Assay of angiotensin I-converting enzyme-inhibiting activity based on the detection of 3-hydroxybutyrate with water-soluble tetrazolium salt.
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.
Lam
Le Hoang
le H
Department of Bioresources Science, Faculty of Agriculture, Kochi University, Monobe B-200, Nankoku 783-8502, Japan.
Shimamura
Tomoko
T
Manabe
Sachiyo
S
Ishiyama
Munetaka
M
Ukeda
Hiroyuki
H
D020155:3-Hydroxybutyric Acid / Q000032:analysis*; D000806:Angiotensin-Converting Enzyme Inhibitors / Q000032:analysis* / Q000737:chemistry; D001681:Biological Assay / Q000379:methods*; D004492:Edetic Acid / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D006884:Hydroxybutyrate Dehydrogenase / Q000737:chemistry; D006885:Hydroxybutyrates; D009243:NAD / Q000737:chemistry; D009245:NADH Dehydrogenase / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012995:Solubility; D012997:Solvents / Q000737:chemistry; D013778:Tetrazolium Salts / Q000737:chemistry*; D014867:Water / Q000737:chemistry*
D000806:Angiotensin-Converting Enzyme Inhibitors; D006885:Hydroxybutyrates; D012997:Solvents; D013778:Tetrazolium Salts; D014867:Water; D009243:NAD; D004492:Edetic Acid; D006884:Hydroxybutyrate Dehydrogenase; D009245:NADH Dehydrogenase; D020155:3-Hydroxybutyric Acid