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Performance of two commonly used angiotensin-converting enzyme inhibition assays using FA-PGG and HHL as substrates

Shalaby, S.M., Zakora, M., Otte, J.
Journal of dairy research 2006 v.73 no.2 pp. 178-186
food analysis, dairy products, fermented foods, functional foods, food quality, assays, reversed-phase high performance liquid chromatography, peptides, angiotensins, enzyme activity, reaction inhibition, hydrolysis, whey protein, antihypertensive agents, enzyme substrates, synthetic peptides, hippuric acid, rapid methods, accuracy, reliability, sampling, peptidyl-dipeptidase A
In development of fermented dairy products and protein hydrolysates with high inhibitory activity towards angiotensin-converting enzyme (ACE), it is crucial to have a reliable assay for measuring the ACE activity. In the present study, the performance of two commonly used assays based on synthetic N-derivates of tripeptides as substrates were tested with respect to reliability in determination of ACE activity per se and to the inhibitory effect of a tryptic whey protein digest and captopril. In one test, the ACE activity was calculated from the amount of hippuric acid liberated from hippuryl-His-Leu (HHL) during 30 min of incubation with ACE, as quantified after HPLC separation of reaction products. In the other assay, the ACE activity was measured directly as the rate of decrease in the absorbance at 340 nm during the first 30 min of ACE catalysed hydrolysis of furanacroloyl-Phe-Glu-Glu (FA-PGG). Both assays, in the absence of inhibitor, showed a good performance with relative standard deviation between replicate samples around 7%. In the presence of inhibitor solutions, relative standard deviations for both assays varied between 1 and 18% for the variously diluted inhibitors. Both assays gave values for the concentration of inhibitor needed to inhibit ACE by 50% similar to those previously reported for whey protein digests and captopril. Different results from the two assays, however, emphasize the importance of controlling the actual ACE-activity for comparison between assays. The limitations of each assay are discussed. Considering the fewer steps in the assay using FA-PGG as substrate, and thus less time and chemicals consumed per sample, and the simpler equipment needed, this assay is recommended for the screening of clear peptide samples.