Microplate Assay of α-Glucosidase and Its Inhibitors Based on the Direct Reduction of Molybdosilicate by Glucose

Abstract

A colorimetric method for monosaccharide determination (Anal. Sci., 2013, 29, 1021) was optimized for the high-throughput screening of α-glucosidase, which hydrolyzes an α-1,4-glycosidic bond of starch and related oligo- and polysaccharides, followed by the release of D-glucose from the non-reducing ends. In a microplate, 40 μL of a sample solution was mixed with 160 μL of a 50 mM Na₂SiO₃, 600 mM Na₂MoO₄, 1.5 M CH₃COOH, and 20% (v/v) dimethyl sulfoxide solution, which was yellowish due to the formation of a yellow molybdosilicate. The mixture was kept at 80°C for 60 min. In the mixture, glucose reduced the Mo(VI) species directly to form a blue heteropolymolybdate(V/VI). Thus, 0.1 mM level glucose can be determined by the color change from yellow to blue. Since maltose cannot render the mixture blue as strongly as glucose, the present method has been successfully applied to a microtiter plate assay of α-glucosidase with the disaccharide. Also, the method has been applied to an assay of α-glucosidase inhibitors, acarbose and quercetin.