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 comp onents and the proportion of gliadins, high and low molecular weight g lutenin subunits (HMW-GS, LMW-GS). A prediction of dough behaviour is possible when the concentration of each prolamin group is known. A met hod 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-pro lamin proteins and lipids were extracted with phosphate buffer and pho sphate buffer containing Triton X114 respectively and 70% (v/v) ethano l was used to extract gliadins. Several solvent systems were tested to exhaustively extract glutenins and a buffer containing 0.05 M tetrabo rate 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 w as compatible with RP-HPLC analysis of extracts. The quantities of ext racted prolamins were estimated from RP-HPLC profiles using peak area determination. The concentration of different classes of prolamins obt ained by RP-HPLC was compared with their determination by nitrogen (N) content. The average yield of extractions performed on 45 cultivars w as 0.99 (coefficient of variation (CV) = 7.6%) for gliadins and 0.89 ( CV = 9.1%) for glutenins. Quantifications of proteins by N determinati on 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 b e used to quantify prolamin groups without using N determination. (C) 1998 SCI.