Flow injection analysis of angiotensin I-converting enzyme inhibitory activity with enzymatic reactors.
Assay of angiotensin I-converting enzyme (ACE) inhibitory activity always draws much attention because of diverse applications in the field of antihypertension and related pathogenesis. Recently, the use of a new synthetic substrate, 3-hydroxybutyrylglycyl-glycyl-glycine (3HB-GGG), for the assay of ACE inhibitory activity has been confirmed. To construct a rapid, economical, and automatic determination system of ACE inhibitory activity using 3HB-GGG, a flow injection analysis (FIA) system with enzymatic reactors was developed in this study. Enzyme reactors were composed of aminoacylase and 3-hydroxybutyrate dehydrogenase immobilized separately on CNBr-activated Sepharose 4B. The assay condition was optimized in terms of the conversion of 3HB-G into NADH by the enzymatic reactors when the reaction solution containing 3HB-G generated from 3HB-GGG (after the incubation with ACE) was repetitively injected into the FIA system. Under the optimized conditions, 3HB-G was converted to 3HB, and then 3HB was oxidized by NAD(+) to form NADH. The developed system successfully detected practical ACE inhibitors with a great sensitivity, high sampling frequency (10 samples h(-1)) and a durable stability of the enzymatic reactors.
Lam
Le Hoang
le H
Faculty of Agriculture, Kochi University, Monobe B-200, Nankoku 783-8502, Japan.
Shimamura
Tomoko
T
Ishiyama
Munetaka
M
Ukeda
Hiroyuki
H
D020155:3-Hydroxybutyric Acid / Q000378:metabolism; D000090:Acetoacetates / Q000378:metabolism; D000581:Amidohydrolases / Q000737:chemistry / Q000378:metabolism; D000806:Angiotensin-Converting Enzyme Inhibitors / Q000032:analysis* / Q000494:pharmacology*; D055162:Biocatalysis; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism; D017022:Flow Injection Analysis / Q000191:economics / Q000379:methods*; D006868:Hydrolysis; D006884:Hydroxybutyrate Dehydrogenase / Q000737:chemistry / Q000378:metabolism; D006885:Hydroxybutyrates; D020128:Inhibitory Concentration 50; D009243:NAD / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism; D010084:Oxidation-Reduction; D007703:Peptidyl-Dipeptidase A / Q000378:metabolism*; D011549:Pseudomonas / Q000201:enzymology; D013053:Spectrophotometry; D013997:Time Factors
C522037:3-hydroxybutyrylglycyl-glycyl-glycine; D000090:Acetoacetates; D000806:Angiotensin-Converting Enzyme Inhibitors; D004800:Enzymes, Immobilized; D006885:Hydroxybutyrates; D009842:Oligopeptides; D009243:NAD; C016635:acetoacetic acid; D006884:Hydroxybutyrate Dehydrogenase; D007703:Peptidyl-Dipeptidase A; D000581:Amidohydrolases; C019672:aminoacylase I; D020155:3-Hydroxybutyric Acid