IV. KHAINIEM VE ENZIM vA AXIT NUCLEIC
c) Phuomg phdp thuy phan khong hodn toan bdng enzim aminopeptitdaza
Enzim aminopeptitdaza xiic tac cho phan ung thiiy phan tao ra amino axit "dau N" va phan tir peptit nho ban
H2 N -CH-CO-NH-CH-CO-NH-CH-CO-... + H2O Ri R2 R3 aminopeptitdaza + H3 N -CH-COO + H3 N - C H - C O - N H - C H - C O - Ri R2 R3
Xac dinh amino axit "duoi C"
Phuang phap nay dua tren su thuy phan peptit nho chat xuc tac la enzim cacboxipeptitdaza. Day la loai enzim xuc tac, phan cat chgn Igc lien ket amit ciia amino axit cuoi cimg c6 nhom COO" tu do.
Vi du: ... - N H - C H - C O - N H - C H - C O - N H - C H - C O O ~ cacboxipeptitdaza ^ Ri R2 R3 ^ ...-NH-CH-CO-NH-CH-COO" + H j N - C H - C O O " I I I Ri R2 R3 Amino axit thu dugc dem phan tich bing phuong phap sac ki. Aminoaxit nay chinh la amino axit "duoi C". Tuy nhien phuong phap nay con de lai ban che la enzim cacboxipeptitdaza khong tach dugc amino axit "duoi C"cua prolin ra khoi mach peptit
Thuy phan timg phan mach peptit
De tao thanh timg doan peptit ngin, nguoi ta thuong dimg cac enzim tripsin, chimotripsin va pepsin dl xiic tac cho su phan cit peptit a nhirng vi tri xac dinh. Chang ban, enzim tripsin phan cit lien kit khi nhom cacboxyl la ciia lysin hay argimin, con enzim chimotripsin xiic tac thiiy phan phenylalanin, tryptophan, tyrosin, leuxin, axit aspactic va axit glutamic, nghia la loai enzim pepsin phan cat co tinh chgn Igc kem hon. Dem phan tich cac doan peptit ngan bang phuong phap sac ki, roi sau do xac dinh trat tu sap xep cac goc amino axit trong moi doan peptit do theo cac phuong phap trinh bay a tren. Ngoai ra co the dimg bromxian (BrCN) de phan cat mach peptit 6 sau goc methionin: ... - H N - C H - C O - N H - C H - C O — N H - C H - C O - N H - C H - C O - . . . _ 5 l £ ^ R, CH2CH2SCH3 R2 R3 H 2 N - C H - C O - N H - C H — C O \ Q + H2N-CH-CO-NH-CH-CO-... + CH3SCN CH2 - C H { 1 Ri R2 R3 homoserinlacton
Nhu vay, neu dimg xiic tac khac nhau de phan cat mat se thu dugc nhirng doan peptit khac nhau.
Vidu:
Ala —X Gly — Tyr - - Trp — Ser — Lys
A
Aminopeptitdaza
Gly — Leu — Met
Tripsin
Glu - ^ G l u
BrCN
Chimotripsin Cacboxipeptitdaza Khi biet dugc trat tu sap xep ciia cac goc amino axit trong tung doan peptit