Characterization of the polymeric proteins of sorghum

Abstract

The role of sorghum protein cross-linking into high M[subscript w] polymeric groups in grain hardness was investigated using a number of protein analytical techniques to study the protein composition (reduced and non-reduced) of isolated vitreous and floury endosperm. The relative molecular weight distributions of polymeric proteins within two of three differentially extracted fractions were determined by size exclusion chromatography (SEC). The proteins in vitreous endosperm showed more protein cross-linking and a larger M[subscript w] distribution than found in the floury endosperm. An improved method for fractionating sorghum proteins designed to obtain intact disulfide linked protein polymers was developed. Three protein fractions obtained by application of the method represented proportionally different protein polymer contents as evidenced by comparative SEC and provides an improved tool for polymeric protein content comparison and measurement. The improved method was applied to a highly diverse non-tannin wild-type sorghum sample set spanning a range of in-vitro protein digestibility (IVPD) values to determine polymers involved with and influencing IVPD. Grain traits other than cross-linked proteins were also investigated for significant relationships to IVPD. Three protein fractions (F1, F2, F3) containing intact protein polymers were obtained for analysis by SEC and RP-HPLC. Proteins represented by four of five individual SEC peaks from F3 were significantly negatively correlated to IVPD, with three of the correlated peaks being polymeric. A 2-dimensional (2-D) technique involving peak collection after size exclusion chromatography followed by reverse phase high performance liquid chromatography (SEC x RP-HPLC) of the collected peaks was applied to protein polymers previously determined to be correlated with IVPD. RP-HPLC chromatogram patterns unique to each collected SEC peak from three selectively extracted protein fractions allowed qualitative and quantitative comparisons of protein polymer components. A pair of early eluting peaks appearing in the [gamma]-kafirin region of 2nd-dimension RP-HPLC chromatograms from a protein fraction with the largest M[subscript w] distribution were significantly correlated to IVPD. The correlated peak of interest was collected and characterized using SDS-PAGE and was preliminarily identified as 27kDa [gamma]-kafirin. By combining techniques using differing selectivity’s (solvent based, molecular size based, hydrophobicity based), it was possible to disassemble and compare components of protein polymers significantly correlated to IVPD.