A rapid, simple and sensitive fluorescence method for the assay of angiotensin-I converting enzyme

A fluorescent assay for angiotensin-I converting enzyme (ACE; EC 3.4.15.1) is described. It is based in the hydrolysis of the internally quenched fluorescent substrate o-aminobenzoylglycyl-p-nitrophenylalanylproline by the action of ACE. The fluorescence generated by the liberation of the product (the o-aminobenzoylglycine group) is read in a microtiter-plate multiscan fluorometer. The different conditions for the assay have been optimised for linearity, sensitivity and precision. Maximal enzyme activity was reached in the pH range 8.0–8.5 and 0.5–0.75 M NaCl concentration in the assay mixture. Kinetics of the enzyme reaction displayed a Km = 109 μM, obtaining an optimal substrate concentration of 0.3 mM in the assay mixture. The assay was adequate for the study of ACE inhibition by captopril and several peptides and it also showed a very good correlation with a well-established method [Biochemical Pharmacology, 20(7) (1971) 1637]. The method has important advantages, being the availability of reagents, its simplicity and the capacity to process a high number of samples in a short time, most outstanding.