Analytical Sciences

Assay of Angiotensin I-converting Enzyme-inhibiting Activity Based on the Detection of 3-Hydroxybutyrate with Water-soluble Tetrazolium Salt

Le Hoang LAM,* Tomoko SHIMAMURA,* Sachiyo MANABE,** Munetaka ISHIYAMA,** and Hiroyuki UKEDA*

*Department of Bioresources Science, Faculty of Agriculture, Kochi University, Monobe B-200, Nankoku 783-8502, Japan
**Dojindo Laboratories, 2025-5 Tabaru, Mashiki-machi, Kumamoto 861-2202, Japan

A newly synthesized substrate, 3-hydroxybutyrylglycyl-glycyl-glycine (3HB-GGG), was applied to the assay of ACE-inhibiting activity to overcome the smaller selectivity and sensitivity of the conventional method. In this study, an ACE-inhibiting assay was improved by the use of a water-soluble tetrazolium salt, 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate sodium salt (WST-1), for the detection of 3-hydroxybutyrate, derived from 3HB-GGG. The optimized conditions were as follows: 0.333 mM NAD⁺, 0.333 mM WST-1, 0.1 mM EDTA, 0.633 U ml^-1 diaphorase, and 0.700 U ml^-1 3-hydroxybutyrate dehydrogenase. The developed assay was efficiently applicable to evaluate the ACE-inhibiting activity of practical ACE inhibitors.

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