1.

Phng php xc nh hm lng protein theo Bradford Trong cc phng php xc nh hm lng protein th y l phng php c nhy cao, c th xc nh ti 1g, ho cht n gin t tn thi gian. Mt u im ln ca phng php ny l t b cn tr bi cc ho cht s dng trong nghin cu protein, nht l amoniumsufate. Nguyn tc: Phng php ny da trn s thay i bc sng hp thu cc i ca thuc nhum Coomassie Brillant Blue khi to phc hp vi protein. Trong dung dch mang tnh acid khi cha kt ni vi protein th thuc nhum dng mu c bc sng thp thu cc i l 465 nm v khi kt hp vi protein th thuc nhum chuyn sang dng mu xanh dng v hp thu cc i mc cc i bc sng 595 nm. hp thu bc sng 596 nm c lin h mt cch trc tip vi nng protein. xc nh protein trong mu, u tin ta xy dng mt ng chun vi dung dch protein chun bit c nng . Sau khi cho dung dch protein vo thuc nhum mu, mu s xut hin sau 2 pht v bn ti 1gi. Tin hnh o dung dch bng my quang ph k (Spectrophotometer) ta c ODx, hp th s t l vi lng protein trong mu. Thc hin mt i chng vi HCl (ODo). Ly gi tr OD = ODX ODO. Lng protein trong mu dung dch o c xc nh bng cch da vo ng chun t gi tr OD trc tung, t suy ra gi tr nng protein tng ng trn trc honh. Ha cht Dung dch albumine 0.1%. Nc ct Dung dch thuc th Bradford: thnh phn thuc th trong 100 ml nh sau o o o Coomassie Brilliant Blue: 0.001g. Ethanol tuyt i: Phosphoric acid: 4.7 g. 85%.

Phm mu Coomassie Brilliant Blue c ho tan trong ethnol trong mt chai ng mu ti c np, b sung phosphoric acid v chnh ti 100 ml bng nc ct. Lc u v gi lnh di 4oC. Dng ng chun albumine Pha long dung dch albumine thnh cc nng khc nhau: 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 g/ml. Bng 3.1. Dng ng chun albumine
ng nghim Nng (g/ml) Dung dch albumin chun (l) Nc ct (l) Thuc th Bradford (ml) 2 2 2 2 2 2 2 2 2 2 0 10 20 30 40 50 60 70 80 90 i chng 0 1 10 2 20 3 30 4 40 5 50 6 60 7 70 8 80 9 90

1000

990

980

970

960

950

940

930

920

910

ng s 0 tng ng vi ng i chng cha nc ct. Tt c cc ng sau khi pha ng nng , khong 20 pht. Tin hnh o mt quang ca dung dch bc sng 595 nm. Dng ng chun. nh lng protein trong mu th nghim Ly 1ml mu th nghim cho vo ng nghim, thm vo 2 ml thuc th Bradford. em o mt quang bc sng 595 nm. T suy ra hm lng protein c trong mu th nghim. Mu c pha long sao cho mt quang o c trong khong ng chun. Kt qu tnh ton: T phng trnh ng chun v mt quang ca mu kho st ta suy ra hm lng protein ca mu l X (g/ml) c trong m (g) nguyn liu.

Vy lng protein c trong 1 gam nguyn liu l: Hm lng protein (mg/g) = X 1000. m

Hnh 3.2. ng chun albumine 2. Xc nh hot tnh enzyme protease bng phng php Amano Nguyn tc: Dng protein casein lm c cht xc tc nh hot tnh phn gii protein ca enzyme protease trn c s nh lng sn phm to thnh trong phn ng bng phn ng mu vi thuc th Folin. Da vo th chun tnh lng Tyrosin tng ng vi lng sn phm thu phn di tc dng ca enzyme. Ho cht: HCL 0.1 M Ma2CO3 0.4 M Dung dch Trichloracetic 0.4 M Tyrosin tinh khit

NaOH 0.1 M Thuc th Folin H3PO4 1/30 M m phosphate (pH 7) 0.1 M

Dng c v thit b ng nghim, pipette, bnh nh mc, giy lc. B n nhit My o quang ph UV Vis My o pH

Xy dng ng chun tyrosine Bng 3.2. ng chun tyrosine

ng s Nng (g/ml) Dung dch tyrosine chun (l) Dung dch HCl (l) Na2CO3 (ml) Thuc th Folin (ml) i chng 0 0 1000 5 1 1 10 10 990 5 1 2 20 20 980 5 1 3 30 30 970 5 1 4 40 40 960 5 1 5 50 50 950 5 1 6 60 60 940 5 1 7 70 70 930 5 1 8 80 80 920 5 1 9 90 90 910 5 1

Pha dung dch tyrosine cc nng khc nhau : 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 gtyrosine / mlHCl nh bng 3.3.2. Thm 5 ml dung dch Na2CO3 0,4M vo dung dch tyrosine cc nng khc nhau trn v thm 1 ml thuc th Folin vo dung dch hn hp. Sau khi trn u, n nh dung dch 37 0,50C trong 20 pht. o hp th ca dung dch ny bc sng 660 nm. Ghi nhn kt qu As10, As20, As30, As40, As50, As60, As70, As80, As90. As100 i vi ng i chng dng 1 ml acid HCl 0,1M thay cho tyrosine. o hp thu ca dung dch ny bc sng 660 nm v ghi nhn kt qu ny l Aso.

Tr s mt quang ca cc ng t 1 n 9 sau khi tr i tr s ca ng i chng s c gi tr OD1 n OD9. V th da vo bin thin ca OD theo nng protein. th ny gi l ng chun tyrosine. Xc nh hot tnh enzyme protease Hot tnh enzyme c kho st nhit 370C v pH = 7. Cho 1 ml dung dch c cht casein 1% vo ng nghim, 37 0,50C trong 10 15 pht. Sau thi gian , cho 1 ml dch chit enzyme th vo v lc u. em hn hp ny 37 0,50C trong mt gi. Cho vo 2 ml dung dch TCA 0,4M ngng phn ng enzyme. n nh dung dch trong 25 pht, sau lc dung dch ny qua giy lc loi ta. Cho 5 ml dung dch Na2CO3 vo 1ml dch lc. Thm 1 ml thuc th Folin. Trn u, yn 37 0,50C trong 20 pht. Khi dung dch xut hin mu xanh, em o hp thu bc sng 660 nm (ghi nhn kt qu ny l Am). Mu i chng : ly 1 ml nc ct thay cho 1 ml dung dch enzyme v tin hnh cc bc tng t nh mu th nghim vi cng iu kin. Ghi nhn kt qu hp th l A0. Hm lng tyrosine c gii phng do protease thy phn c tnh bng cch ly Am A0 ri ly kt qu so vi ng chun tyrosine xc nh hot tnhprotease theo tnh ton sau : Mt n v hot tnh (VHT) enzyme protease c xc nh l lng enzyme to ra lng amino acid tng ng vi 100 g tyrosine trong 1 ml dch lc di iu kin th nghim. K hiu l: U Hot tnh enzyme protease (VHT/ml) = (Am A0).F.1/100.n Hot tnh enzyme protease (VHT/g) = (Am A0).F.1/100.n.V m

F = [10/ As10 + 20/ As20 + 30/ As30 + 40/ As40 + 50/ As50 + 60/ As60 + 70/ As70 + 80/ As80 + 90/ As90+100/As100]/10 Am : hp thu ca mu A0 : hp thu ca ng i chng F : H s tng quan gia hm lng tyrosine v hp thu bc sng 660 nm trn ng chun. n : H s pha long ca enzyme 1/100 : H s chuyn i V : Th tch ca dung dch enzyme m : khi lng ch phm enzyme th. Tnh ton hot tnh ring (HTR) ca enzyme protease: t kt qu hm lng protein (mg/ml) v hot tnh enzyme protease (U/ml) tnh hot tnh ring ca enzyme. HTR (U/mg) = S n v hot tnh mg protein enzyme

ng chun tyrosine