Simultaneous Separation of β-Lactamase Isoenzymes by High Performance Hydroxyapatite Chromatography with Selective Postcolumn Enzymatic Reaction

Abstract

A high performance liquid chromatography method for the simultaneous determination of various types of β-lactamase with detection specific to enzyme activity has been developed. Separation was achieved on a hydroxyapatite column utilizing a two-step gradient elution of potassium phosphate buffers, with no loss of enzyme activity. A postcolumn reaction based on an enzyme-substrate reaction with Nitrocefin, a well known chromogenic substrate for β-lactamases, was also optimized to give a highly selective determination using a photometric monitor at 495nm. Using the established method, several β-lactamases, either commercial or clinically isolated, were successfully separated and detected in a single run. The method was applicable for the measurement of various β-lactamases.