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, molecular nitrogen, carbonate, and phenylalanine, in the course of which an intense spectral absorption of the substrate (epsilon(350) = 19,000 M(-1) cm(-1)) disappears completely. This furnishes a sensitive spectrophotometric detection of the protease. Steady-state catalytic velocity depends upon enzyme and substrate concentrations in the normal manner, and 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 substrate, a solution containing 10 nM enzyme produces a spectral absorptivity change at 350 nm of -0.03 a.u/min (1-mm path-length), suitable for assay purposes. Catalysis may alternatively be monitored at wavelengths as long as 400 nm. (C) 1996 Academic Press, Inc.