A HPLC-UV method for the determination of angiotensin I-converting enzyme (ACE) inhibitory activity |
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Authors: | Véronique Lahogue Karine Réhel Laure Taupin Dominique Haras Patrick Allaume |
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Affiliation: | 1. Laboratoire de Biotechnologie et Chimie Marines, Université Européenne de Bretagne, Université de Bretagne Sud, BP 92116, 56321 Lorient, France;2. IDMER, 2 rue Batelière, 56100 Lorient, France |
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Abstract: | To determine the angiotensin-converting enzyme (ACE) inhibitory activity of a fish hydrolysate, different methods were tested. Finally, a sensitive, extraction-free HPLC method using N-(3-2-furylacryloyl)-Phe-Gly-Gly (FAPGG) as substrate was preferred. This method relies on the UV-titration of the peptide 2-furylacryloyl-l-Phe (FAP) resulting from the hydrolysis of the FAPGG after a chromatographic separation on a reverse phase column. The experimental conditions (enzyme/substrate ratio, incubation time, NaCl concentration) were optimised for linearity, sensitivity and precision. The assay was adequate for the study of ACE inhibition by Captopril, used as reference, and several peptides. Captopril and the fish hydrolysate had IC50 values, respectively of 0.19 ng and 43 μg with standard deviations of 0.09 ng and 5 μg. Afterwards, the determination of the Hill coefficient sustained the hypothesis that active peptides present in the fish hydrolysate were low-molecular weight molecules. This result was confirmed by the activity measurement of the fish hydrolysate fractions obtained by gel filtration. |
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Keywords: | ACE angiotensin-converting enzyme FAPGG N-(3-[2-furylacryloyl)-Phe-Gly-Gly FAP 2-furylacryloyl-l-Phe HHL hippuryl-l-histidyl-l-leucine HA hippuric acid TNBS 2 4 6-trinitrobenzene sulphonate HPLC high-performance liquid chromatography |
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