Kinetic analyis of the carboxypeptidase a hydrolysis of oligopeptides by reversed-phase high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic (HPLC)-based method was developed to follow the time course of the hydrolytic action of pancreatic carboxypeptidase A on oligopeptide substrates of the general formula benzoyl-(glycyl)n-l-Phe, n being in the range 1-5. The proposed procedure is sensitive at the nanomolar level of substrate, but also allows the kinetics of substrate hydrolysis to be investigated over a wide range of concentrations. The prior quenching of the carboxypeptidase activity and the use of isocratic conditions for the rapid separation of the substrates and their products (in less than 8 min for the slowest one), allow the automation of the procedure, which could become an easy and versatile alternative to the commonly used spectrophotometric methods. A comparative evaluation of the use of a spectrophotometric method to measure carboxypeptidase A activity on the same substrates indicated that the latter is not valid for long oligopeptides (n {slanted equal to or greater-than} 2) at concentrations higher than 5 mM owing to an interference of a physico-chemical nature. For benzoylglycyl-l-Phe (n = 1), the apparent kinetic parameters were calculated by means of the HPLC method and by a well established spectrophotometric procedure, and both yielded similar values. © 1989.