Determination of cephalosporin-C amidohydrolase activity with fluorescamine.
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.
Reyes
F
F
Departmento de Microbiologia Aplicada, Centro de Investigaciones Biológicas (C.S.I.C.), Madrid, Spain.
Martinez
M J
MJ
Soliveri
J
J
D000581:Amidohydrolases / Q000032:analysis*; D002511:Cephalosporins / Q000378:metabolism*; D005450:Fluorescamine; D006863:Hydrogen-Ion Concentration; D013050:Spectrometry, Fluorescence
D002511:Cephalosporins; D005450:Fluorescamine; C025163:cephalosporin C; D000581:Amidohydrolases