Kinetic analysis 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 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 spectrophotometric procedure, and both yielded similar values.
Serra
M A
MA
Departament de Bioquímica i Biologia Molecular, Facultat de Ciències, Universitat Autonòma de Barcelona, Spain.
Avilés
F X
FX
Giralt
E
E
Cuchillo
C M
CM
D000818:Animals; D002268:Carboxypeptidases / Q000378:metabolism*; D043422:Carboxypeptidases A; D002851:Chromatography, High Pressure Liquid; D006868:Hydrolysis; D007202:Indicators and Reagents; D007700:Kinetics; D009842:Oligopeptides / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D013552:Swine
D007202:Indicators and Reagents; D009842:Oligopeptides; D002268:Carboxypeptidases; D043422:Carboxypeptidases A