Epelbaum S, Chipman D M, Barak Z
Department of Biology, Ben-Gurion University, Beer-Sheva, Israel.
Anal Biochem. 1990 Nov 15;191(1):96-9. doi: 10.1016/0003-2697(90)90393-n.
The enzyme acetohydroxy acid synthase (AHAS, EC 4.1.3.18) catalyzes two competing reactions of physiological importance: condensation of two molecules of pyruvate to form acetolactate (AL) or condensation of pyruvate and 2-ketobutyrate to form acetohydroxybutyrate (AHB). The activity of AHAS is most frequently analyzed using the Westerfeld method, in which the acetoin formed upon decarboxylation of AL is determined by colorimetric reaction with creatine and alpha-naphthol. However, there has been confusion as to the interpretation of the results of this assay in the presence of both substrates, conditions which lead to formation of both AL and AHB. By applying this assay to enzymatically prepared samples of AL and AHB which have also been analyzed by two other independent methods, we show here that the color yield for AHB in the commonly used assay is 35-40% that for equivalent amounts of acetoin or AL. The relative color yield is not significantly affected by varying the time or temperature of various steps in the color-forming reaction. This information could in principle be used, together with an independent specific assay for AHB, to determine the composition of an AHAS product mixture; it would, however, be less accurate than a simultaneous chromatographic method.
乙酰羟酸合酶（AHAS，EC 4.1.3.18）催化两个具有重要生理意义的竞争反应：两分子丙酮酸缩合形成乙酰乳酸（AL），或丙酮酸与2-酮丁酸缩合形成乙酰羟丁酸（AHB）。AHAS的活性最常采用韦斯特费尔德方法进行分析，该方法中，AL脱羧形成的乙偶姻通过与肌酸和α-萘酚的比色反应来测定。然而，在两种底物都存在的情况下，对于该测定结果的解读存在混淆，这种情况会导致同时形成AL和AHB。通过将该测定应用于酶促制备的AL和AHB样品，这些样品也通过另外两种独立方法进行了分析，我们在此表明，在常用测定中，AHB的显色产率是等量乙偶姻或AL显色产率的35%-40%。改变显色反应中各个步骤的时间或温度，相对显色产率不会受到显著影响。原则上，该信息可与AHB的独立特异性测定一起用于确定AHAS产物混合物的组成；然而，它不如同时进行的色谱法准确。
