Determination of cephalosporin-C amidohydrolase activity with fluorescamine

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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 μg mL−1. The same method can be used as a fluorometric procedure 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.