Donato H, Mani R S, Kay C M
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Biochem J. 1991 May 15;276 ( Pt 1)(Pt 1):13-8. doi: 10.1042/bj2760013.
The effect of Cd2+ binding on bovine brain S-100b protein was studied using c.d. u.v. difference spectroscopy and fluorescence measurements. At pH 7.5, S-100b protein binds two Cd2+ ions per monomer with a Kd value of 3 x 10(-5) M. Addition of Cd2+ resulted in perturbing the single tyrosine residue (Tyr17) in the protein as indicated by u.v. difference spectroscopy and aromatic c.d. measurements. In the presence of Cd2+, the tyrosine residue moves to a more non-polar environment, since a red shift was observed in the u.v. difference spectrum. When the protein was excited at 278 nm, the tyrosine fluorescence emission maximum was centred at 306 nm. Cd2+ addition resulted in an increase in intrinsic fluorescence intensity. Fluorescence titration with Cd2+ indicated the protein binds Cd2+ with a Kd value of 3 x 10(-5) M. 2-p-Toluidinylnaphthalene-6-sulphonate-labelled protein, when excited at 345 nm, had a fluorescence emission maximum at 440 nm. Addition of Cd2+ to labelled protein resulted in a 5-fold increase in fluorescence intensity accompanied by a 5 nm blue shift in the emission maximum, suggesting that the probe, in the presence of Cd2+, moves to a hydrophobic domain. U.v. difference spectroscopic studies indicated a unique Cd2(+)-binding site on the protein, since Cd2+ addition yielded a large positive absorption band in the 240 nm region that is not found with either Ca2+ or Zn2- ions. Similar absorption bands have been observed in Cd-protein complexes such as Cd-metallothionein [Vasak, Kagi & Hill (1981) Biochemistry 20, 2852-2856] and also in model complexes of Cd2+ with 2-mercaptoethanol. This absorption band is believed to arise as a result of charge-transfer transitions between the thiolate and Cd2+. Of the two Cd2- -binding sites on the beta-chain, one must be located at the N-terminal end near the single tyrosine residue, since Cd2- and Zn2+ produced similar effects on the intrinsic protein fluorescence. The other Cd2+ site which is unique to Cd2+ must be Cys84, located at the C-terminal end.
采用圆二色（c.d.）紫外差光谱法和荧光测量法研究了Cd2+结合对牛脑S-100b蛋白的影响。在pH 7.5时，S-100b蛋白每个单体结合两个Cd2+离子，解离常数（Kd）值为3×10−5 M。紫外差光谱法和芳香族圆二色测量结果表明，添加Cd2+会扰乱该蛋白中的单个酪氨酸残基（Tyr17）。在Cd2+存在的情况下，酪氨酸残基转移到一个极性更低的环境中，因为在紫外差光谱中观察到了红移。当该蛋白在278 nm处激发时，酪氨酸荧光发射最大值集中在306 nm处。添加Cd2+导致固有荧光强度增加。用Cd2+进行荧光滴定表明该蛋白结合Cd2+的Kd值为3×10−5 M。用2-对甲苯胺基萘-6-磺酸盐标记的蛋白在345 nm处激发时，荧光发射最大值在440 nm处。向标记蛋白中添加Cd2+导致荧光强度增加5倍，同时发射最大值发生5 nm的蓝移，这表明在Cd2+存在的情况下，探针转移到了一个疏水区域。紫外差光谱研究表明该蛋白上有一个独特的Cd2+结合位点，因为添加Cd2+在240 nm区域产生了一个大的正吸收带，而Ca2+或Zn2+离子不会产生这种吸收带。在Cd-金属硫蛋白等Cd-蛋白复合物中[瓦萨克、卡吉和希尔（1981年）《生物化学》20，2852 - 2856]以及Cd2+与2-巯基乙醇的模型复合物中也观察到了类似的吸收带。据信这种吸收带是由于硫醇盐与Cd2+之间的电荷转移跃迁产生的。在β链上的两个Cd2+结合位点中，一个必定位于靠近单个酪氨酸残基的N末端，因为Cd2+和Zn2+对蛋白固有荧光产生了类似的影响。另一个Cd2+特有的位点必定是位于C末端的Cys84。
