Cereal Chem 68:95-99   |  VIEW ARTICLE

Reversed-Phase High-Performance Liquid Chromatographic Separation of Wheat Proteinaceous Inhibitors of Insect and Mammalian Alpha-Amylases.

G.-H. Feng, M. Chen, K. J. Kramer, and G. R. Reeck. Copyright 1991 by the American Association of Cereal Chemists, Inc. 

A heat-treated 0.15M NaCl extract of wheat flour was separated into four proteinaceous fractions by ammonium sulfate precipitation. Each fraction had a distinct composition of polypeptides with apparent molecular mass of approximately 14 kDa and a distinct inhibitory spectrum of activity against human, porcine, and insect (confused flour beetle and rice weevil) alpha-amylases. More than 10 peaks in each fraction were resolved by reversed-phase high-performance liquid chromatograph (RP-HPLC) with mixtures of acetonitrile and H2O as the mobile phase. RP-HPLC separations revealed more heterogeneity in wheat alpha-amylase inhibitors than was apparent from either nondenaturing or denaturing sodium dodecyl sulfate gel electrophoresis. RP-HPLC fractions were characterized further by polyacrylamide gel electrophoresis and by assaying inhibitory activity against the four different alpha-amylases. At least five different HPLC fractions were found to be selective inhibitors of insect alpha-amylases, some of which belong to the 0.28 family of inhibitors. Combining certain RP-HPLC fractions resulted in dramatic increases in inhibitory activity. These results suggest that RP-HPLC resolves the oligomeric inhibitors into their monomeric forms and that both the activity and selectivity of the inhibitors can be influenced by recombination of individual polypeptides.