Định nghĩa đơn vị hoạt tính Anson

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

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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