Purification and characteristics of an endogenous alpha-amylase inhibitor from barley kernels.

An inhibitor of malted barley (Hordeum vulgare cv Conquest) alpha-amylase II was purified 125-fold from a crude extract of barley kernels by (NH(4))(2)SO(4) fractionation, ion exchange chromatography on DEAE-Sephacel, and gel filtration on Bio-Gel P 60. The inhibitor was a protein with an approximate molecular weight of 20,000 daltons and an isoelectric point of 7.3. The protein was homogeneous, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino acid analysis indicated the presence of about 9 half-cystine residues per mole. The neutral isoelectric point of the inhibitor suggested that some of the apparently acidic residues (glutamic and aspartic) existed in the amide form. The first twenty N-terminal amino acids were sequenced. Some homology appeared to exist between the alpha-amylase II inhibitor and trypsin inhibitor from barley. Complex formation between alpha-amylase II and the inhibitor was detected by the appearance of a new molecular weight species after gel filtration on Bio-Gel P 100. Enzyme and inhibitor had to be preincubated for 5 min, prior to assaying for enzyme activity before maximum inhibition was attained. Inhibition increased at higher pH values. At pH 5.5, an approximately 1100 molar excess of inhibitor over alpha-amylase II produced 40% inhibition, whereas, at pH 8.0, a 1:1 molar ratio of inhibitor to enzyme produced the same degree of inhibition.