Simplified electrophoretic assay for human salivary alpha-amylase inhibitor detection in cereal seed flours.
Alpha-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 alpha-amylase-inhibiting proteins after incubation of the gel in an alpha-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 x 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 alpha-amylase inhibitors.
Fontanini
Debora
D
Department of Biology, University of Pisa, Via L. Ghini 5, 56126 Pisa, Italy. dfontanini@biologia.unipi.it
Capocchi
Antonella
A
Saviozzi
Franco
F
Galleschi
Luciano
L
D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004791:Enzyme Inhibitors / Q000032:analysis*; D005433:Flour; D006801:Humans; D012463:Saliva / Q000201:enzymology; D012639:Seeds / Q000737:chemistry; D014908:Triticum / Q000737:chemistry*; D000516:alpha-Amylases / Q000037:antagonists & inhibitors*
D004791:Enzyme Inhibitors; D000516:alpha-Amylases