High-performance liquid chromatography of α-amylases from germinating wheat and complexes with the α-amylase inhibitor from barley

Abstract

Wheat α-amylases were separated into about eight major components in less than 10 min by high-performance liquid chromatography (HPLC) on non-porous ion exchange columns. This allowed rapid quantitative estimation of α-amylase isoenzyme groups. The proportions of group 1 (low pI) and group 2 (high pI) isoenzymes changed very little from 2 days (74 % group 2) to 4 days (83 % group 2) of germination at 20 °C despite a five-fold increase in total α-amylase activity. Addition of the α-amylase inhibitor from barley resulted in an apparent increase in the molecular weight of wheat α-amylase on gel filtration. Wheat α-amylases eluted from the HPLC earlier in the presence of the barley inhibitor probably because of the formation of enzyme/inhibitor complexes with higher pI. This suggests that the barley α-amylase inhibitor may be useful in the utilization of wheat damaged by preharvest sprouting. This technique has potential for use in rapid quantitative analysis of genetic variations in α-amylases and α-amylase/inhibitor complexes.