ni 10. Copeland. Robert A., 2000. Enzymes; A Practical Introduction To Structure; Mechanism & Data Analysis. Willey-VCH. A John Willey & Sons, INC., Pub. 2nd ed. 11. Dennison Clive . 2002. A Guide To Protein Isolation. Kluwer Academic Publishers. New York, Boston, Dordrecht, Lodon, Moscow. 12. Fersht Alan, 1998, Structure and Mechanism in Protein Science, W. H. Freeman, 3rd Rev Edit. 13. Hans U. B., 1974. Methods of Enzymatic Analysis. Second English Edition Acdemic Press, Inc., New York San Francisco London, Vol., 4. 14.Liebler Daniel C., 2002. Introduction to proteomics. Humana Press Inc. Totuwa, New Jersey. 15. Reseachers Asociates, 1996. Vaccine Handbook. The national Institue of Health. Tokyo, Japan 16.Walker John M., 1996. The Protein Protocols Hand book. 2nd ed. Humana Press Inc. Totuwa, New Jersey. 17. http://www.ivac.com.vn/Default.asx

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