Holloway C J, Tegtmeier F, Brunner G, Trautschold I
J Chromatogr. 1980 Dec 26;202(3):423-9. doi: 10.1016/s0021-9673(00)91827-2.
A method is described for the quantitative assay of the methylation of alkane-thiols from the methyl-donor S-adenosylmethionine, catalysed by the microsomal enzyme S-adenosyl-L-methionine:thiol S-methyltransferase (E.C. 2.1.1.9). The reaction is carried out in sealed vials, one fifth of whose volume is taken up by an aqueous phase containing the enzyme and reactants. The volatile substrates and products of the reaction, thiols and thioethers, respectively, are present in equilibrium both in the liquid and gas phases in the reaction vessels. Aliquots of the gas phase are removed at intervals in gas-tight syringes, and analysis is performed directly on a gas chromatograph fitted with a flame-ionization detector. The amounts of thiol and thioether detected are then related to the total amounts of substance in the reaction vessels from calibration measurements, so that the kinetics of the enzymatic process can be evaluated. This technique offers distinct advantages over previously reported methods, in that no radioactively labelled compounds are required. Furthermore, decreases in substrate and increases in product can be assayed simultaneously, and the methylation of a mixture of thiols can be monitored in a single set of analyses.
本文描述了一种定量测定由微粒体酶S-腺苷-L-甲硫氨酸：硫醇S-甲基转移酶（E.C. 2.1.1.9）催化、从甲基供体S-腺苷甲硫氨酸生成烷硫醇甲基化的方法。反应在密封小瓶中进行，小瓶体积的五分之一被含有酶和反应物的水相占据。反应的挥发性底物和产物，即硫醇和硫醚，分别在反应容器的液相和气相中处于平衡状态。每隔一段时间用气密注射器取出气相的等分试样，并直接在配备火焰离子化检测器的气相色谱仪上进行分析。然后，根据校准测量结果，将检测到的硫醇和硫醚的量与反应容器中物质的总量相关联，从而可以评估酶促过程的动力学。该技术相对于先前报道的方法具有明显优势，因为不需要放射性标记化合物。此外，可以同时测定底物的减少和产物的增加，并且可以在一组分析中监测硫醇混合物的甲基化。
