The synthesis, kinetic characterization and application of a novel biotinylated affinity label for cathepsin B.
In this study we report on the synthesis, kinetic characterization and application of a novel biotinylated and active-site-directed inactivator of cathepsin B. Thus the peptidyldiazomethane biotinyl-Phe-Ala-diazomethane has been synthesized by a combination of solid-phase and solution methodologies and has been shown to be a very efficient inactivator of bovine and human cathepsin B. The respective apparent second-order rate constants (k0bs./[I]) for the inactivation of the human and bovine enzymes by this reagent, namely approximately 5.4 x 10(4) M-1.min-1 and approximately 7.8 x 10(4) M-1.min-1, compare very favourably with those values determined for the urethane-protected analogue benzyloxycarbonyl-Phe-Ala-chloromethane first described by Green & Shaw [(1981) J. Biol. Chem. 256, 1923-1928], thus demonstrating that the presence of the biotin moiety at the P3 position is compatible with inhibitor effectiveness. The utilization of this reagent for the detection of cathepsin B in electrophoretic gels, using Western blotting and in combination with a streptavidin/alkaline phosphatase detection system, is also demonstrated. Given that the peptidyldiazomethanes exhibit a pronounced reactivity towards cysteine proteinases, we feel that the present label may well constitute the archetypal example of a wide range of reagents for the selective labelling of this class of proteinase, even in a complex biological milieu containing additional classes of proteinases.
Walker
B
B
Division of Biochemistry, School of Biology and Biochemistry, Queen's University of Belfast, Northern Ireland, U.K.
Cullen
B M
BM
Kay
G
G
Halliday
I M
IM
McGinty
A
A
Nelson
J
J
D000345:Affinity Labels / Q000138:chemical synthesis*; D000818:Animals; D001665:Binding Sites; D001710:Biotin / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000494:pharmacology; D015153:Blotting, Western; D002401:Cathepsin B / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D002417:Cattle; D003978:Diazomethane / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000494:pharmacology; D004151:Dipeptides / Q000138:chemical synthesis* / Q000494:pharmacology; D006801:Humans; D007202:Indicators and Reagents; D007700:Kinetics
D000345:Affinity Labels; D004151:Dipeptides; D007202:Indicators and Reagents; C064862:biotin-phenylalanyl-alanine diazomethyl ketone; D003978:Diazomethane; D001710:Biotin; D002401:Cathepsin B