Sep 22, 1989

Kinetic analysis of the carboxypeptidase A hydrolysis of oligopeptides by reversed-phase high-performance liquid chromatography

Journal of Chromatography
M A SerraC M Cuchillo


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 greater than or equal to 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 spectroph...Continue Reading

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Mentioned in this Paper

Carboxypeptidase A
Carboxypeptidase A5
Pancreatic Carboxypeptidase A Activity
Carboxypeptidase A Activity
Carboxypeptidase Activity
Spectrophotometry, Ultraviolet
High Pressure Liquid Chromatography Procedure
CPA1 gene

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