Singh B K, Stidham M A, Shaner D L
American Cyanamid Company, Princeton, New Jersey 08540.
Anal Biochem. 1988 May 15;171(1):173-9. doi: 10.1016/0003-2697(88)90139-x.
Acetohydroxyacid synthase (AHAS), also known as acetolactate synthase, has received attention recently because of the finding that it is the site of action of several new herbicides. The most commonly used assay for detecting the enzyme is spectrophotometric involving an indirect detection of the product acetolactate. The assay involves the conversion of the end product acetolactate to acetoin and the detection of acetoin via the formation of a creatine and naphthol complex. There is considerable variability in the literature as to the details of this assay. We have investigated a number of factors involved in detecting AHAS in crude ammonium sulfate precipitates using this spectrophotometric method. Substrate and cofactor saturation levels, pH optimum, and temperature optimum have been determined. We have also optimized a number of factors involved in the generation and the detection of acetoin from acetolactate. The results of these experiments can serve as a reference for new investigators in the study of AHAS.
乙酰羟酸合酶（AHAS），也被称为乙酰乳酸合酶，由于发现它是几种新型除草剂的作用位点，最近受到了关注。检测该酶最常用的方法是分光光度法，涉及对产物乙酰乳酸的间接检测。该检测方法包括将终产物乙酰乳酸转化为乙偶姻，并通过形成肌酸和萘酚复合物来检测乙偶姻。关于该检测方法的细节，文献中存在相当大的差异。我们使用这种分光光度法研究了在粗硫酸铵沉淀中检测AHAS所涉及的一些因素。确定了底物和辅因子的饱和水平、最适pH值和最适温度。我们还优化了从乙酰乳酸生成和检测乙偶姻所涉及的一些因素。这些实验结果可为AHAS研究的新研究者提供参考。
