Eberhard M, Erne P
Department of Research, Kantonsspital, Basel, Switzerland.
Eur J Biochem. 1994 May 15;222(1):21-6. doi: 10.1111/j.1432-1033.1994.tb18836.x.
Ca2+ and Mg2+ binding to rat parvalbumin was measured by means of the fluorescent Ca2+ indicator fluo-3 using a method developed earlier [Eberhard, M. & Erne, P. (1991) Eur. J. Biochem. 202, 1333-1338]. We demonstrate that rat parvalbumin contains two equivalent Ca2+/Mg2+ binding sites and that Ca2+ and Mg2+ compete for the same sites. Dissociation constants (Kd) for Ca2+ and Mg2+ in Hepes buffer containing 150 mM K+ at 35 degrees C and pH 7.2 are 11.0 +/- 1.8 nM and 41 +/- 8 microM, respectively. At an ionic strength below 0.2 M, Kd values of Ca2+ binding to rat parvalbumin are approximately proportional to the ion concentration. Kd values of Ca2+ binding were found to be about fourfold larger in the presence of Na+ as compared with K+, indicating that Na+ distinctly influences Ca2+ binding to rat parvalbumin. Both Ca2+ and Mg2+ binding to parvalbumin are exothermic whereas Ca2+ and Mg2+ binding to fluo-3 are endothermic entropy-driven processes.
采用先前开发的方法[埃伯哈德，M. & 厄内，P.（1991年）《欧洲生物化学杂志》202卷，1333 - 1338页]，通过荧光Ca²⁺指示剂fluo - 3测定了Ca²⁺和Mg²⁺与大鼠小清蛋白的结合。我们证明大鼠小清蛋白含有两个等效的Ca²⁺/Mg²⁺结合位点，且Ca²⁺和Mg²⁺竞争相同的位点。在35℃、pH 7.2且含有150 mM K⁺的Hepes缓冲液中，Ca²⁺和Mg²⁺的解离常数（Kd）分别为11.0±1.8 nM和41±8 μM。在离子强度低于0.2 M时，Ca²⁺与大鼠小清蛋白结合的Kd值大约与离子浓度成正比。发现与K⁺存在时相比，在Na⁺存在下Ca²⁺结合的Kd值大约大四倍，表明Na⁺明显影响Ca²⁺与大鼠小清蛋白的结合。Ca²⁺和Mg²⁺与小清蛋白的结合都是放热过程，而Ca²⁺和Mg²⁺与fluo - 3的结合是吸热的熵驱动过程。
