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Photometric Method for Estimation of Amidase Activity of Proteases Using 4-Methylcoumaryl-7-Amide Substrates
V. F. Pozdnev, S. E. Rabinovich, T. S. Paskhina
Institute of Biological and Medical Chemistry, Academy of Medical Sciences of the USSR, Moscow
Abstract: It was shown that 7-amino-4-methylcoumarin (MC-amin), resulted from the enzymatic hydrolysis of 4-methylcoumaryl-7-amide (MC-amide) peptide substrates, may be estimated not only fluorometrically but also photometrically. A photometric method for estimating activity of tissue kallikrein (EC 3.4.21.35) and urokinase (EC 3.4.21.31) is suggested using Z-Phe-Arg-NHMC and Z-Gly-Gly-Arg-NHMC, respectively, as substrates. Kinetic parameters of the enzymatic hydrolysis, as obtained by photometric and fluorometric detection of the MC-amine formed, were in good agreement. The differential coefficient of molar extinction of the substrates and MC-amine at 360 nm was found to be 10 800 M-1 cm-1.
Russian Journal of Bioorganic Chemistry 1990, 16 (5):590-594