Nagahara Noriyuki, Koike Shin, Nirasawa Takashi, Kimura Hideo, Ogasawara Yuki
Isotope Research Center, Nippon Medical School, Japan.
Department of Analytical Biochemistry, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose-shi, Tokyo, 204-8588, Japan.
Biochem Biophys Res Commun. 2018 Feb 5;496(2):648-653. doi: 10.1016/j.bbrc.2018.01.056. Epub 2018 Jan 10.
It has been known that hydrogen sulfide and/or polysulfides are produced from a (poly)sulfurated sulfur-acceptor substrate of 3-mercaptopyruvate sulfurtransferase (MST) via thioredoxin (Trx) reduction in vitro. In this study, we used thiosulfate as the donor substrate and the catalytic reaction was terminated on the formation of a persulfide or polysulfides. We can present alternative pathway of production of hydrogen sulfide and/or polysulfides from (poly)sulfurated catalytic-site cysteine of reaction intermediates of MST via Trx reduction. Matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometric analysis revealed that after prolonged incubation of MST with thiosulfate, a trisulfide adduct becomes predominant at the sulfurated catalytic-site cysteine. When these adducts were reduced by Trx with reducing system (MST:Escherichia coli Trx:E. coli Trx reductase:NADPH = 1:5:0.02:12.5 molar ratio), liquid chromatography with tandem mass spectrometric analysis for monobromobimane-derivatized HS revealed that HS first appeared, and then HS and HS did later. The results were confirmed by high-performance liquid chromatography-fluorescence analysis.
已知在体外，硫化氢和/或多硫化物是由3-巯基丙酮酸硫转移酶（MST）的（多）硫化硫受体底物通过硫氧还蛋白（Trx）还原产生的。在本研究中，我们使用硫代硫酸盐作为供体底物，催化反应在过硫化物或多硫化物形成时终止。我们可以提出通过Trx还原从MST反应中间体的（多）硫化催化位点半胱氨酸产生硫化氢和/或多硫化物的替代途径。基质辅助激光解吸电离串联飞行时间质谱分析表明，MST与硫代硫酸盐长时间孵育后，三硫化物加合物在硫化催化位点半胱氨酸处占主导地位。当这些加合物通过还原系统（MST：大肠杆菌Trx：大肠杆菌Trx还原酶：NADPH = 1：5：0.02：12.5摩尔比）被Trx还原时，对单溴联苯胺衍生的HS进行液相色谱-串联质谱分析表明，首先出现了HS，随后出现了HS和HS。结果通过高效液相色谱-荧光分析得到证实。
