小麦贮藏蛋白反相高效液相色谱分析体系研究

 【目的】反相高效液相色谱（RP-HPLC）是定性和定量分析小麦贮藏蛋白的有效方法，在国外的应用始于20世纪80年代初，但在国内一直没有利用，也无开展系统的研究。本研究的目的是填补国内这一研究的空白。【方法】选用2个代表性品种中优9507（强筋）和京411（弱筋），系统研究了柱温、洗脱梯度、样品量、提取时间和进样体积对贮藏蛋白分离效果和量化的影响，并验证了分析重复性。【结果】结果表明，45 mg面粉样品，使用50%正丙醇（v/v）和含有50%正丙醇（v/v）、1%二硫苏糖醇（w/v）的弱酸缓冲液（Tris-HCl，pH6.6），分别对醇溶蛋白和谷蛋白提取45 min，可以使提取率达到90%。醇溶蛋白和谷蛋白分别进样10～15 µl和15～20 µl，可以使实际量化值接近理论量化值。针对我国小麦品种贮藏蛋白组成的特殊性对洗脱梯度进行优化后，提高了高分子量谷蛋白亚基与低分子量谷蛋白亚基之间的分离度。【结论】配合聚丙烯酰氨凝胶电泳（PAGE）可以对贮藏蛋白各组分进行准确定性定量分析，为深入研究小麦贮藏蛋白和加工品质的关系提供了工具。

Protocol Establishment of Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) for Analyzing Wheat Gluten Protein

Reversed-phase high-performance liquid chromatography (RP-HPLC) is an effective method in separating wheat gluten protein, which has been widely used in wheat quality study. Two cultivars, Zhongyou 9507 and Jing 411，were chosen to study the effect of temperature, elution gradient, sample weight, extraction duration, injection volume on gluten protein resolution and the repeat ability of these analyses. The results indicated that the gliadin and glutenin subunits extraction rate can reach 90% when 45mg of flour was extracted with 1ml 50% aqueous 1-propanol (v/v) and glutenin extraction buffer, respectively. The optimal sample injection volume of gliadin and glutenin was 10~15 ?l and 15~20 ?l, respectively. The modified elution gradient can separate all types of gliadin and glutenin subunits. This new protocol can qualify and quantify the components of gliadin and glutenin accurately, by which we can predicate the wheat end-use quality.