Beckmann J D
Department of Internal Medicine, University of Nebraska Medical Center, Omaha 68198.
Anal Biochem. 1991 Sep 2;197(2):408-11. doi: 10.1016/0003-2697(91)90412-m.
Phenol sulfotransferases (EC 2.8.2.1) catalyze the sulfation of the acceptor hydroxyl group using 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as the donor substrate. Previous assays of these enzymes, which exhibit varied acceptor substrate specificities, have required termination of the catalysis followed by isolation and quantitation of formed sulfate ester. In this report, the sulfation of the fluorescent compound, resorufin, is investigated. Reaction of PAPS with resorufin, catalyzed by bovine lung phenol sulfotransferase, bleaches the emission of this acceptor at the pH of the reaction (pH 6.4 optimum). It is thereby possible to continuously record the sulfation reaction. Analysis of single progress curves by integrated replot can be used to determine the initial velocities and also indicates the formation of a product inhibitor, probably resorufin sulfate ester, with Ki less than Km. Sensitivity of the reaction is less than 1 pmol/min. The maximal rate of resorufin sulfation by the bovine lung enzyme is estimated at 57 nmol/mg/min, which is 10% of the rate with an optimal substrate 2-naphthol. This assay may be most sensitive for phenol sulfotransferases with optimal activities at greater than pH 6, due to the acid-base properties of resorufin (pK alpha 6), which becomes nonfluorescent upon protonation.
酚磺基转移酶（EC 2.8.2.1）以3'-磷酸腺苷5'-磷酸硫酸酯（PAPS）作为供体底物，催化受体羟基的硫酸化反应。这些酶表现出不同的受体底物特异性，以往对它们的测定需要终止催化反应，然后分离并定量生成的硫酸酯。在本报告中，对荧光化合物试卤灵的硫酸化反应进行了研究。在反应pH值（最适pH 6.4）下，牛肺酚磺基转移酶催化PAPS与试卤灵反应，使该受体的荧光发射减弱。因此，可以连续记录硫酸化反应。通过积分重绘分析单条进程曲线可用于确定初始速度，同时也表明形成了一种产物抑制剂，可能是试卤灵硫酸酯，其抑制常数（Ki）小于米氏常数（Km）。该反应的灵敏度小于1皮摩尔/分钟。牛肺酶催化试卤灵硫酸化的最大速率估计为57纳摩尔/毫克/分钟，这是其对最佳底物2-萘酚催化速率的10%。由于试卤灵的酸碱性质（pKα6），质子化后会变为非荧光状态，因此该测定方法对于在pH大于6时具有最佳活性的酚磺基转移酶可能最为灵敏。
