Trng Cao ng Kinh t Cng ngh TPHCM

Khoa Cng ngh Sinh hc

***************





Bi ging:


CNG NGH PROTEIN






CBGD: ThS. L Thanh Hi








TPHCM, thng 01 nm 2013
(Ti liu lu hnh ni b)

i
MC LC

MC LC ........................................................................................................................................i
PHN I: CNG NGH PROTEIN............................................... Error! Bookmark not defined.
CHNG 1: M U ................................................................................................................... 4
I/ Khi qut chung v protein ................................................................................................... 4
1/ c trng chung ca nhm cht protein ....................................................................... 4
2/ ngha khoa hc v thc tin ca protein .................................................................... 4
II/ Phn loi protein ............................................................................................................... 5
1/ Protein n gin ............................................................................................................ 5
2/ Protein phc tp ............................................................................................................ 5
III/ Chc nng sinh hc ca protein ........................................................................................ 6
1/ Chc nng xc tc ......................................................................................................... 6
2/ Chc nng v y hc ....................................................................................................... 7
3/ Chc nng khc ............................................................................................................. 9
CHNG 2: AMINO ACID ........................................................................................................ 10
I/ Thnh phn, tnh cht l ha ca amino acid .................................................................. 10
1/ Thnh phn v cu to ca amino acid ....................................................................... 10
2/ Mu sc v mi v ca amino acid .............................................................................. 12
3/ Tnh tan ca amino acid .............................................................................................. 12
4/ Tnh quang hc ca amino acid ................................................................................... 13
5/ Tnh lng cc ca amino acid ................................................................................... 13
II/ Thu nhn amino acid bng thy phn protein ................................................................. 14
1/ Thy phn bng acid ................................................................................................... 14
2/ Thy phn bng kim .................................................................................................. 14
3/ Thy phn bng enzyme ............................................................................................. 14
III/ Cc phng php phn tch amino acid .......................................................................... 15
1/ Sc k giy .................................................................................................................. 15
2/ Sc k bn mng ......................................................................................................... 16
3/ Sc k kh .................................................................................................................... 16
4/ Phng php in di .................................................................................................... 17
5/ Phng php quang ph khi ...................................................................................... 17
6/ Phng php ng v .................................................................................................. 17
7/ Phng php enzyme .................................................................................................. 17
8/ Phng php vi sinh vt .............................................................................................. 18
CHNG 3: PEPTIDE ................................................................................................................. 19

ii
I/ Tnh cht chung ca peptide ............................................................................................... 19
1/ nh ngha ................................................................................................................... 19
2/ Cu to ........................................................................................................................ 19
3/ Cch gi tn ................................................................................................................ 19
4/ Phn ng c trng ca peptide .................................................................................. 20
II/ Cc phng php tch v xc nh peptide .................................................................... 20
CHNG 4: CU TRC V TNH CHT HA L CA PROTEIN .................................... 22
I/ Cc kiu lin kt trong cu trc ca protein ....................................................................... 22
1/ Lin kt cng ha tr ................................................................................................... 22
2/ Cc lin kt yu lm n nh cu trc ca protein ...................................................... 22
II/ Hnh dng, kch thc v cu trc ca protein ............................................................... 23
1/ Hnh dng, kch thc ca protein .............................................................................. 23
2/ Cu trc ca protein .................................................................................................... 24
III/ Tnh cht ha l ca protein ........................................................................................... 26
1/ Tnh tan ....................................................................................................................... 26
2/ Tnh ngm nc ca protein ....................................................................................... 26
3/ nht ca dung dch ................................................................................................ 26
4/ Tnh cht in ly ca protein....................................................................................... 26
5/ S kt mui ca dung dch protein ............................................................................. 27
6/ Biu hin quang hc ca protein ................................................................................. 27
7/ Kt ta thun nghch v khng thun nghch .............................................................. 28
8/ Cc phn ng ha hc ca protein .............................................................................. 28
IV/ Bin tnh protein ............................................................................................................. 28
1/ Khi nim chung ......................................................................................................... 28
2/ Cc yu t gy bin tnh protein ................................................................................. 29
3/ Tnh cht ca protein bin tnh ................................................................................... 29
CHNG 5: CC PHNG PHP CHIT RT TINH SCH V XC NH PROTEIN .. 30
I/ Cc bin php thu nhn protein nguyn th ....................................................................... 30
1/ Nhit ....................................................................................................................... 30
2/ pH ................................................................................................................................ 30
3/ Tc nhn ha hc ........................................................................................................ 31
II/ Ph v t bo v chit rt protein ................................................................................... 31
1/ Ph v t bo ............................................................................................................... 31
2/ Chit rt protein .......................................................................................................... 32
III/ Tinh sch protein ............................................................................................................ 32
1/ Cc loi tp cht .......................................................................................................... 32
2/ Cc phng php tinh sch protein ............................................................................. 32

iii
IV/ nh lng v nh gi tinh sch ca ch phm protein ........................................... 35
1/ Cc phng php xc nh hm lng protein ........................................................... 35
2/ Phng php nh gi tinh sch ca ch phm protein ......................................... 36
TI LIU THAM KHO ............................................................................................................. 38
Ti liu Ting Vit ..................................................................................................................... 38
Ti liu Ting Anh ..................................................................................................................... 38
TI TIU LUN .................................................................................................................... 39
Cng ngh Protein
4
CHNG 1: M U
I/ Khi qut chung v protein
1/ c trng chung ca nhm cht protein
Protein c pht hin ln u tin th k XVIII; mi u c gi l albumin. Mi
n nm 1838, Mulder ln u tin a ra thut ng protein.
V mt s lng, protein 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.
Trong cu trc ca protein gm bn nguyn t chnh l C, H, O, N vi t l C 50%, H
7%, O 23% v N 16%. Ngoi ra trong protein cn gp mt s nguyn t khc nh
S 0-3% v P, Fe, Zn, Cu...
Khi lng phn t c k hiu l Mr (c tnh bng Dalton - Da). Khi lng phn
t ca cc loi protein thay i trong nhng gii hn rt rng, thng thng t hng trm
cho n hng triu.
VD: Insulin c Mr = 5.733Da = 5,733kDa
Glutamate dehydrogengenase trong gan b c Mr = 1.000.000Da = 1.000kDa
Bng 1. Khi lng phn t v cu trc ca mt s loi protein
Protein
Khi lng
(Dalton)
S gc
amino acid
S chui
polypeptide
Glucagon 3.482 29 1
Insulin 5.733 51 2
Ribonuclease (ty b) 12.640 124 1
Lysozyme (lng trng trng) 13.930 129 1
Myoglobin (tim nga) 16.890 153 1
Chymotripsin (ty b) 22.600 241 3
Hemoglobin (ngi) 64.500 754 4
Albumin (huyt thanh ngi) 68.500 550 1
Hexokinase (men bia) 96.000 800 4
Tryptophan synthetase (E.coli) 117.000 975 4
-globulin (nga) 149.000 1.250 4
Glycogen phosphorylase (c th) 495.000 4.100 4
Glutamate dehydrogengenase (b) 1.000.000 8.300 40
Synthetase ca acid bo (men bia) 2.300.000 20.000 21
Virus khm thuc l 40.000.000 336.500 2130
2/ ngha khoa hc v thc tin ca protein
ngha khoa hc: Protein tham gia mi hot ng sng trong c th sinh vt (xy dng
t bo, m, tham gia hot ng xc tc v nhiu chc nng khc)
Cng ngh Protein
5
ngha thc tin: 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 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:
Protein n gin
Protein phc tp
1/ Protein n gin
Protein n gin l nhng phn t m thnh phn cu to ca n gm hon ton amino acid.
VD: Ribonuclease gm 124 gc amino acid, Mr = 12.640Da
Chymotripsin gm 241 gc amino acid, Mr = 22.600Da
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 (NH
4
)
2
SO
4
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, Na
2
SO
4
..., v b kt ta nng mui (NH
4
)
2
SO
4
bn
bo ho.
Prolamin: khng tan trong nc hoc dung dch mui long, tan trong ethanol, isopanol
70-80%.
Glutelin: 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/ Protein phc tp
Protein phc tp l nhng protein m thnh phn phn t ca n ngoi cc -amino acid cn c
thm thnh phn khc c bn cht khng phi protein (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)...
Metalloprotein: nhm ngoi l kim loi

Cng ngh Protein
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III/ Chc nng sinh hc ca protein
1/ Chc nng xc tc
Quan im v xc tc enzyme:
Hu ht 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.
Enzyme l nhng protein c kh nng xc tc c hiu cho cc phn ng ho hc, l cht
xc tc sinh hc 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.
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:
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 Fe
3+
ch xc tc phn ly c 10
-6
mol H
2
O
2
/pht. Trong khi mt phn t
catalase c mt nguyn t Fe xc tc phn ly 5.10
6
mol H
2
O
2
/pht;
1 gram pepsin trong 2 gi thu phn c 5 kg protein trng luc nhit bnh thng.
1 gram 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.
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:
o 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...
o 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
V d: Enzym urease ch phn ho urea ch khng nh hng ti
metylurea

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
V d: Nhm esterase c lipase ct mch este gia acid bo v ru (acid
bo c th di ngn khc nhau, ru c th glycerin hoc ru vng...).
Tuy vy tc phn ng c thay i.
Cng ngh Protein
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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
V d: carboxypeptidase c kh nng phn ct lin kt peptide gn nhm
carboxyl t do
c hiu quang hc: l enzyme ch tc dng ln mt trong hai dng ng
phn quang hc ca cc cht
V d: L- arginin b phn ho bi L- arginase thnh omitin v urea, cn D-
arginin enzym ny khng phn ha.
Enzym lactat-dehydrogenase ca bp tht ch xc tc chuyn acid lactic
kiu L (+) thnh acid pyruvic nhng khng tc ng ln kiu D (-)
Cc yu t nh hng n hot tnh ca enzyme
Nng enzyme
Nng c cht
Nhit
pH
Cc ion kim loi
S phn b ca 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, nicotinic-mononucleotide
adenylyltransferase, 5nucleotidase.
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
2/ Chc nng v y hc
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.
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.
Cng ngh Protein
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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:
o 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.
o Nhng bin i v cht lng protein: l nhng ri lon v cu trc protein do
gene b 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...
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 dch c nhc n nhiu hn c l
cc khng th, b th v cc cytokine.
o 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, c th chia globulin thnh 5 loi, bao gm: IgG, IgA, IgM,
IgD, IgE.
o 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
o 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 on
p ng min dch cp tnh.
Protein vn chuyn: Trong c th c nhng protein lm nhim v vn chuyn nh
hemoglobin, mioglobin, hemocianin vn chuyn O
2
, CO
2
v H
+
i khp cc m, cc c
Cng ngh Protein
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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.
3/ Chc nng khc
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
Nhiu protein trc tip tham gia trong qu trnh chuyn ng nh: c c, dui c, chuyn
v tr ca nhim sc th trong qu trnh phn bo. Cc protein lm nhim v co rt nh
myosin, actin si c
Protein cn l cht dinh dng quan trng cung cp cc acid amin cho phi pht trin.
Cc protein lm nhim v d tr nh casein ca sa, ovalbumin ca trng, gliadin trong
ht la m, zein ca ng, feritin (d tr st) trong l, v.v...


Cng ngh Protein
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CHNG 2: AMINO ACID
I/ Thnh phn, tnh cht l ha ca amino acid
1/ Thnh phn v cu to ca amino acid
Amino acid l n v cu to protein
Amino acid l cht hu c, phn t c cha nhm: carboxyl (COOH) v nhm amino (NH
2
) gn
vi carbon v tr
Hu ht amino acid thu nhn c khi thy 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)
Da vo tnh cht ca gc R cc amino acid c chia lm 5 nhm:
Nhm I: gm 7 amino acid c R khng phn cc, k nc: glycine, alanine, proline, valine,
leucine, isoleucine v methionine

Nhm II: gm 3 amino acid c gc R cha nhn thm: phenylalanine, tyrosine v tryptophan


Cng ngh Protein
11
Nhm III: gm 5 amino acid c gc R phn cc, khng tch in: serine, threonine, cysteine,
aspargine v glutamine

Nhm IV: gm 3 amino acid c R tch in dng: lysine, histidine v arginine

Nhm V: gm 2 amino acid c gc R tch in m: aspartate v glutamate

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.

Cng ngh Protein
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Bng 2: Cc amino acid thng gp
Tn amino
acid
Tn amino acid gi theo php danh ha
hc
Tn
vit tt
K
hiu
Khi lng
(Mr - Dalton)
Glycine -aminoacetic Gly G 75
Alanine -aminopropionic Ala A 89
Proline -pirolidincarboxylic Pro P 115
Valine -aminoiaovaleric Val V 117
Leucine -aminonoisocaproic Leu L 131
Isoleucine -amino--metylvaleric Ile I 131
Methionine -amino-metyltiobutiric Met M 149
Phenylalanine -amino--phenylpropionic Phe F 165
Tyrosine -amino--hydrogengenxyphenylpropionic Tyr Y 181
Tryptophan -amino-idolylpropionic Trp W 204
Serine -amino--hydroxypropionic Ser S 105
Threonine -amino--hydrogengenxybutiric Thr T 119
Cysteine -amino--tiopropionic Cys C 121
Aspargine Amid ca aspartate Asn B 132
Glutamine Amid ca glutamate Gln Q 146
Lysine ,-diaminocaproic Lys K 146
Histidine -amino--imidazolpropionic His H 155
Arginine -amino--guanidinvaleric Arg R 174
Aspartate -aminosucinic Asp D 133
Glutamate -aminoglutarate Glu E 147
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: Methionine, Valine, Leucine, Isoleucine,
Threonine, Phenylalanine, Triptophan, Lysine, Arginine v Histidine, ngy nay ngi ta cn
xem Cysteine cng l mt amino acid khng thay th.
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.
2/ 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)
3/ 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).
Cng ngh Protein
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4/ 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, gi l ng phn lp th. S ng phn lp th c tnh
theo 2n (n l s carbon bt i)
Hu ht cc amino acid khc hp th tia cc tm bc sng () khong t 220 - 280 nm. c
bit cng nng 10
-3
M, trong bc sng khong 280 nm tryptophan hp th nh sng cc tm
mnh nht, gp 4 ln kh nng hp th ca tyrosine v phenylalanine l yu nht. Phn ln cc
protein u cha tyrosine nn ngi ta s dng tnh cht ny nh lng protein.

5/ Tnh lng cc 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 -NH
2
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 in), v tip tc tng
pH amino acid s nhng proton th hai chuyn thnh dng anion (tch in m). V vy i khi
ngi ta coi n nh mt di-acid.

Tng ng vi phn ly H
+
ca cc nhm COOH v
NH
3+
c cc tr s pK
1
v pK
2
. T ngi ta xc
nh c pI = (pK
1
+ pK
2
)/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.

Cng ngh Protein
14
Bng 3: Cc tr s pK ca cc amino acid thng gp
Tn cc amino
acid
Cc tr s pH
pK
1
(ca COOH) pK
2
(ca NH
3+
) pK
R
(ca R) pI
Glycine 2,34 9,60 5,97
Alanine 2,34 9,60 6,01
Proline 1,99 10,96 6,48
Valine 2,32 9,62 5,97
Leucine 2,36 9,60 5,98
Isoleucine 2,36 9,68 6,02
Methionine 2,28 9,21 5,74
Phenylalanine 1,83 9,13 5,48
Tyrosine 2,20 9,11 10,07 5,66
Tryptophan 2,38 9,39 5,89
Serine 2,21 9,15 5,68
Threonine 2,11 9,62 5,87
Cysteine 1,96 10,28 8,18 5,07
Aspargine 2,02 8,80 5,41
Glutamine 2,17 9,13 5,65
Lysine 2,18 8,95 10.53 9,74
Histidine 1,83 9,17 6,00 7,59
Arginine 2,17 9,04 12,48 10,76
Aspartate 1,88 9,60 3,65 2,77
Glutamate 2,19 9,67 4,25 3,22
II/ Thu nhn amino acid bng thy phn protein
1/ Thy phn bng acid
thu nhn cc amino acid phng php thng c dng nhiu nht l thu phn bng acid
HCl 6N d nhit 100-120
o
C 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 NH
4
+

2/ Thy 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.
3/ Thy 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.
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:
Peptidhydrogenlase ct t lin kt peptid u tn cng ca chui polypeptide c gi
l exopeptidase nh:
o Carboxypeptidase phn gii lin kt peptide u C tn cng
o Aminopeptidase phn gii lin kt peptide u N tn cng

Cng ngh Protein
15
Peptidhydrogenlase ct t cc lin kt peptide gia ca chui polypeptide c gi l
cc endopeptidase nh:
o Pepsin
o Tripsin, v.v...
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:
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.
Xc nh thi gian cn thit thu nhn c mt lng bin thin nht nh ca c cht
hay sn phm ng vi mt lng enzyme nht nh.
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.
III/ Cc phng php phn tch amino acid
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 vo 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 NaBH
4
, hoc s dng enzyme carboxypepitdase v.v...
1/ Sc k giy
Nguyn tc: 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 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
Rf = a/b
Trong : - a l khong cch chuyn dch ca cht phn tch
- b l khong cch chuyn dch ca dung mi
Cc kiu sc k giy:
Sc k mt chiu i ln
Sc k mt chiu i xung
Sc k vng nm ngang
Sc k hai chiu

Cng ngh Protein
16
Loi giy thng dng:
Whatman s 1
Schleicher-Schull 2044 a v b
Dung mi:
Dung mi dng cho chiu th nht: Butanol : Acetic acid : Nc (4:1:5)
Dung mi dng cho chiu th hai: Phenol : Nc (3:1)
2/ Sc k bn mng
Nguyn tc: 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: silicagel, alumin oxyt, sephadex ,v.v...c kt hp vi thch
cao dn vo phin knh.

3/ Sc k kh
Nguyn tc: da vo tnh cht kh bay hi ca cc amino acid nn c th s dng 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 anhydrid acetic.
Ct thng dng l loi Chromosorb W (60-80 mesh) c mt lp polyetylenglycol 1%
(carbowax 1564 hoc 6000). Thng s nhit : 125
o
C - 155
o
C, tc dng chy 60-240
ml/pht.


Cng ngh Protein
17
4/ Phng php in di
Nguyn tc: da vo tnh cht tch in ca cc amino acid trong mi trng c pH nht nh.
Di tc dng ca in trng cc amino acid tch in dng (+) s chy v pha cc (-), cc
amino acid tch in m s chy v cc (+). Do kh nng tch in khng ging nhau gia cc
amino acid v vy tc di chuyn ca cc amino acid khng ging nhau trong in trng. Kt
qu cc amino acid phn b tri ra trn gi th (giy hoc polyamide).

5/ Phng php quang ph khi
Nguyn tc: dng mt chm electron bn vo mt lng cht th rt nh, cc phn t cht th
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 peak 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

S hot ng ca quang ph khi
6/ Phng php ng v
Nguyn tc: 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.
ng 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.
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
Cng ngh Protein
18
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 CO
2
trong mi trng. Xc nh nng CO
2
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-carboxylyase (glutamate decarboxylase - EC 4.1.1.15),
EC 4.1.1.15 xc tc chuyn ho L-glutamic 4-aminobutanoate (-aminobutyrate) +
CO
2

- pH 5,5 to ra L-tyrosine carboxylyase (EC 4.1.1.25), EC 4.1.1.25 xc tc chuyn ho
L-tyrosine Tyramine + CO
2


Cng ngh Protein
19
CHNG 3: PEPTIDE
I/ Tnh cht chung ca peptide
1/ nh ngha
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.
2/ Cu to
Cc amino acid l nhng n phn t xy dng nn cc chui polypeptide. Trong cc chui
polypeptide cc amino acid c lin kt vi nhau thng qua lin kt peptide.

u ca mt chui peptide l amino acid c nhm -amino (-NH
2
) 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.
3/ Cch gi tn
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.
VD: Cch gi tn ca mt pentapeptide: Seryl-Glycyl-Tyrosyl-Alanyl-Leucine (SGYAL)

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, 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
Cng ngh Protein
l u N-tn cng hay u C- tn cng v nh v
hay Leu-COOH. Trong trng h
cha xc nh c r ngi ta c th
(Ala,Leu, Val) Glu-Arg-...
4/ Phn ng c trng c
Ngoi phn ng ca nhm NH
2
v COOH
phn ng mu c trng ca cc amino acid t
c trng nht cho lin kt peptide l ph
acid t do. Trong mi trng ki
cht mu tm v c kh nng h
dng rng ri nh lng protein. Phng php xc
nguyn tc ca phn ng ny b
ca phn ng sau khi thc hin ph
th to phc mu xanh da tr
II/ Cc phng php tch v xc
C mt s phng php tch phn l
trong peptide. Nguyn tc chung c
bn cng nh i vi protein. Tuy nhin peptide l nh
th c th b qua giai on ct chu
ngay bng phng php in di hay s
peptide ngi ta tin hnh thu phn n hon ton thnh cc amio acid t
amino acid u N-tn cng v amino acid
c so snh i chiu v tng h
V d, Puppy v Bodo phn tch m
d kin sau y:
Thnh phn amino acid c
1 Ala, 2 Glu,1His, 1Thr, 1Val,v 1Lys.
Dng phng php Sanger xc
php carboxypeptidase xc
Cu to ca peptide nh (b
acid, amino acid u N-tn cng v amino acid
Cys- Ala Glu- Cys (Val
Cys-(Ala,Glu) Cys
Ala- Glu Glu (Cys, His) Glu
Thr (Val, Glu, Lys)
Tng hp cc d kin trn, h
nghin cu l:
H
2
N-Cys-Ala-Glu-
20
n cng v nh vy cng pentapeptide trn c th
ng hp c nhng amino acid mt on no trong chu
i ta c th cc amino acid trong ngoc chng h
c trng ca peptide
v COOH u tn cng, cc gc R ca peptide c
a cc amino acid t do tng ng. Mt trong nh
t peptide l phn ng Biure, phn ng ny khng x
m mnh, lin kt peptide phn ng vi CuSO
nng hp th cc i bc sng 540 nm. y l ph
ng protein. Phng php xc nh protein theo Lowry c
ng cch thm thuc th Folin-Ciocalteau
n phn ng biure, ng thi da vo cc gc Tyr, Trp nh
c mu xanh da tri.

Cc phng php tch v xc nh peptide
phng php tch phn lp v xc nh thnh phn, s lng v trnh t
c chung ca cc phng php tch phn lp v xc
i protein. Tuy nhin peptide l nhng on ngn ca chu
t chui polypeptide thnh cc peptide nh m c th
n di hay sc k tch ring tng peptide. Sau khi
phn n hon ton thnh cc amio acid t do. T
n cng v amino acid u C-tn cng. Cc d liu thu
p li.
, Puppy v Bodo phn tch mt peptide ca dch khi thu phn Cytocrom C thu
a peptide sau khi c thu phn hon ton v s
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 Ly
(bng cch thu phn tng phn ban u v xc
n cng v amino acid u C-tn cng ca m
Cys (Val- Glu)
(Ala,Glu) Cys- His Thr (Val, Glu)
(Cys, His) Glu- Lys
Thr (Val, Glu, Lys)
kin trn, h xc nh c trnh t cc amino acid c
-Cys-His-Thr-Val-Glu-Lys-COOH.
trn c th vit H
2
N-Ser
n no trong chui peptide
ng hn Ala- Ser-Gly-
a peptide cng cho nhng
ng phn ng mu
ng ny khng xy ra vi amino
i CuSO
4
to thnh phc
c sng 540 nm. y l phn ng c s
nh protein theo Lowry cng da trn
lm tng nhy
c Tyr, Trp nh thuc
ng v trnh t amino acid
p v xc nh peptide v c
a chui polypeptide v
m c th tch phn lp
ng peptide. Sau khi tch ring cc
do. T xc nh cc
c qua phn tch s
phn Cytocrom C thu c cc
phn hon ton v sc k l 2Cys,
n cng l Cys v phng
n cng l Lys.
u v xc nh cc amino
i peptide nh):
cc amino acid ca peptide
Cng ngh Protein
21
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.

Cng ngh Protein
22
CHNG 4: CU TRC V TNH CHT HA L CA
PROTEIN
I/ Cc kiu lin kt trong cu trc ca protein
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, 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.
Cc kiu lin kt trong cu trc ca protein:
- Cc lin kt cng ha tr
- Cc lin kt yu lm n nh cu trc ca protein
1/ Lin kt cng ha 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-).
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. 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.

S hnh thnh cu disunfua gia hai phn t cystein
2/ Cc lin kt yu lm n nh cu trc ca protein
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 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.
Cng ngh Protein
23

Mt s lin kt hydro quan trng trong h thng sng
a) gia hydro ca mt ancohol v oxy ca nc; b) gia nhm carbonyl keto v nc;
c) gia nhm peptide trong polypeptide
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 in t
lin kt khng c 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 H
2
O to thnh mt
lp v bc ngoi nn khng th lin kt trc tip vi cc anion v cation khc. Do , tng tc
tnh in ny khng ng vai tr quan trong quyt nh cu hnh khng gian ca cc phn t hu
c.
Lin kt Vander 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.
Lin kt 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 H
2
O (y phn t H
2
O ra ngoi) c gi l lin kt k nc. y l khuynh hng loi tr
cc nhm khng phn cc ra khi mng li nc. 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.
II/ Hnh dng, kch thc v cu trc ca protein
1/ Hnh dng, kch thc ca protein
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. 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 3000A
o
, ng knh khong 15A
o
.
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...

Cng ngh Protein
24
2/ Cu trc ca protein
Cu trc bc 1 (cu trc s cp)
Cu trc bc I ca protein 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.
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.

Cu trc bc 1 ca ribonucleseas b
Cu trc bc 2 (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.

Cc kiu xon trong cu trc bc 2 ca protein

Cng ngh Protein
25
Bng 4: S lng xon v phin gp trong chui n ca mt s protein
Protein (s gc)
S gc (%)
Xon Phin gp
Chymotrypsin (247) 14 45
Ribonuclease (124) 26 35
Carboxypeptidase (397) 38 17
Cytochrom C (104) 39 0
Lysozyme (129) 40 12
Myoglobin (153) 78 0
Cu trc khng gian ca protein
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.

S cu trc khng gian ca 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 protein 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 Cystein
to nn lin kt disulfua gia cc gc Cystein 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.
Nh vy, 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.

Cng ngh Protein
26
III/ Tnh cht ha l ca protein
1/ Tnh tan
Cc loi protein khc nhau c kh nng ho tan d dng trong mt s loi dung mi nht nh.
V d: - 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...
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 phn
t protein nh -NH
2
, -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.
3/ nht ca dung dch
Khi protein ho tan trong dung dch, mi loi dung dch ca nhng protein khc nhau c nht
khc nhau. 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 5: nht ca mt s dung dch protein
Protein Nng % (trong nc) nht tng i (ca nc = 1)
Gelatin 3,0 4,54
Albumin trng 3,0 1,20
Gelatin 8,0 14,2
Albumin trng 8,0 1,57
4/ Tnh cht in ly 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 NH
2
ca Lys; nhm OH ca Ser, Thr, Tyr v.v...
Trng thi tch in ca cc nhm ny 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.
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.

Cng ngh Protein
27
Bng 6: Gi tr pHi ca mt s protein
Protein pHi Protein pHi
Pepsin 1,0 Globulin sa 5,2
Albumin trng 4,6 Hemoglobin 6,8
Casein 4,7 Ribonuclease 7,8
Albumin huyt thanh 4,9 Tripsin 10,5
Gelatin 4,9 Prolamin 12,0
5/ 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. Cc mui c ion ho tr
2 (MgCl
2
, MgSO
4
...) lm tng ng k tan ca protein hn cc mui c ion ho tr 1 (NaCl,
NH
4
Cl, 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).
V d dng mui (NH
4
)
2
SO
4
50% bo ho kt ta globulin v dung dch (NH
4
)
2
SO
4
bo ho
kt ta albumin t huyt thanh.
6/ Biu hin quang hc ca protein
Cng nh nhiu cht ho hc khc, protein c kh nng hp th v bc 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. 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.
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.
Cng ngh Protein
28
7/ Kt ta thun nghch v khng thun nghch
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.
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.
8/ Cc phn ng ha 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, -NH
2
, gc R v phn ng to mu c trng ca lin kt peptide nh
phn ng biure. 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:
Phn ng vi thuc th Folin-Ciocalteau
Thuc th Folin-Ciocalteau c cha acid phosphomolipdic v acid phosphovolframic. 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.
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.
Di tc dng ca ninhydrin nhit cao, amino acid to thnh NH
3
, CO
2
v aldehit, mch
polypeptide ngn i 1 carbon; ng thi ninhydrin chuyn thnh dicetooxyhindrien.
Dicetooxyhindrien, NH
3
mi to thnh tip tc phn ng vi mt phn t ninhidrin khc to
thnh phc cht mu xanh tm
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
HNO
3
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 HNO
3
c to thnh kt
ta mu nu t v.v...
IV/ Bin tnh protein
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
o
C. Song nhit 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
Cng ngh Protein
29
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.
2/ Cc yu t gy bin tnh protein
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.
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
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, v 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
o
C, 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.

Cng ngh Protein
30
CHNG 5: CC PHNG PHP CHIT RT TINH SCH V
XC NH PROTEIN
I/ Cc bin php thu 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.
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 c gi 50 - 70
0
C 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 0
o
C trnh
bin tnh c bit l protein enzyme.
2/ pH
Protein l cc cht lng tnh, v vy, trong cc dung dch acid v kim chng s b phn ly:

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 amino acid, OH ca
Tyrosine, -NH
2
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 H
+
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 c gi
Cng ngh Protein
31
pH 5 trong thi gian xc nh. Protein tp b bin tnh cng c loi b bng cch lc hoc ly
tm.
3/ Tc nhn ha hc
C th dng mui trung tnh hoc cc dung mi hu c tch chit cc protein. 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 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 5
o
C tr xung). Dng
dung mi hu c c th tin hnh tch phn on di 0
o
C v c th n -20
o
C, nh vy c tc
dng tt n n nh ca protein.
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
)
2
SO
4
, Na
2
SO
4
, MgSO
4
... Tuy nhin, ngi ta nhn
thy mui (NH
4
)
2
SO
4
l tt nht v n khng lm hi m lm n nh (lm bn) hu ht cc loi
protein. Loi mui ny li r tin v ph bin. ha tan ca n li rt ln (bo ha 767g/l
25
o
C)
Ngoi ra, nng (NH
4
)
2
SO
4
cn thit kt ta protein khc nhau th khc nhau nhiu.
V d: Protease ca nm mc d b kt ta 70% ca (NH
4
)
2
SO
4
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 (NH
4
)
2
SO
4
cao hn cc mui khc.
C th dng (NH
4
)
2
SO
4
c 2 dng: dng bt v dng dung dch bo ha. Khi dng bt, ngi ta
cho tng t mt vo dch chit protein. 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 nim v s phn trm ca bo ha hon ton c cp n. Protein c th b kt
ta 50% (0,5) hoc 70% (0,7) ca bo ha hon ton ca (NH
4
)
2
SO
4
. Khi cho dung dch
(NH
4
)
2
SO
4
bo ha vo dch chit protein th nng (NH
4
)
2
SO
4
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. Kt ta c ly ra bng cch ly tm hoc lc. Khi ha tan kt ta li ngi ta thng thm
ion Ca
2+
lm bn protein (dng CaCl
2
hoc Ca(CH
3
COO)
2
).
II/ Ph v t bo v chit rt protein
1/ Ph v t bo
Protein c trong tt c cc c th ng vt, thc vt v vi sinh vt. Nhiu protein lin kt rt cht
ch vi cc bo quan ca t bo. Do c th chit rt cc protein, bc u tin l phi ph
v cu trc ca cc t bo c cha protein 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 motor 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 (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,
Cng ngh Protein
32
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.
2/ Chit rt protein
Sau khi ph v cu trc ca cc t bo tin hnh chit xut cc protein bng cc dng dch
m thch hp, dung dch mui trung tnh hoc bng nc hoc bng ly tm: vic chn phng
php tch chit protein ty thuc vo tnh cht ca protein cn nghin cu.
III/ Tinh sch protein
1/ Cc loi tp cht
Trong dch chit th thu c ngoi protein nghin cu cn c cc protein tp, cc cht cao phn
t khc nh polysaccharide, 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,
cc phng php sc k trao i ion, in di, phng php lc gel.
2/ Cc phng php tinh sch protein
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. Mt s phng php s dng cho vic tinh sch
cc protein:
a) 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 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.
b) 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 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 cellophane. 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
Cng ngh Protein
l nhng i phn t khng th
i thng xuyn dung dch r
thm tch, n l dung dch
nh th khi tin hnh thm tch d
p sut thy tnh y , dng th
tn ca cc phn t vo dung d
Phng php thm tch thng th
(khng qu 2/3 th tch ti). Cho thu
thm tch lu, protein c th b
ming chu nc gi ti
i nc thng xuyn. Mui (NH
c th t 24 - 48 gi, lm thm tch l
C th tng tc thm tch khi khu
hoc l quay chm ti nh
hnh tt c cc thao tc mi tr
c) Phng php sc k l
Sc k lc gel l mt trong nh
tinh sch protein. Dch chit protein
m bo ng nht cn thi
C s ca phng php lc gel l d
t lng ca protein c trong h
m bo cho vic tch protein
protein. Cht ny cng khng h
hc. Ngoi ra cht c s d
(khng co) v phi l cht a n
Gel sephadex l cht tha mn cc y
vt khc nhau l Leuconostoc
Trng lng phn t ca dextran c th
cc chui do cc gc glucose t
bng cch x l ha hc (do tc d
ngang gi l "sng phn t" v ch
cng nhiu, kch thc ca l sng phn t
Phng php lc phn t trn Sephadex
di v cn bng bng dung d
lc v chit bng dung mi thch h
mui) s khuch tn chm qua cc l
trng lng phn t ln hn (
nhanh qua cc ht sephadex v s
c trng lng phn t cao hn thot ra kh
33
khng th vt qua ti thm tch v c gi li trong ti. B
ch ra c th ty sch mui ra khi protein, m
c pha long hn. C th lm gim bt hoc lo
m tch di p sut, c ngha l khi dung dch
dng thy ng hc ca nc t dung dch s
vo dung dch. Phng php ny thng i hi c thi
m tch thng thng l cho dung dch c kt ta protein vo ti th
Cho thuc st trng hoc toluen bo qun protein (v th
thi hng). Buc ti vo mt que thy tinh, gc que th
gia chu. Cho vi nc chy nh lin tc vo y ch
i (NH
2
)SO
4
ha tan trong nc v b loi dn. Th
m tch ln cui cng bng nc ct.
m tch khi khuy dung dch ra bng my trn c h
ng c khng ln. Khi thm tch cc protein th
mi trng lnh
c k lc gel
t trong nhng k thut sc k ct c dng ph bi
t protein c loi b phn ln cc protein t
t c tip tc lm sch bng phng php s
c gel l da vo s khc nhau nhau v kch th
a protein c trong hn hp tch chng ra.
c tch protein c tt, cht ry phn t phi l cht tr, khng ph
ng khng ha tan v tng i bn vi cc yu t v
ng cho mc ch lc phn t phi l cht khng c tnh n h
t a nc (hidrofil).
a mn cc yu t trn. Sephadex l ch phm dextran do cc loi vi sinh
to ra khi chng c nui cy trn mi tr
a dextran c th t ti hng triu v ln hn. Phn t
c glucose to thnh cc lin kt 1,6 glucoside. Sephadex
c (do tc dng ca epichlohidrin) to ra cc li phn nhnh c lin k
" v cht ny tr thnh khng tan trong nc. S
sng phn t cng nh.

trn Sephadex c tin hnh nh sau: cho sephadex vo c
ch m c pH nht nh. Sau cho dung dch protein ln c
ng dung mi thch hp, cc phn t c trng lng phn
m qua cc l nh ca cc ht Sephadex b trng ph
n hn ( trng hp ny l protein) khng c kh
t sephadex v s c chit nhanh ra khi ct. V vy ta c th
cao hn thot ra khi ct gel trc so vi cht c phn t
i trong ti. Bng cch thay
c du trong qu trnh
c loi tr s pha long
c x l nm di mt
cn bng s khuch
t b c bit.
a protein vo ti thm tch
n protein (v thi gian
y tinh, gc que thy tinh ln
c vo y chu thay
n. Thi gian thm tch
n c hc hay my trn t
m tch cc protein thng ngi ta tin
n trong tch chit v
n cc protein tp nhng vn cha
ng phng php sc k ct.
c, hnh dng v phn
t tr, khng phn ng vi
c hc cng nh sinh
t khng c tnh n hi
m dextran do cc loi vi sinh
y trn mi trng cha saccharose.
n hn. Phn t dextran bao gm
. Sephadex nhn t dextran
i phn nhnh c lin kt
lin kt ngang to ra

n hnh nh sau: cho sephadex vo ct thy tinh
ch protein ln ct. Khi
t nh ( y l cc
trng phng, cn cht c
nng i vo m lch
y ta c th tch c cht
t c phn t lng nh.
Cng ngh Protein
34
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, chng cn c s dng c c dung dch protein.
d) 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. 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 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) 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 thng thng c dng tch protein,
cn cc cht trao i ion c cht gi l polistirol 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.
Anionit DEAE - cellulose (diethylamino - ethyl - cellulose) l dn xut este ca cellulose)
e) Phng php sc k i lc
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 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 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
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 khi ct trng thi sch.
f) Kt tinh protein
y l phng php c hiu tt nht tch tng phn protein giai on tinh ch cui cng.
Khi protein c lm tinh khit hon ton, trong nhng trng hp ring bit, ngi ta c th
tin hnh kt tinh chng. Cc tinh th protein kt tinh ln u i khi c sch khng vt qu
50% v c th cha cc protein khc. Ngi ta thng tin hnh kt tinh protein trong dung dch
(NH
4
)
2
SO
4
. 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 (NH
4
)
2
SO
4
vo dung dch protein
kh m c cho n khi lm vn c nh nhng dung dch. Sau tng rt t t nng mui
trong dung dch. Trong qu trnh kt tinh c th thay i ch s pH hoc nhit . kt tinh
protein c d dng, nhng giai on trc , ngi ta thng tch tng phn cc protein
bng cc dung mi hu c.
g) 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 b kh nhit trong phng th a s protein b bin tnh. Protein ch c th
c gi dng 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
Cng ngh Protein
35
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 mmHg (c to ra nh my ht chn khng mnh). Hi nc c to ra u c ng
bng trong bu d tr nhit thp hn (-70, -80
o
C). 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 ny, huyt thanh kh
ny c th c s dng trong mc ch truyn mu.
IV/ nh lng v nh gi tinh sch ca ch phm protein
1/ Cc phng php xc nh hm lng protein
Protein sau khi c tch chit v lm sch c th nh lng c. 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 nghin cu l enzyme th vic nh lng thng qua xc nh hot ca enzyme.
Nu protein khng phi l enzyme th ta c th nh lng chng bng cc phng php khc.
Nu l protein vn chuyn th c th xc nh chng thng qua nng cht m chng vn
chuyn. 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: hormon sinh trng (growth hormone) s kch thch s sinh trng ca cc t bo ch
nui cy.
Mt s phng php thng c s dng xc nh hm lng protein:
nh lng nitrogen theo phng php kjeldahl:
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 H
2
SO
4
m c v cht xc tc. Dng
mt kim mnh (NaOH hoc KOH) y NH
3
t mui (NH
4
)
2
SO
4
. Sau hng NH
3
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
Cng ngh Protein
36
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 1g/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 theo 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 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)=1,55xA
280
- 0,77xA
260

Trong : A
280
: hp th bc sng 280nm
A
260
: hp th bc sng 260nm
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 huyn ph ch khng phi trong dung dch. (V
d, trong mng hoc cc phc hp c trng lng phn t ln). Nu t l A
280
/A
260
thp hn 0,6
ngha l dung dch protein cha sch, b ln cc cht khc.
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.
2/ Phng php nh gi tinh sch ca ch phm 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.
Cc phng php kim tra tnh ng th (hoc cn gi l tnh ng nht) ca protein
Xy dng ng biu din v ho tan: y l phng php n gin v nhy nht
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
Cng ngh Protein
37
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 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.
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 loi 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

38
TI LIU THAM KHO
Ti liu Ting Vit
[1] Ha sinh cng nghip, L Ngc T, NXB KHKT HN, 2002.
[2] Cng ngh enzyme, Nguyn c Lng, NXB HQG TPHCM, 2004
Ti liu Ting Anh
[3] Enzymes in Food technology, Robert J.Whitehurst Barry A.Law, Sheffield Academic Press
CRC Press, 2002
[4] Protein in Food processing, R. Y. Yada, Woodhead Publishing Limited and CRC Press
LLC, 2004

39
TI TIU LUN
1/ Cc phng php bin tnh protein
2/ Protein trong sa (casein, whey protein)
3/ Protein trong thc vt (u nnh, ht c du)
4/ Protein trong c tht v trong ng cc
5/ Thu nhn v tinh sch enzyme amylase t ht ny mm
6/ Thu nhn v tinh sch enzyme protease t vi sinh vt
7/ Protein to gel, mng n c
8/ ng dng protein lm ph gia trong thc phm
9/ Cc phng php c nh enzyme
10/ ng dng enzyme trong sn xut nc p tri cy
11/ ng dng enzyme trong sn xut tinh bt bin tnh
12/ ng dng enzyme trong sn xut ru vang
13/ ng dng enzyme trong ln men
14/ ng dng enzyme trong sn xut bnh m, pasta v m n lin
15/ Protein t to bin v ng dng trong cng nghip
16/ Thu nhn v tinh sch enzyme pepsin t d dy heo
17/ ng dng enzyme trong sn xut bia
18/ Cc phng php trch ly v tinh sch protein