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 (epsilon350 = 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 Km value of 0.11 mM and a kcat 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 pathlength), suitable for assay purposes. Catalysis may alternatively be monitored at wavelengths as long as 400 nm.