三硝基苯磺酸(trinitrobrnzen sulfonic acid,TNBS)法是测定蛋白质中游离氨基含量的方法。该文比较了该方法的2种终止反应条件及分析方法,探究了该方法对不同蛋白质(牛血清蛋白、胰蛋白酶、溶菌酶)的适用性。结果表明,碱性条件下的响应值显著高于酸性(P<0.01);紫外可见分光光度计和酶标仪2种测定方法对蛋白质游离氨基含量的测定结果在一定范围内没有显著差异(P>0.05),紫外可见分光光度计测定结果精确度较高,对浓度变化的响应更灵敏。蛋白质种类不仅影响到该方法的取样浓度,而且蛋白质中的巯基含量会干扰该方法的准确性。
The trinitrobenzenesulfonic acid (TNBS) method is a kind of method for determining the content of free amino groups in proteins. This paper compared two termination reaction conditions and analytical methods, and explored the applicability of the method for different proteins (bovine serum albumin, trypsin, lysozyme). The results showed that (a) the response value under alkaline conditions was significantly higher than that of acidity (P<0.01); (b) the results of UV-visible spectrophotometer and microplate reader showed no significant difference in a certain range(P>0.05); (c) the accuracy of the ultraviolet-visible spectrophotometer was higher and the response to the changes in concentration was more sensitive than control group. Now only did protein species affect the sampling concentration, but also the sulfhydryl content in the proteins interfered with the accuracy of the method.
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