Simplified Electrophoretic Assay for Human Salivary α-Amylase Inhibitor Detection in Cereal Seed Flours

α-Amylase inhibitors are antinutritional proteins largely found in cereal seeds. An in-gel assay was developed that allowed the rapid screening of these compounds in complex seed extracts. The assay was based on the electrophoretic separation of the extract proteins on starch-containing gels, followed by the detection of α-amylase-inhibiting proteins after incubation of the gel in an α-amylase solution; inhibitors were revealed by a staining method based on iodine binding to nondigested starch. The one-dimensional method can be useful to test inhibitory activity of purified proteins or to assay fractions recovered during a purification procedure. A two-dimensional (IEF × PAGE) non-denaturing system with second-dimension separation on starch-PAGE was also developed; the technique allowed the screening of complex protein mixtures for multiple inhibitory proteins. The newly developed assay method was used to test the presence of inhibitory activity in a crude extract from wheat flour, and it was validated by comparing in-gel and in-solution assays of commercially available α-amylase inhibitors.

Keywords: α-Amylase inhibitor; in-gel assay; starch-PAGE; non-denaturing 2-DE