小麦谷蛋白大聚合体亚基定量分析技术研究

A Method of Determing Subunit Quantitation of Wheat Glutenin Macropolymer

本文在Fu等(1996 )和Bean(1998)蛋白提取方法的基础上,对面粉细度、提取温度、蛋白酶抑制剂以及正丙醇浓度等方面进行了研究,建立了一种提取彻底,并且蛋白各组分间交叉污染较少,适于谷蛋白大聚合体亚基准确定量的提取方法。具体步骤如下:选用2 0 0目的面粉,首先用0 2 5mol/L乙二胺四乙酸钠盐在6 0℃下预处理1h ,然后再在6 0℃下用4 5 %正丙醇提取单体蛋白,最后用样品提取液进行谷蛋白大聚合体的提取。谷蛋白大聚合体亚基的定量采用AlphaInnotech公司的ChemilmagerTM4 4 0 0型凝胶成像扫描仪,并与Photoshop 7 0处理软件相结合,研究了稳定的谱带面积测定方法和稳定的灰度获取方法。建立了待测亚基含量的计算公式CX=CSSX/SS×[1+(WS-WX) / (2 5 5 -WS-G) ]。

Protein is an important factor for bread quality, the variety with high quality subunit often brings about good bread quality. But only the type of subunit cannot explain all variation of bread quality, other factors, such as the content and ratio of subunit also have a great influence to flour processing quality. So far, there are many studies on the relation between relative content of subunit and processing quality, however, but no reports about the relation among subunit content, ratio and wheat quality. The purpose of this experiment was to set up a method that subunit of wheat glutenin macropolymer(GMP) could be quantified precisely, so the function could be studied thoroughly. Based on Fu's (1996) [13] and Bean's (1998) [14] methods, the effect of granular diameter of flour, extraction temperature (Fig.1), proteinase inhibitor(Fig.2-B) and concentration of 1-propanol to extraction were studied. A new method suited to quantifying GMP was set up, by which protein could be extracted thoroughly and GMP could avoid the contamination with other protein fractions. The procedure was as follows: firstly, flour grinded to 200 mesh sieve was disposed with 0.25mol/L EDTA Na + for 1 h at 60℃; secondly, extracting monomeric proteins with 45% 1-propanol at 60℃; thirdly, extracting GMP from 45% 1-propanol insoluble fractions. After electrophoresis of GMP, the peak area and grey value of band were determined with Chemilmager TM 4400 and Photoshop 7.0 software. The content of each subunit could be calculated with the equation C X = C S S X/ S S×[1+( W S- W X)/(255- W S- G )]. In order to verify the method set up, six wheat varieties were used to measure GMP content. The range of GMP content of six varieties was from 1.84% to 5.66%, and this range was consistent with that measured with biuret method. It proves that the method we have set up is applicable and precise.