Syntheses of argininal semicarbazone containing peptides and their applications in the affinity chromatography of serine proteinases.
Eight argininal semicarbazone containing peptides prepared by liquid phase synthesis were all found to be reversible inhibitors of model serine proteinases including trypsin and plasma kallikrein (PK). Among the peptides tested, those having a Lys residue at position P2 displayed the maximum binding potency towards PK. One of the peptides, Leu-enkephalin-argininal semicarbazone, a comparatively weak inhibitor, was chosen in order to develop an affinity-based purification protocol for PK. The affinity column was prepared by covalent attachment of the NH2-terminal moiety of the peptidyl semicarbazone to a solid-phase matrix bearing a spacer group. For efficient binding of PK, it was found necessary to optimize parameters like the concentration of inhibitor linked to the solid matrix, the ionic strength of the buffer used, the temperature and the pH. The majority of the bound enzyme could be recovered following elution with guanidine hydrochloride or benzamidine hydrochloride in a high salt buffer at pH 6.0. The usefulness of the affinity procedure towards the purification of other serine proteinases is also discussed.
Basak
A
A
J.A. de Sève Laboratory of Molecular, Clinical Research Institute of Montreal, Quèbec, Canada.
Gong
Y T
YT
Cromlish
J A
JA
Paquin
J A
JA
Jean
F
F
Seidah
N G
NG
Lazure
C
C
Chrétien
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives*; D002846:Chromatography, Affinity; D007610:Kallikreins / Q000037:antagonists & inhibitors / Q000302:isolation & purification; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis* / Q000494:pharmacology; D051381:Rats; D012697:Serine Endopeptidases / Q000302:isolation & purification*; D015842:Serine Proteinase Inhibitors / Q000138:chemical synthesis*; D014357:Trypsin / Q000302:isolation & purification; D014361:Trypsin Inhibitors / Q000138:chemical synthesis
D010455:Peptides; D015842:Serine Proteinase Inhibitors; D014361:Trypsin Inhibitors; D001120:Arginine; D007610:Kallikreins; D012697:Serine Endopeptidases; D014357:Trypsin