Extractability and chromatographic separation of rice endosperm proteins

Abstract

The molecular weight (MW) distribution of proteins extracted with different solvents from defatted rice endosperm was examined by size exclusion-high performance liquid chromatography (SE-HPLC) with 2.0% sodium dodecyl sulfate (SDS) (w/v) as mobile phase. The resulting protein peaks were further characterized by SDS-PAGE. Under the experimental conditions, 2.0% SDS extracted 64% of the proteins. Adding 6.0 M urea resulted in a 15% increase in extractability (up to 79%). With using 20–100 mM NaOH, 70–81% of the proteins were extractable. Maximum extractability was reached with 2.0% SDS, 6.0 M urea and 0.5–1.5% dithiothreitol (DTT). Apparent MW profiles of rice endosperm proteins allowed classification into six fractions of decreasing apparent MW. Fraction VI contained the low MW albumin, globulin, and prolamin protein material. Fractions IV and V originated from α and β glutelin subunits, respectively. The polypeptides of fraction III consisted of an α and a β subunit linked by an intermolecular disulfide bond. The polypeptides of fractions I and II were dimers, trimers or more highly polymerized forms of the (α–β) glutelin subunit dimer in fraction III. While the work confirmed that rice glutelin is composed of polymers of α and β subunits, remarkably, higher MW glutelin aggregates (fractions I–III) only partly dissociated on reduction. Low MW protein material (fraction VI) was entrapped in the aggregated protein network and was released on reduction. The rapid and reproducible SE-HPLC method developed for rice protein separation allows a more quantitative approach than SDS-PAGE.