Horowitz P M, Bowman S
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284.
J Biol Chem. 1989 Feb 25;264(6):3311-6.
Controlled conditions have been found that give complete reactivation and long term stabilization of rhodanese (EC 2.8.1.1) after oxidative inactivation by hydrogen peroxide. Inactivated rhodanese was completely reactivated by reductants such as thioglycolic acid (TGA) (100 mM) and dithiothreitol (DTT) (100 mM) or the substrate thiosulfate (100 mM) if these reagents were added soon after inactivation. Reactivability fell in a biphasic first order process. At pH 7.5, in the presence of DTT inactive rhodanese lost 40% of its reactivability in less than 5 min, and the remaining 60% was lost more gradually (t 1/2 = 3.5 h). TGA reactivated better than DTT, and the rapid phase was much less prominent. If excess reagents were removed by gel filtration immediately after inactivation, there was time-independent and complete reactivability with TGA for at least 24 h, and the resulting samples were stable. Reactivable enzyme was resistant to proteolysis and had a fluorescence maximum at 335 nm, just as the native protein. Oxidized rhodanese, Partially reactivated by DTT, was unstable and lost activity upon further incubation. This inactive enzyme was fully reactivated by 200 mM TGA. Also, the enzyme could be reactivated by arsenite and high concentrations of cyanide. Addition of hydrogen peroxide (40-fold molar excess) to inactive rhodanese after column chromatography initiated a time-dependent loss of reactivability. This inactivation was a single first order process (t 1/2 = 25 min). Sulfhydryl titers showed that enzyme could be fully reactivated after the loss of either one or two sulfhydryl groups. Irreversibly inactivated enzyme showed the loss of one sulfhydryl group even after extensive reduction with TGA. The results are consistent with a two-stage oxidation of rhodanese. In the first stage there can form sulfenyl and/or disulfide derivative(s) at the active site sulfhydryl that are reducible by thioglycolate. A second stage could give alternate or additional oxidation states that are not easily reducible by reagents tried to date.
已发现一些可控条件，能使经过氧化氢氧化失活后的硫氰酸酶（EC 2.8.1.1）完全重新激活并实现长期稳定。如果在失活后不久添加诸如巯基乙酸（TGA）（100 mM）、二硫苏糖醇（DTT）（100 mM）等还原剂或底物硫代硫酸盐（100 mM），失活的硫氰酸酶可被完全重新激活。重新激活能力呈双相一级过程下降。在pH 7.5、存在DTT的情况下，失活的硫氰酸酶在不到5分钟内失去40%的重新激活能力，其余60%则逐渐丧失（半衰期 = 3.5小时）。TGA比DTT的重新激活效果更好，且快速阶段不那么明显。如果在失活后立即通过凝胶过滤去除过量试剂，用TGA处理至少24小时可实现与时间无关的完全重新激活能力，且所得样品稳定。可重新激活的酶对蛋白水解有抗性，且与天然蛋白一样，在335 nm处有最大荧光。经DTT部分重新激活的氧化型硫氰酸酶不稳定，进一步孵育会失去活性。这种失活的酶可被200 mM TGA完全重新激活。此外，该酶可被亚砷酸盐和高浓度氰化物重新激活。柱色谱后向失活的硫氰酸酶中添加过氧化氢（摩尔过量40倍）会引发重新激活能力随时间的丧失。这种失活是单一的一级过程（半衰期 = 25分钟）。巯基滴定表明，在失去一个或两个巯基后，酶仍可被完全重新激活。即使在用TGA大量还原后，不可逆失活的酶仍显示失去了一个巯基。结果与硫氰酸酶的两阶段氧化过程一致。在第一阶段，活性位点巯基处可形成可被巯基乙酸还原的亚磺酰基和/或二硫键衍生物。第二阶段可能产生迄今尝试的试剂不易还原的交替或额外氧化态。
