Chen P, Munoz A, Nettesheim D, Shaw C F, Petering D H
Department of Chemistry, University of Wisconsin-Milwaukee 53201, USA.
Biochem J. 1996 Jul 15;317 ( Pt 2)(Pt 2):395-402. doi: 10.1042/bj3170395.
Experiments were done to define the stoichiometry of binding of Cu(I) to metallothionein (MT) and to determine its sites of binding in mixed-metal species. Spectrophotometric titrations of rabbit liver Cd7-MT 2, apoMT, and Cd4-alpha-domain with Cu(I) revealed endpoints of 3-4, 4 and 8, and 4 and 6-7 added Cu(I)/mol of MT for the three species respectively. Observed endpoints depended on conditions of the titration and the wavelength chosen for absorbance measurement. Nevertheless, from metal and sulphydryl analyses of titrated proteins that were pretreated with Chelex-100 to remove metal ions from solution, almost all of the cadmium was displaced from Cd7-MT by the addition of 7 Cu(I)/mol of MT. Similarly, 4 Cu(I)/mol of Cd4-alpha-domain completely displaced bound cadmium. The Cu4-alpha-domain was converted into a Cu6-alpha species upon addition of two equivalents of Cu(I)/mol of alpha-domain. Reaction of Cd7-MT with 7, 12 and 20 Cu/mol of MT, followed by reaction with Chelex resin, generated protein samples in each case with about 7 Cu/mol of MT. 111Cd-NMR analysis of the reaction of 111Cd7-MT with Cu(I) showed that nearly co-operative one-for-one replacement of 111Cd occurred and that the beta-domain cluster reacted before the alpha-domain cluster. Two mixed-metal MTs with Cu to Zn ratios approximating 3 to 4 and 6 to 4 were isolated from calf liver. After substitution of Zn with 111Cd, NMR spectra of each protein showed that 111Cd was confined almost completely to the alpha-domain. By inference, about 3 or 6 Cu were bound in the beta-domain of these proteins. Supporting this segregation of metal ions into domains, reaction of Cu6, Zn4-MT with nitrilotriacetate removed zinc exclusively, whereas reaction of Cu6,Cd4-MT with 4,7-phenylsulphonyl-2,9-dimethyl-1,10-phenanthroline extracted only Cu(I). Proteolytic digestion of both products followed by gel filtration demonstrated that Cu(I) and Cd were bound to fragments of the intact protein. Finally, reaction of rabbit liver 111Cd7-MT 2 with Cu10-MT 2 resulted in interprotein metal exchange in which 111Cd-moved from the beta- to the alpha-domain according to NMR analysis. In contrast with the prevalent view that six Cu(I) bind to each domain of MT, the present results show that Cu(I) binds to MT with a minimum stoichiometry of about 7 Cu(I)/mol of MT and can bind to the alpha-domain with stoichiometries of 4 or 6 Cu(I)/mol of MT. Although MTs interacting with 12 or 20 Cu(I)/mol of MT are less stable than that binding about 7 Cu(I)/mol, it appears that MT can bind Cu(I) in multiple stoichiometries.
开展实验以确定Cu(I)与金属硫蛋白(MT)结合的化学计量关系，并确定其在混合金属物种中的结合位点。用Cu(I)对兔肝Cd7-MT 2、脱金属硫蛋白(apoMT)和Cd4-α结构域进行分光光度滴定，结果显示这三种物种分别达到终点时，每摩尔MT添加的Cu(I)量为3 - 4、4和8，以及4和6 - 7。观察到的终点取决于滴定条件和用于吸光度测量的波长。然而，通过对用Chelex-100预处理以从溶液中去除金属离子的滴定蛋白质进行金属和巯基分析，添加每摩尔MT 7个Cu(I)时，几乎所有镉都从Cd7-MT中被置换出来。同样，每摩尔Cd4-α结构域添加4个Cu(I)可完全置换结合的镉。添加每摩尔α结构域两当量的Cu(I)后，Cu4-α结构域转变为Cu6-α物种。Cd7-MT与每摩尔MT 7、12和20个Cu反应，随后与Chelex树脂反应，每种情况下生成的蛋白质样品中每摩尔MT约有7个Cu。对111Cd7-MT与Cu(I)反应的111Cd-NMR分析表明，111Cd几乎以一对一的协同方式被置换，且β结构域簇比α结构域簇先发生反应。从小牛肝中分离出两种铜锌比约为3比4和6比4的混合金属MT。用111Cd替代锌后，每种蛋白质的NMR光谱显示111Cd几乎完全局限于α结构域。由此推断，这些蛋白质的β结构域中约结合有3个或6个Cu。支持金属离子分隔到不同结构域的是，Cu6,Zn4-MT与次氮基三乙酸反应仅去除锌，而Cu6,Cd4-MT与4,7-苯基磺酰基-2,9-二甲基-1,10-菲咯啉反应仅萃取出Cu(I)。对两种产物进行蛋白水解消化，然后进行凝胶过滤，结果表明Cu(I)和Cd与完整蛋白质的片段结合。最后，兔肝111Cd7-MT 2与Cu10-MT 2反应导致蛋白质间金属交换，根据NMR分析，111Cd从β结构域转移到α结构域。与普遍认为六个Cu(I)结合到MT的每个结构域的观点相反，目前的结果表明，Cu(I)与MT结合的最小化学计量约为每摩尔MT 7个Cu(I)，并且可以以每摩尔MT 4个或6个Cu(I)的化学计量结合到α结构域。尽管与每摩尔MT与12个或20个Cu(I)相互作用的MT相比，结合约每摩尔MT 7个Cu(I)的MT稳定性较差，但似乎MT可以以多种化学计量结合Cu(I)。
