Toth L A, Kao G, Elchisak M A
Life Sci. 1987 Feb 2;40(5):473-80. doi: 10.1016/0024-3205(87)90113-5.
Phenol sulfotransferase activity is often measured by incubating dopamine with a source of the enzyme and the sulfate donor 35S-3'-phosphoadensine-5'-phosphosulfate and then separating the product, 35S-dopamine sulfate, from the substrates using precipitation with barium hydroxide and zinc sulfate. Using dopamine sulfate standards and high performance liquid chromatography with dual-electrode electrochemical detection, we investigated the effects of several parameters on product recovery obtained with this procedure. Amounts of precipitants needed to produce maximal sample-to-blank ratios were determined using crude enzyme preparations from rat brain and human platelets incubated under conditions generating approximately 3 nM dopamine sulfate. Under these conditions, recovery of 3H-DAS standards was 70-80%. Increasing either the concentration of dopamine sulfate or the amounts of precipitants used resulted in concentration-dependent decreases in dopamine sulfate recovery. These data indicate that the recovery of product in this assay may vary with assay conditions, and should be carefully monitored and optimized. Caution should be exercised when using substrates other than dopamine in this assay procedure, since similar factors might also contribute to variable recovery of other products.
酚磺基转移酶活性的测定通常是将多巴胺与酶源及硫酸盐供体35S-3'-磷酸腺苷-5'-磷酸硫酸一起孵育，然后使用氢氧化钡和硫酸锌沉淀，将产物35S-多巴胺硫酸盐与底物分离。我们使用多巴胺硫酸盐标准品以及配备双电极电化学检测的高效液相色谱法，研究了几个参数对该方法产物回收率的影响。使用大鼠脑和人血小板的粗酶制剂，在产生约3 nM多巴胺硫酸盐的条件下孵育，确定产生最大样品与空白比值所需的沉淀剂用量。在这些条件下，3H-DAS标准品的回收率为70-80%。增加多巴胺硫酸盐浓度或所用沉淀剂的量会导致多巴胺硫酸盐回收率呈浓度依赖性下降。这些数据表明，该测定中产物的回收率可能因测定条件而异，应仔细监测并优化。在此测定过程中使用多巴胺以外的底物时应谨慎，因为类似因素也可能导致其他产物的回收率变化。
