Purification and partial characterization of wheat alcohol dehydrogenase

Abstract

Alcohol dehydrogenase (ADH) has been purified to homogeneity from embryo of tetraploid wheat (T. turgidum) and whole grain of diploid wheat (T. monococcum). The embryo ADH was purified 22 fold and the ADH from whole grain was purified 103 fold. Purification was achieved using streptomycin sulfate precipitation, gel filtration chromatography, DEAE-cellulose anion exchange chromatography, and preparative isoelectric focusing. This purification scheme effectively removes the wheat storage proteins, which were a particular problem, and produces a stable ADH. The development of a purification procedure for ADH from whole grain wheat allowed the diploid wheat, monococcum, which is of limited supply, to be effectively utilized to obtain a single isozyme ADH preparation. The three ADH isozymes of the tetraploid wheat T. turgidum were purified and then resolved by ion exchange chromatography. The isozymes were present in an approximate ratio of 1:2:1 which is the expected ratio for randomly dimerizing subunits. The molecular weight of the native enzyme was determined by gel filtration chromatography to be 116,000 and the subunit molecular weight was determined by SDS polyacrylamide gel electrophoresis to be 60,000. Wheat ADH was shown by atomic absorption analysis to contain zinc. ADH is stabilized by dithiotheritol and mercaptoethanol which suggests the presence of SH groups in the enzyme. ...