Zelazowski A J, Gasyna Z, Stillman M J
Department of Chemistry, University of Western Ontario, London, Canada.
J Biol Chem. 1989 Oct 15;264(29):17091-9.
We report new spectroscopic properties for a range of silver-metallothionein species. The binding reactions that take place following addition of Ag+ to rabbit liver apoMT 2, and the apo alpha and -beta fragments have been studied using the techniques of circular dichroism (CD) and emission spectroscopy. Titrations carried out at 20 degrees C and 55 degrees C reveal for the first time the formation of a sequence of clusters (Ag6-MT, Ag12-MT and, finally, Ag18-MT) as Ag+ is added to rabbit apoMT 2. (The division of mammalian metallothioneins into two major subforms, MT 1 and MT 2, is based on differences in molecular charge, which results from differences in the sequence of amino acids that do not involve the cysteines.) It is proposed that the novel Ag18-MT complex forms with a structure that involves a well defined three-dimensional structure, in the same manner as that recently reported for the Hg18-MT complex (Cai, W. and Stillman, M. J., (1988) J. Am. Chem. Soc. 110, 7872-7873). Addition of silver in excess of 20 mol equivalents leads to the collapse of this structure. At the elevated temperatures, it is suggested that the protein can exert cooperativity so that completely filled domains are formed rather than mixtures of complexes. This contrasts with the kinetic product in which metals are bound across the peptide chain forming more random "cross-linked" regions in place of the cluster structure. CD spectra were recorded as Ag+ was added to the alpha and beta fragments formed from rabbit liver MT 1. The silver-containing fragments are less stable than the Ag-MT. The alpha and beta fragments exhibit CD spectral patterns indicative of stoichiometrically defined species. The presence of Ag3- alpha MT 1 and Ag6- alpha MT 1 is suggested by the spectral data obtained at 20 and 55 degrees C. Formation of Ag3- beta MT 1 is suggested by the spectral data recorded at 20 degrees C for the beta fragment. We also report that silver-containing metallothioneins are luminescent. Both the position of the band maximum in the 460-600 nm region and the emission intensity are strongly dependent on the stoichiometry of silver to protein. In the range of molar ratios for silver:MT of 1-12, bands at 465 and 520 nm intensify to a maximum for Ag10-MT 2. A band at 575 nm reaches a maximum for Ag16-MT 2. Analysis of the emission data suggests that Ag+ binds in a domain specific mechanism to apoMT 2.
我们报告了一系列银 - 金属硫蛋白物种的新光谱性质。利用圆二色性（CD）和发射光谱技术，研究了向兔肝脱金属硫蛋白2（apoMT 2）以及脱金属α和β片段中添加Ag⁺后发生的结合反应。分别在20℃和55℃下进行的滴定首次揭示了随着Ag⁺添加到兔apoMT 2中，会形成一系列簇（Ag₆ - MT、Ag₁₂ - MT，最终是Ag₁₈ - MT）。（哺乳动物金属硫蛋白分为两个主要亚型MT 1和MT 2，是基于分子电荷的差异，这是由不涉及半胱氨酸的氨基酸序列差异导致的。）有人提出，新型Ag₁₈ - MT配合物形成的结构具有明确的三维结构，与最近报道的Hg₁₈ - MT配合物的结构方式相同（蔡，W.和斯蒂尔曼，M. J.，（1988年）《美国化学会志》110，7872 - 7873）。添加超过20摩尔当量的银会导致这种结构崩溃。在升高的温度下，表明蛋白质可以发挥协同作用，从而形成完全填充的结构域，而不是配合物的混合物。这与动力学产物形成对比，在动力学产物中金属跨肽链结合，形成更随机的“交联”区域，而不是簇结构。当向由兔肝MT 1形成的α和β片段中添加Ag⁺时，记录了CD光谱。含银片段比Ag - MT稳定性差。α和β片段表现出指示化学计量定义物种的CD光谱模式。在20℃和55℃下获得的光谱数据表明存在Ag₃ - αMT 1和Ag₆ - αMT 1。在20℃下记录的β片段光谱数据表明形成了Ag₃ - βMT 1。我们还报告含银金属硫蛋白是发光的。在460 - 600 nm区域内带最大值的位置和发射强度都强烈依赖于银与蛋白质的化学计量。在银与MT的摩尔比为1 - 12的范围内，465和520 nm处的带对于Ag₁₀ - MT 2增强到最大值。575 nm处的带对于Ag₁₆ - MT 2达到最大值。对发射数据的分析表明，Ag⁺以结构域特异性机制与apoMT 2结合。
