CHNG 3.

CC PHNG PHP TINH CH ENZIM

1. LCH S PHAT TRIN CA TINH CH ENZIM
Giai on 1: da trn cc tnh cht l ho hc tc ng ln cc
cht phn t lng ln.

Giai on 2:
Khai thc cc tnh cht c trng khc nhau ca protein cu. Phn tch da trn cc thng s ring nh trng lng phn t, in tch ca protein.

Giai on 3: da trn nhng tng tc c hiu v c tnh rhun

nghch gia enzim v: C cht. Coenzim. Cht gn. Cht hot ho.

2. NGUYN TC CHN PHNG PHP TINH CH ENZIM

Xc nh enzim mong mun l g v chn nguyn liu giu enzim. Xc nh cc c trng ca nguyn liu: Bn cht ca NL: lng, rn Dng chung ca NL: ng vt, thc vt, VSV.

Xc nh vng cha enzim: ty th, t bo cht, mng t bo. Xc nh cc tnh cht cu trc v l ho ca enzim: trng lng PT, cu trc PT, hng s ng hc, tnh n nh PT, pH ti u, im ng in, cht hot ho, cht km hm Phi c phng php gii phng protein di dng ho tan m khng lm mt hot tnh ca n. X l phi thc hin T thp (+4OC); mi trng c cht m; tc nhn bo v (nu cn nh EDTA, 2-mecaptoetanol); thi gian x l ngn.

Cu trc sinh hc ca NL: t bo VSV, t bo thc vt.

3. CC PHNG PHP KT TA PHN ON ENZIM

1. Kt ta phn on bng mui trung tnh Nguyn tc:
Protein ho tan trong cc dung dch mui c pH xa vi im ng in. ho tan ca P ch tng cng vi lc ion MT trong mt khong nht nh ca [mui], Nng mui cao hn protein enzim c th kt ta. Mi enzim c mt khong [mui] m enzim kt ta hon ton, gi l nng mui tch. Da vo tnh tan khc nhau m phn on enzim.

Cch tin hnh

B sung vo dch cha enzim mt th tch mui bo ho hoc mt lng mui trung tnh dng rn t c phn trm bo ho no ca mui ny. Nu kt ta l nhng phn t khng phi enzim mong mun th tch hai pha lng rn v loi b kt ta thu hi dch trong. Nu enzim cn tinh ch kt ta th sau khi phn tch hai pha lng - rn ho tan kt ta trong mt lng nh dung dch m.

 Loi mui v cch tnh lng mui s dng

Mui sulfat amn v mui sulfat natri: gi r, kh nng kt ta cao, ho tan ln, protein t b bin tnh, thao tc n gin. Cc cng thc tnh lng mui cn thit a vo dung dch nh sau:

100.(S 2 S1 ) Y= 1 S2

A=

0,1.( S 2 S1 ).G Vg 1 S2 1000

Trong :G s gam (NH4)2SO4 trong 1000ml dung dch bo ho (G = 515g 0oC; 530,7g 15oC; 536g 20oC. Vg th tch ring biu kin ca dung dch (NH4)2SO4 bo ho (Vg/1000 = 0,271 0oC; Vg/1000 = 0,288 15oC; Vg/1000 = 0,29 20oC. Y: dung dch mui bo ho (ml) b sung vo 100ml dch enzim A: trng lng mui tinh th (g) b sung vo 100ml dch enzim S1: bo ho ban u ca dung dch S2: bo ho ca dung dch cui nhn c

 Kt tinh enzim
Kt ta enzim thu hi c phn tch nh dung dch mui sulfat amn c nng gim dn 0oC. Cc protein nhim tp ho tan nng mui cao hn enzim s c loi ra. V d: mt enzim kt ta trong khong bo ho 45 60% s c tch chit vi cc dung dch mui bo ho 65; 58; 52%. Nng nhit ca cc phn phn on nhn c ti nhit mi trng v protein enzim kt tinh. Qa trnh kt tinh c th thc hin ch trong mt vi pht hoc vi gi. Cc on protein enzim thu c sau phi c loi b mui bng phng php thm tch hoc phng php lc gel.

u nhc im ca phng php

K thut ny cho php nhn c mt phn phn on c lm giu enzim t th tch ln: loi b cc protein, enzim lin kt hy cc tp cht khc khng ho tan trc v sau phn phn on c cha enzim cn tch chit. Thao tc n gin, d p dng nn c dng ph bin, nhng khi lm vic vi th tch ln c nhiu kh khn do i hi thit b phi chu n mn ca mui cao (inox). Khong nng mui tch ca cc protein thng hay trng nhau nn khng bao gi c th thu c cc phn phn on enzim tinh khit hon ton. Thng s dng phng php ny vo giai on u ca qu trnh tinh ch enzim.

2. Kt ta ngin Nguyn tc:

Enzim c ho tan ti thiu xung quanh im ng in (pI), v phn t protein enzim c tng in tch bng khng. Khng c lc y tnh in nn cc phn t protein enzim s kt hp vi nhau to nn kt ta. Mi protein c mt pI khc nhau ph thuc thnh phn axit amin trong chui mch bn ion ho, do kt ta c enzim mong mun hoc cc protein tp khc.

Cch tin hnh

iu chnh pH ca dch hn hp cha enzim ti im ng in ca enzim cn phn tch hoc ca cc protein tp. Lc hay ly tm thu hoc loi b cc kt ta.

u nhc im ca phng php

K thut n gin, d p dng. Qu trnh iu chnh pH mi trng c th dn ti bin tnh protein enzim mt vi pH.

3. Tc dng ca nhit Nguyn tc:

Enzim c ho tan tng cng vi nhit trong gii hn di 40 50oC. Vt qua ngng gii hn ny protein enzim tr nn khng n nh v bin tnh. Da vo s khc nhau v n nh ca protein enzim nhit cao phn hu mt cch chn lc mt s enzim nhim tp.

u nhc im ca phng php

K thut i hi gi thnh cao. Cn phi kim sot cht ch trnh tn tht enzim mong mun.

4. Kt ta bng dung mi hu c Nguyn tc:

Dung mi hu c trung tnh ho tan trong nc s lm thay i hng s in mi ca dung dch, lm tng lc ht tnh in gia cc phn t protein. Khi b sung lng dung mi tng dn th phc hp gia cc phn t protein enzim tch in tri du ln dn cho ti khi kt ta. Tu tnh cht tng loi protein enzim, dung mi hu c v iu kin kt ta m mi enzim c kt ta c kt ta nng dung mi hu c khc nhau. Protease kt ta vi etanol 76 78% (v/v) -amylase kt ta vi etanol 70%; hoc izopropanol 55%; hoc axeton 60%. Glucoamylase kt ta vi etanol 45%.

u nhc im ca phng php

Mt s dung mi sau khi s dng c th chng ct v thu hi li c. X l phi c thc hin nhit thp (trn im lnh ng) v dung mi l cc cht d chy (-5oC). Phng php chr s dng i vi enzim t b bin tnh bi dung mi hu c. Dng vi qui m nh do chi ph cao.

5. Kt ta bng cc polyme c khi lng phn t ln

Cc polyme nh dextran, polyetylen glycol thng rt ho nc s lm mt lp v solvat ho ca cc phn t protein enzim, ko theo s thay i hng s in mi ca mi trng xung quanh, thay i cc tng tc khng gian ca cc nhm ho nc trong cc phn t protein enzim. Polyetylen glycol nng 5% (theo trng lng) trong nc c th lm kt ta phn ln cc protein nng 6 20%. Hiu sut kt ta ph thuc vo nhit , lc ion, pH, nng protein v khi lng phn t ca cc enzim. Lin kt ion kim loi protein lm gim ho tan ca protein. K thut ny khng p dng to kt ta enzim mong mun v phc to nn thng khng phi l thun nghch (dn sut sulhydryl) v bin tnh protein enzim. K thut ny c p dng tch axit nucleic ra khi hn hp sau khi ph v t bo VSV. Ion kim loi (Mn+2) to mui kt ta vi axit nucleic v c loi ra ngoi. Axit nucleic chim khong 7% E. coli, c nht cao nh hng ln ti qu trnh tinh ch enzim. Axit nucliec c nhm axit mnh lin kt vi nhm amin cua protein enzim trong mi trng axit lm kt ta chng. B sung protein kim tnh nh protamin s thay th c v tr ca protein trong phc hp v enzim s tn ti trong dung dch. Ly tm lai b phc ny. Cc cht phn ng thng s dng l mui mangan, sulfat streptomycin, sulfat protamin, bromcetytrimetylamn, polyetylenimin (polyme cationic). Kinh t nht l s dng ch phm enzim nuclease (ribo v desoxyribo nuclease).

6. Kt ta bng ion kim loi v cc tc nhn c hiu

3. CC PHNG PHP PHN TCH LNG - LNG V LNG - RN

1.Phn tch lng - lng
Phn tch trong dung dch c hai polyme khng trn ln nhau
Hai polyme t phn thnh hai pha phn bit nhau, mi pha ho tan c mt trong cc protein. S phn tch ph thuc vo khi lng phn t v nng ca cc polyme; khi lng phn t ca protein; nhit v lc ion. H dung mi s dng c th l polyetylen glycol/ dextran hay polyetylen glycol/ sulfat amn. Phng php ny t c s dng trong cng nghip do gi thnh cao.

Phn tch da vo s thay i tnh ho tan

Thay i pH: kt ta im ng in. Thay i lc ion: kt ta bng mui trung tnh. Lm gim hng s in mi: dung mi hu c ho tan trong nc.

Phn tch da vo in tch: in di, sc k trao i ion. Phn tch da vo khi lng phn t: ly tm, lc gel, thm tch, siu lc. Phn tch da vo v tr lin kt c hiu b mt: sc k i lc.

2.Phn tch lng - rn

Thu nhn enzim hoc loi b cc phn t rn (mnh t bo hay cc phn t khc), sau tin hnh cc x l khc. S dng cc phng php lc (lc p) hay ly tm: cn ch ti cc yu t nh hng ti enzim (s to bt, oxy ho enzim, nng lng v ma st sinh ra tc ln.

3.Phn tch s dng mng

Loi b cc phn t ho tan nh khng mong mun ra khi phn kt ta enzim thu nhn c (sulfat amn) bng cc phng php khc nhau: Phng php thm tch: Ti thm tch cha dch chit enzim. Ti c cu to bng mt mng bn thm (selophan). t ti trong th tch ln dung dch m khng cha cht ho tan cn loi b. Cht ho tan khuch tn ra khi mng theo chiu gradient nng cho n khi t trng thi cn bng. Thay dung dich m mi cho ti khi t tinh sch mong mun. Phng php trng in: cht ho tan dch chuyn do trng in. Phng php tc ng p sut ln dung dch pha trc mng lc: dung mi i qua (thm thu ngc); cc phn t nh i qua (siu lc). Phng php s dng vo cui QT tinh ch loi b cc tc nhn tch ra (sc k).

4. CC PHNG PHP sc k
1.Cc khi nim v sc k
Nguyn tc : S phn tch c c nh s chuyn dch khc nhau trong pha
ng ca cc cht ho tan ban u c gn trn pha tnh trng thi rn.

Pha tnh: kh nng gn cc cht ho tan phi ln. Tng tc gia CHT v pha
tnh l kt qu ca c ch hp ph, phn chia, tng tc ion, ry lc phn t, tng tc k nc hay tng tc sinh hc c hiu. SK hp ph: CHT gn vo pha tnh bng LK k nc; lc Van der Waals. SK trao i ion: CHT gn vo pha tnh bng LK ion gia cc in tch ca CHT v pha tnh. SK lc gel: CHT khuch tn vo trong pha tnh c dng ht xp tu theo kch thc cc phn t. SK k nc: Phn k nc trong CHT to LK vi phn k nc ca pha tnh. SK c hiu sinh hc: CHT v pha tnh c lin kt sinh hc c hiu enzim c cht; khng nguyn khng th.

Pha ng: cht lng c kh nng lm thay i dn tng tc gia CHT v pha
tnh, bng cch li cun CHT cng vi n.

2.Sc k lc gel
Nguyn tc : Khi dung dch hn hp protein chy qua ct tu theo khi lng,
kch thc v hnh dng ca CHT chng s xm nhp nhiu hay t vo trong pha tnh xp (l cc ht gel xp cha y trong ct). Cc phn t c ng knh ln hn l xp ca pha tnh, khng th kuch tn vo trong nn b loi tr, chng ra khi ct cng vi th tch ra bng th tch gia cc ht gel Vo. Cc phn t b nht s kuch tn ti a vo trong ht gel. Th tch dch ra ca chng s bng tng th tch pha lng ca ct (Vt). Cc phn t c kch thc trung gian s khuch tn hn ch vo trong ht.th b qua nu cc protein trong hn hp u l hnh cu.

Phn on hn hp protein theo khi lng, kch thc v hnh dng phn t Mc phn tch rng hp khc nhau tu theo mc to mng ca gel. Kch thc phn t cng nh, ng i cng ln, th tch dch ra cng ln. Cht ra ct sc k thu hi trong cc bnh cha c kt ni vi detecteur UV.

 Gel Sephadex
Gel Sephadex l mt loi bt kh, khng tan trong nc, nhng khi ngm trng ra v to thnh gel.

Gel Sephadex bn trong MT axit yu, kim yu. Lin kt glucozit trong gel s b thu phn trong MT axit / hoc kim mnh.
c tnh gel ca Sephadex nh sau:

gel dextran c lin kt ngang gia cc mch polysacarit vi nhau to thnh mng li ba chiu. Gel trung ho v in tch nn khng c tng tc anion v cation. Mc lin kt ngang quyt nh kch thc mt li. Nu t lin kt th mt li ln, ngm noc nhiu v ngc li. Tu thuc mc lin kt chia gel Sephadex lm cc loi Loi Gii hn phn t b Phm vi phn tch cc cht c Sephadex loi tr hon ton M t G-25 G-50 G-75 G-100 G-200 5.000 10.000 50.000 100.000 200.000 100 5.000 5.000 10.000 10.000 50.000 50.000 100.000 100.000 200.000

 Mt s gel khc
Gel Sepharose v Superose: dn sut ca agarose. Gel Sephacryl: dn sut ca dextran. Gel Gel Ultrogel: hn hp dn sut ca agarose v polyacrylamit c lin kt ngang.

Kch thc l ln tch enzim c khi lng PT cao trn 75.000 Da vi vn tc dng cao. Gel chu nn km nn khng s dng vi chiu cao ct ln.

3.

Sc k trao i ion Nguyn tc

S dng tnh cht lng tnh ca protein enzim i phn t vi nhiu nhm chc tch in v c tnh cht nh polyelectrode. Nu s dng vt liu mang in tch tri du vi in tch ca protein cn tch s gi li c protein cn tch. Ngc li s gi protein nhim tp. Cc protein ca hn hp c cc nhm bn ion ho khc nhau, do mt pH nht nh cc protein s c in tch khng ging nhau. Kh nng c gi trn nha khc nhau.
Nha trao i ion: l khung vt liu rn khng ho tan, trn c gn bng lin kt ng ho tr vi cc nhm ion ho c. Cc nhm ny li lin kt vi cc ion i v cc ion i li c th trao i thun nghch vi cc ion tri du. Khung mang nhm tch in dng v ion i in tch m c gop l nha trao i anion. Khung mang nhm tch in m v ion i tch in dng gi l nha trao i cation. Nha trao i ion c bn cht ho hc khc nhau nhm p ng mt s c tnh cn thit: xp ln, ho nc, chu lc cao, kh nng gi nc cao, phn tch cao, c tnh n nh ho hc v nhit, kt qu phn tch n nh, bn ct cao.

Cht mang

Nha trao i Bn cht ho hc khung nha

DEAE-Sephadex AmbelitE-120 Cc mch dextrin lin kt cho Polystyren lin kt cho bng divinylbeen Agarose lin kt cho bng 2,3dipromopropanol

Nhm ion ho

Kh nng trao i

Dietylamin etyl Trao i anion yu Sulfonat Trao i cation mnh

CM-Sepharose

Cacboxymetyl Trao i cation yu

Nha trao i ion t dn xut ca xenlulose: Kch thc l ln; kh nng gi thp hn. Cho php ra ct trong K mm du hn. Dng si hay vi ht. H s nn cao nn gy kh khn khi tin hnh. Nha trao i cation: cacboxylmetyl xenlulose; photpho xenlulose; sulfo xenlulose. Dng tinh sch enzim kim tnh. Ion trao i l H+ theo s : X-CH2-COOH X-CH2COO- + H+ X l gc xenlulose Nha trao i anion: dietylamino etyl xenlulose (DEAE-xenlulose); trietylamino etyl xenlulose. Dng tinh sch enzim axit tnh. Ion trao i l H+ theo s : X-C2H4N(C2H5)2 + H2O X-C2H4N+H(C2H5)2 + OHX l gc xenlulose

 Hi lu dung dch ra (dung dch sc k c cng pH v lc ion ca vo v ra ca ct) qua ct nha trc khi hot ng: lm trng n xenlulose, ui ht bt kh trong ct, loi cc phn t nh, cn bng ct gel c kh nng hp ph tt. Nha dn xut ca agarose lin kt cho (Sepharose CL-6B) v nha polyme tng hp (Trisacryl): cng sut cao; h s nn khng ng k; khng thay i th tch theo pH v cng ion; ti to li nha m khng cn tch khi ct. Qu trnh phn tch trn ct Giai on 1: hp ph thun nghch protein enzim cn tinh sch vo nha trao i i0n. Giai on 2: kh hp ph cc protein c hp ph bng mt trong cc cch: Thay i pH ca dch ra: thay i mc ion ho, do tahy i in tch tng ca protein. Tng lc ion v tng nng ion i cnh tranh. Cc phng thc ra Vi ct ngn CHT khng nm trn ct: ra bng dung dch m ban u. Vi ct di hn (loi b c tp cht; cc cht c tnh cht sc k gn ging enzim c hp ph: ra thc hin nh gradient pH v/ hay lc ion (b sung KCl, NaCl...) tng hay gim lin tc hoc gin on, tuyn tnh hay khng tuyn tnh. C th dng cc dung dch m photphat, axetat, borat, xitrat. Loi ra c bit: thc hin nh c cht, cht ging c cht hay coenzim c nng nht nh.

4.

Sc k tng tc k nc Nguyn tc
Cc protein c nhm k nc trn b mt, cht mang c cha nhm k nc v dung mi a nc to thnh h ba thnh phn v tng tc vi nhau. Cc phn t c phn tch tu theo mc tng tc ca chng vi cht mang. H thng ba thnh phn c th b ri lon khi thay i nhit , pH, lc ion ... Nng cao lc ion (dung dch NaCl 4M) s gi cc protein li. Tin hnh ra gii chn lc bng mt trong cc cch: Gim lc ion Gim phn cc ca dung mi ra (b sung etylen glycol; b sung cht ty ra hay tng pH dch ra.

Cht mang
Cht mang octylsepharose cc nhm octyl v phenyl c nh ln cc n v monosaccarit ca agarose bng lin kt este khng tch in v bn ho hc

Cht mang Ccphenylsepharose s dng cc nhm a bo khc nhau. cht mang khc:

5.

Sc k hp ph khng c hiu Nguyn tc

Cc protein c gi li

Cht hp ph khng cc Tng tc k nc Lc VanderWan

Cht hp ph c cc Tng tc ion Lin kt hydrogen

Phn tch da trn s khc nhau v i lc b mt ca cc phn t vi cht hp ph. Cc phn t c gi tt hn nu b mt v trong lng phn t ca chng ln hn. Cht hp ph c s dng rng ri nht l photphat canxi tinh th-hydroxy apatit Cht hp ph r nht l bn Kieselguhr hay Xelit (thnh phn gm CaCO3 v vi loi silicat.

6.

Sc k i lc

Nguyn tc
Enzim c kh nng lin kt c hiu v thun nghch vi mt phn t khc (phi t), c gn bng LK ng ho tr vo mt cht mang khng ho tan cha trong ct, cho php gi li mt cch c hiu enzim cn tch t hn hp protein, trong khi tt c cc protein khng tng tc vi phi t s tri ra khi ct. Cui cn enzim c ra gii ra bng cc phng php khc nhau.

Cht mang

Cc cht mang thng s dng Dn xut ca xenlulose Gel dextran Thu tinh

Cc tiu ch la chn cht mang Hon ton khng ho tan trong pha ng. Bn v tnh cht ho hc v sinh hc. Bn vng c hc, ho nc v thm thu.

Agarose hoc hn hp agarose v Khng c tng tc phi c hiu. polyacrylamit Cha nhiu nhm chc c kh nng bin i khi hot ho trong K m du.

Phi t

Nhng cht tng t c cht; cht km hm; cofact (t c hiu) ca enzim mun lm sch. Phi c hiu vi enzim v c i lc trung bnh vi enzim. Nng phi t thch hp.

Cnh tay n

Cu to t mt mch hydrocacbon nh chc c chiu di 6 8 cacbon. Cnh tay n ngn nht l axit aminocaproic v hexametylamin.

Hot ho cht mang

Phi t v cnh tay n phi c gn ln cht mang. Cht mang trc tin phi c hot ho. Chn phng php hot ho tu thuc vo phi t v bn cht ca nhm chc ho hc mun gn phi t.

Phng php loi ra

S dng mt cht cnh tranh ra gii enzim khi ct do phc hp gia enzim - phi t c bn cht phi ng ho tr v thun nghch. Dung dch ra gii phi c mt trong nhng tnh cht sau:

Cha phi t ( dng t do) c th cnh tranh vi phi t ng LK vi enzim. pH ca dung dch gy bin tnh thun nghch enzim, lm bin dng TTH enzim. Lm yu tng tc c hiu sinh hc bng cch thay i lc ion.

7.

Mt s sc k bin th ca sc k i lc
Sc k vi phi t l thuc nhum
Phi t l thuc nhum si tng hp c gn ng ho tr vo agarose c kh nng hp ph cc enzim c cofact nucleic. Cc thuc nhum thng s dng l cibacron xanh, procion , da cam A, xanh l cy A, xanh lc B. C ch cha r rng, xong c s km hm cnh tranh gia thuc nhum v cc enzim c cofact ny. K thut ny c phi t rt r tin v cht mang c th s dng nhiu ln.

Sc k i lc chelat kim loi

Phi t l cc ion kim loi nng tng tc vi cc gc cystein (nhm tiol) v histidin (nhm imidazol) ca protein. Phi t l cc ion Zn+2 v Cu+2 to phc cng cua vi axit iminoaxetic vn c kt gn vo cht mang agarose. Nh hp ph thc hin bng cch gim pH hoc tng lc ion ca dch m ra gii.