A RAPID KINETIC ASSAY METHOD FOR ALPHA-CHYMOTRYPSIN AND ITS APPLICATION

A rapid kinetic assay method has been developed for α-chymotrypsin which in principle can be applied to other enzyme systems when the appropriate substrates are available. The method is capable of measuring irreversible or slowly reversible changes in enzyme conformation in terms of their effects on kinetic parameters. By combining a continuous optical assay for chymotrypsin with the use of the integrated Michaelis-Menten equation we have been able to study the effect of hydrogen peroxide oxidation of the enzyme on the kinetic parameters. Approximately a 15% decrease is observed in Vmax, while K0(app) changes by a factor of 3, suggesting that only the binding efficiency of the enzyme changes on oxidation. Since the observed change in K0(app) is linear with time it must depend only on the ratio of Eoxidized to Etotal, a ratio independent of enzyme concentration if the concentration of hydrogen peroxide is much greater than the concentration of enzyme. © 1969.