Quantification Of Individual High And Low Molecular Weight Glutenin Subunits Of One Wheat Kernel Using SDS-PAGE And Scanning Densitometry

1 Variation of extract yield of both high molecular weight(HMW-GS)and low molecular weight glutenin subunits(LMW-GS)with temperature conditions and storage duration was observed.At high temperature(90 and 65℃)the yield of both HMW-GS and LMW-GS soon got to it's maximum(in 5 and 30 min respectively),and prolonged duration only reduced the yield.At lower temperature conditions(-20,4,0,15,18,23,35 or 50℃) the variation was complicate.In 18h the yield of HMW-GS increased with time,and the higher temperature the more yield could be got,but when the duration last for 7 days the most yield was got at -20℃followed sequentially by at 0 and 4℃and the yield at 18,23, 35 and 50℃decreased to so low that they almost could not be detected with Coomassie Brilliant Blue R250.While the yield of LMW-GS increased with time as HMW-GS in 27h, the increased temperature(from -20 to 35℃)didn't increase the yield and they appeared to be the same,and the lower temperature conditions were all more favorable than 90and 65℃for extracting LMW-GS.23℃was determined to be the best temperature condition for extracting LMW-GS when duration last for 7 days.Times moving samples into -70℃for 1h also favored the extraction of both HMW-GS and LMW-GS.2 Based on the extract characters,a procedure which combine 90,23 and -70℃and 48h was established for completely extracting both HMW-GS and LMW-GS from a wholemeal of wheat(Triticum aeativum L.)at one-time extraction.Temperature and duration condition for removal of monomeric proteins were optimized to obtain maximum yield and purity of glutenin subunits as was the separation of the HMW and LMW glutenin subunits by SDS-PAGE,which was necessary for accurate quantification of the individual subunit by scanning densitometry.3 A monomeric protein was used as quantification standard for quantifying subunits with the unitμg.The method developed has several important advantages over methods which quantify HMW-GS and LMW-GS subunits seperately.First,the relative quantity of HMW-GS and/or LMW-GS of whole glutenin fraction or wholemeal of wheat can be obtained.Second,the weight of individual HMW-GS and LMW-GS on gels of samples and of one wheat kernel can be got and arraged.4 All weight of individual glutenin subunits of 180 wheat varieties was screened by the method above.According to the glutenin subunits molecular weight range,the sum of glutenin subunits weight of group A,group B,group C and group D was calculated,and the distribution and variation of them was analysed.The variation range of the sum of glutenin subunits of all glutenin,group A,group B,group C and group D was 21.79～352.36μg,4.12～85.01μg,5.91～102.52μg,4.82～113.75μg and 8.86～135.52μg,and the variation coefficient was 35.07%,39.32%,37.02%,39.83%and 42.66%,respectively.