Spectral titration of active site of carboxypeptidase A.

In the carboxypeptidase A-catalyzed hydrolysis of O-[trans-alpha-(benzoylamino)-cinnamoyl]-L-beta-phenyllactate (BACPL) or O-[trans-alpha-(benzoylamino)-p-(phenylazo)cinnamoyl]-L-beta-phenyllacta te (BAPACPL), biphasic kinetic behavior was observed due to the accumulation of an intermediate. At -12 degrees C, conversion of the intermediate into the product was much slower than the formation of the intermediate, which accumulated in quantitative amounts. From the absorbance changes observed during the formation process of the intermediate, the concentration of active site of the enzyme was determined.