Analysis of wheat storage proteins by exhaustive sequential extraction followed by RP-HPLC and nitrogen determination

Wheat storage proteins are responsible for the viscoelastic properties of dough. Their effect on dough rheology depends on the glutenin components and the proportion of gliadins, high and low molecular weight glutenin subunits (HMW-GS, LMW-GS). A prediction of dough behaviour is possible when the concentration of each prolamin group is known. A method of sequential extraction and quantification of wheat hour proteins was developed. Reversed-phase high-performance liquid chromatography (RP-HPLC) was used to quantify gliadins and HMW-GS and LMW-GS. Non-prolamin proteins and lipids were extracted with phosphate buffer and phosphate buffer containing Triton X114 respectively and 70% (v/v) ethanol was used to extract gliadins. Several solvent systems were tested to exhaustively extract glutenins and a buffer containing 0.05 M tetraborate pH 8.5, 2% (v/v) 2-mercaptoethanol, 1 g litre(-1) glycine, and 8 M urea was selected. It facilitated the most complete extraction and was compatible with RP-HPLC analysis of extracts. The quantities of extracted prolamins were estimated from RP-HPLC profiles using peak area determination. The concentration of different classes of prolamins obtained by RP-HPLC was compared with their determination by nitrogen (N) content. The average yield of extractions performed on 45 cultivars was 0.99 (coefficient of variation (CV) = 7.6%) for gliadins and 0.89 (CV = 9.1%) for glutenins. Quantifications of proteins by N determination and RP-HPLC were strongly correlated: regression coefficients were 0.86 for total prolamins and gliadins, and 0.71 for glutenins with the gradient of regression of approximately 1. Therefore, RP-HPLC could be used to quantify prolamin groups without using N determination. (C) 1998 SCI.