ARAZOFORMYL PEPTIDE SURROGATES AS SPECTROPHOTOMETRIC KINETIC ASSAY SUBSTRATES FOR CARBOXYPEPTIDASE A

N-(4-Methoxyphenylazoformyl)-L-phenylalanine is efficiently cleaved by the enzyme bovine carboxypeptidase A into fragments anisole, molecula r nitrogen, carbonate, and phenylalanine, in the course of which an in tense spectral absorption of the substrate (epsilon(350) = 19,000 M(-1 ) cm(-1)) disappears completely. This furnishes a sensitive spectropho tometric detection of the protease. Steady-state catalytic velocity de pends upon enzyme and substrate concentrations in the normal manner, a nd a Michaelis-Menten K-m value of 0.11 mM and a k(cat) value of 44 s( -1) were measured at pH 7.5 in saline solution. These parameters have a pH dependence typical for the enzyme. With saturating amounts of sub strate, a solution containing 10 nM enzyme produces a spectral absorpt ivity change at 350 nm of -0.03 a.u/min (1-mm path-length), suitable f or assay purposes. Catalysis may alternatively be monitored at wavelen gths as long as 400 nm. (C) 1996 Academic Press, Inc.