Phng php Anson xc nh hot tnh enzyme protease
Phng php Anson xc nh hot tnh enzyme protease1. Nguyn l ca phng phpCasein b phn gii trong mi trng kim di tc dng ca protease to sn phm l cc on peptide ngn ha tan trong tricloroacetic acid [TCA], xc nh lng tyrosine v tryptophan ha tan bi thuc th Folin.2. i tng p dng ca phng phpPhng php ny p dng xc nh hot enzyme trong thc phm, thc n chn nui, ch phm sinh hc.3. Dng c v ho cht3.1 Dng c, thit b - My lc - My o mt quang - Bp n nhit - Bnh tam gic 250 ml. - Bnh nh mc 10 ml, 100 ml, 500ml - ng nghim - Pipet 1 ml, 5 ml, 10 ml 3.2 Ho cht- Dung dch Na2HPO4 1/15M: ha tan 5,9690gNa2HPO4.12H2O trong nc va 250 ml.- Dung dch KH2PO4 1/15M : ha tan 0,9072 g KH2PO4 trong nc va 100 ml- Dung dch m Sorensen pH 7,6: trn chung 88,5 ml dung dch Na2HPO4 1/15M vi 11,5 ml dung dch KH2PO4 1/15M n khi pH t 7,6.- Dung dch casein 1%:pha 1g Casein trong 100 ml dung dch m Sorensen - Dung dch TCA 5%: ha tan 5 g TCA trong nc cho 100 ml- Dung dich NaOH 0,5N: ha tan 10 g NaOH trong nc cho 500 ml.- Thuc th Folin[ pha long vi nc ct theo t l 1:4].- Dung dich HCl 0,2N: trn 4,25 ml HCl m c vi nc cho 250 ml - Dung dch Tyrosine 20 M/l : khuy nghin 1,8119 g Tyrosin trong dung dch HCl 0,2N va 500 ml- Dung dch Tyrosin chun 1 M/ltrong dung dch HCl 0,2N: pha long 5 ml dung dch Tyrosine 20 M/l trong dung dch HCl 0,2N thnh 100 ml 3.3 Chun b ng chun 3.3.1 Chun b dung dch tyrosinchun- T dung dch tyrosin chun 1 M/l, xy dng ng chun vi dung dch tyrosin c nng t 0,2 - 1,0 M/l . Dung dch ha chtng nghim
123456
Dung dch tyrosine chun [ml]0,20,40,60,81,00
Lng tyrosine tng ng [M]0,20,40,60,81,05,0
Dung dch HCl 0,2N [ml]4,84,64,44,24,05,0
Dung dch NaOH 0,5N [ml]101010101010
Thuc th Folin [ml]333333
Lc mnh, sau 10 pht o OD bc sng 660nm
ng s 6 l ng kim chng [KC], cc ng cn li l ng th nghim [TN]. V ng chun tyrosine tng quan tng quan gia lng tyrosine [M] v OD [OD = ODTN - ODKC ].4. Phng php tin hnh4.1. Chit muMu cn chnh xc 10g [ i vi mu rn] hoc 10 ml [i vi mu lng] cho vo becher, nh mc va 100ml bng nc ct em lc trn my lc trong vng 15 pht. em ly tm lnh vi 4000 vng/ pht trong 10 pht, thu dch, b b [i vi mu lng khng cn ly tm, ch lc ng nht mu]. 4.2 Tin hnh Ly 6 ng nghim sch, kh, tin hnh lm 3 ng th tht, 3 ng th khngDung dch ha chtng nghim
Th thtTh khng
Dung dch casein 1% [ml]55
Dung dch TCA 5% [ml]010
Dung dch enzyme mu [ml]11
Lc u v gi 35,5oC trong 20 pht
Dung dch TCA 5% [ml]100
yn 30 pht, lc ly dch bn di
Ly 2 ng nghim mi khc,cho vo ng th nht 5ml dch lc ca ng th tht v cho vo ng th hai 5ml dch lc ca ng th khng.Tip tc thm vo mi ng 10ml NaOH 0,5N v 3ml thuc th Folin, lc mnh, sau 10 pht o OD bc sng 660nm. OD = ODTT ODTK, sau da vo th chun suy ra c M tyrosine.4.3 Tnh kt qunh ngha n v Anson: mt n v Anson l lng enzyme ti thiu trong iu kin th nghim [ 35,5oC: pH 7,6 ] thy phn casein trong 1 pht to thnh sn phm ha tan trong TCA, phn ng vi thuc th Folin cho ta hp thu OD bc sng 660nm tng ng vi 1 M tyrosine trong ng chun. H Protease [UI/g]= H Protease [UI/g]/ t * m * v
Vi:H Protease : hot enzyme protease [ UI/g]V : tng th tch hn hp trong ng th tht hoc ng th khng[ml]v : th tch dch lc em phn tch [ml]t: thi gian thy phn [pht]m : khi lng mu enzyme em i xc nh hot tnh [g]L: pha long enzymeM tyrosine: lng M tyrosine trong v [ml] suy ra t ng chun
Minh Nam