Separation of alcohol-soluble maize proteins by reversed-phase high performance liquid chromatography

Abstract

Three alcohol-soluble protein fractions from maize endosperm [zein; water-soluble, alcohol-soluble reduced glutelin (wsASG); and water-insoluble, alcohol-soluble reduced glutelin (wiASG)] were extracted simultaneously with a mixture of ethanol and water (70:30 v/v) containing 5% (v/v) 2-mercaptoethanol and 0·5% (w/v) Na acetate (70% EtOH-0·5% Na acetate-5% ME) from near-isogenic sugary-1 (su1), floury-2 (fl2), opaque-2 (o2) and normal (+) maize. Subsequent fractionation by reversed-phase high performance liquid chromatography (RP-HPLC) on a C18 column using an aqueous acetonitrile (CH3CN)-trifluoroacetic acid (CF3COOH) solvent gradient gave good resolution, and accurate quantification was achieved by monitoring the absorbance of column eluates at 210 nm. The relative amounts of these three alcohol-soluble protein fractions varied significantly in both normal and mutant lines, and RP-HPLC patterns were specific for each inbred and genotype. A correlation coefficient of -0·90 was found between combined peak absorbance per gram protein and the lysine content of the protein. Two su1 maize inbreds, having more methionine than normal counterparts due to increased ASG, also had more 15,000 mol.wt high methionine ASG subunits as revealed by RP-HPLC, indicating that the amounts of high methionine (wi) and high histidine, high proline (ws) ASG vary regularly in mutant genotypes. This rapid and simple extraction method, in conjunction with RP-HPLC, may replace sequential extraction techniques. In conjunction with RP-HPLC, this procedure provides another new method for determining protein quality and genotype of maize.