The synthesis, kinetic characterization and application of biotinylated aminoacylchloromethanes for the detection of chymotrypsin and trypsin-like serine proteinases.
The synthesis of two biotinylated affinity labels for chymotrypsin and trypsin-like serine proteinases is described, along with their kinetic characterization and application to the detection of these proteinases after PAGE and Western blotting. Thus the chloromethane analogues biotinylphenylalanylchloromethane (Bio-Phe-CH2Cl; reagent 1) and biotinylarginylchloromethane (Bio-Arg-CH2Cl, reagent 2), have been shown to be potent active-site-directed inactivators of chymotrypsin and trypsin respectively. The apparent overall second-order rate constants (kobs./[I]) for the inactivation of chymotrypsin and trypsin by reagent 1 (approximately 4.9 x 10(3) M-1.min-1) and reagent 2 (approximately 1.0 x 10(5) M-1.min-1) respectively are comparable with those obtained by other workers with simple urethane-protected analogues and demonstrates that the presence of the bulky biotinyl moiety is compatible with inhibitor effectiveness. Samples of chymotrypsin and trypsin that have been inactivated by reagents 1 and 2 respectively and which have been subjected to SDS/PAGE and Western blotting can be revealed with a streptavidin/alkaline phosphatase label. We can presently detect down to 20 ng of inactivated proteinase by using this system. The utility of the arginine derivative for the detection of the plasma trypsin-like proteinases plasmin and thrombin has also been demonstrated, thus holding out the possibility that this reagent may find general application as an active-site-directed label for this class of proteinase.
Kay
G
G
Division of Biochemistry, School of Biology and Biochemistry, Queen's University of Belfast, Northern Ireland, U.K.
Bailie
J R
JR
Halliday
I M
IM
Nelson
J
J
Walker
B
B
D000345:Affinity Labels / Q000138:chemical synthesis*; D000818:Animals; D001665:Binding Sites; D001710:Biotin / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000494:pharmacology; D002417:Cattle; D002918:Chymotrypsin / Q000378:metabolism*; D006801:Humans; D007202:Indicators and Reagents; D007700:Kinetics; D008962:Models, Theoretical; D008970:Molecular Weight; D011485:Protein Binding; D012697:Serine Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000378:metabolism; D014357:Trypsin / Q000302:isolation & purification / Q000378:metabolism*; D014361:Trypsin Inhibitors / Q000138:chemical synthesis
D000345:Affinity Labels; D007202:Indicators and Reagents; D014361:Trypsin Inhibitors; C074296:biotinylarginylchloromethane; C074297:biotinylphenylalanylchloromethane; D001710:Biotin; D012697:Serine Endopeptidases; D002918:Chymotrypsin; D014357:Trypsin; D013917:Thrombin