SDS-聚丙烯酰胺凝胶电泳法同时测定不同产地大蒜药材中的蒜酶与凝集素

目的：建立SDS-聚丙烯酰胺凝胶电泳同时测定不同产地大蒜药材中蒜酶与凝集素相对含量和相对分子质量的方法。方法：通过考察溶媒方法、样品前处理方法、供试品溶液浓度等因素，确定SDS-聚丙烯酰胺凝胶电泳法测定蒜酶与凝集素的最佳条件：以超纯水为溶媒，静置10 min为前处理方法，供试品溶液质量浓度为28 mg·mL^-1；分别配制5%浓缩胶、12%分离胶，凝胶厚度为1 mm，浓缩胶电压为80 V，分离胶电压为100 V，进行不连续体系垂直板电泳，借助Gel-pro Analyzer软件分析凝胶电泳图谱。以标准蛋白为marker，建立标准曲线，将蒜酶和凝集素的比移值代入标准曲线方程，计算蒜酶和凝集素的相对分子质量。结果：标准曲线方程为lgM=-1.369 1X+2.184 7，r=0.966 6。大蒜药材中蒜酶相对分子质量为52.1~54.0 kDa，凝集素相对分子质量为11.0~11.8 kDa。不同产地大蒜药材中蒜酶和凝集素总相对含量为18.1%~77.2%，其中蒜酶的相对含量为5.60%~27.1%，凝集素的相对含量为12.5%~68.2%。新疆巴里坤市大蒜样品中蒜酶的相对含量最高为27.1%，新疆阿克苏拜城县大蒜样品中凝集素的相对含量最高为68.2%。不同产地大蒜药材中蒜酶、凝集素的相对含量存在差异，凝集素相对含量普遍高于蒜酶相对含量。结论：与其他测定蛋白质含量的方法相比，本文建立的方法操作简便，样品用量少，能够准确测定大蒜药材中蒜酶及凝集素的相对分子质量与相对含量。不同产地大蒜中蒜酶与凝集素相对总含量均达50%以上，证明蒜酶和凝集素是大蒜中的主要蛋白质。大蒜中的蒜酶与蒜氨酸相对含量的相关性并不显著。

Objective: To establish a method that simultaneously determine relative content and molecular mass of alliinase and lectin in garlic from the different origins by SDS-polyacrylamide gel electrophoresis.Methods: The optimal conditions of the method were confirmed by investigating the factors of solvent methods,sample pretreatment method and the test solution concentrations. The relative content and the molecular mass of alliinase and lectin in garlic were measured by SDS-polyacrylamide gel electrophoresis. The optimal conditions were as follows:ultra-pure water as solvent,stationary for 10 minutes as pretreatment method,the sample solution concentration was 28 mg·mL^-1. And 12% separation gel,5% concentrated gel were separately prepared. The gel thickness was 1mm. The concentrated gel voltage was 80 V,and the concentrated gel voltage was 100 V. Then the discontinuous system and vertical plate electrophoresis were applied. The map of SDS-polyacrylamide gel electrophoresis was analyzed by gel-pro analyzer software. The standard curve was established with standard protein as a marker. The Rf values of alliinase and lectin were substituted for the standard curve equation to calculate their molecular mass.Results: The standard curve equation was lgM=-1.369 1X+2.184 7,r=0.966 6. In the garlic herbs,the relative molecular mass of alliinase was 52.1-54.0 KDa,lectin was 11.0-11.8 KDa. The total relative content of alliinase and lectin was 18.1%-77.2% in garlic from different origins. The relative content of alliinase was 5.60%-27.1% and lectin was 12.5%-68.2%. The garlic sample from Xinjiang Balikun had the highest relative content of alliinase that was 27.1%. The garlic sample from Xinjiang Akesu Baicheng County had the highest relative content of lectin that was 68.2%. The contents of alliinase and lectin in garlic were different among different origins. The relative content of lectin was generally higher than alliinase.Conclusions: Compared with other reported methods of determining the protein content,this method has the advantages of convenient operation,small amount of required sample and can accurately measure the relative molecular mass and the relative content of alliinase and lectin in the garlic. The total relative contents of alliinase and lectin were more than 50% from difference origins of garlic,which proved that alliianse and lectin were the main proteins in garlic. The preliminary results indicate that there is no significant correlation between the amount of alliinase and alliin.