Determination of cephalosporin-C amidohydrolase activity with fluorescamine

Abstract

A spectrophotometric procedure for the assay of cephalosporin-C amidohydrolase activity, based on the determination of the 7-aminocephalosporanic acid (7-ACA) produced in the hydrolysis of cephalosporin-C by the enzyme, is described. This procedure can be used to detect 7-ACA over a range of 10 to 200 micrograms mL-1. The same method can be used as a fluorometric procedures with a 100-fold greater sensitivity. At pH 4.5 7-ACA produces a strong fluorophor with fluorescamine, detectable spectrophotometrically at 378 nm and fluorometrically at an excitation of 378 nm and emission of 495 nm. At this pH the fluorophors formed with cephalosporin-C, proteins and aminoadipic acid present minimal absorbance values. The conditions for maximal detection of 7-ACA in the presence of proteins, cephalosporin-C and aminoadipic acid have been determined.