Purification and properties of an alpha-amylase protein-inhibitor from Arachis hypogaea seeds

Abstract

A protein showing highly specific inhibitory activity towards hog pancreatic and human salivary alpha-amylases (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1), but not towards plant and bacterial alpha-amylases, has been purified 197-fold from an aqueous extract of peanut cotyledons using heat treatment, (NH4)2SO4 precipitation and ion-exchange chromatography on DEAE- and CM-cellulose. The purified inhibitor was homogeneous by polyacrylamide gel electrophoresis. Its molecular weight, as determined by Sephadex G-100 gel-filtration, and its electrophoretic mobility at pH 8 relative to bromophenol blue, were 25 000 and 0.14, respectively. The inhibitory activity was relatively resistant to thermal treatment and markedly increased when the inhibitor was preincubated with the enzyme before the addition of starch. Further, the inhibition was found to be pH-dependent and non-competitive in nature.