A thermodynamic analysis of the binding of calcium and magnesium ions to parvalbumin.
A thermodynamic analysis of the binding of calcium and magnesium ions to parvalbumin.
复制标题
钙和镁离子与小清蛋白结合的热力学分析。
DOI：
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发表时间：
2005
期刊：
影响因子：
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通讯作者：
J. Cox
中科院分区：
文献类型：
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作者：
H. J. Moeschler;J. Schaer;J. Cox
采用微量热法和平衡透析法测定了pI为4.25的鲤鱼肌异小清蛋白与Ca ~（2+）、Mg ~（2+）相互作用的热力学函数Δ H_0、Δ G_0和Δ S_0。平衡透析研究表明，小清蛋白的两个金属位点对Ca ~（2+）或Mg ~（2+）的亲和力相等，平衡常数KCa = 2.7 × 10 ~（9）M ~（-1），KMg = 9.5 × 10 ~（4）M ~（-1）。两种金属离子的结合是完全竞争性的，没有显示出协同效应。在1 mM Mg ~（2+）存在下，表观Ca ~（2+）亲和常数K ′ Ca为2.8 × 10 ~（7）M ~（-1），Mg ~（2+）-Ca ~（2+）交换平衡常数为2.8 × 10 ~（4）M ~（-1）。微量热分析表明，小清蛋白对Ca ~（2+）结合的金属中心为-37.2 kJ/mol，对Mg ~（2+）-Ca ~（2+）交换的金属中心为-25.1 kJ/mol，对Mg ~（2+）络合物的形成产生Δ H_0 = -12.1 kJ/mol。焓的变化是线性依赖于结合到蛋白质的金属的量，从而证实了两个网站的亲和力相等。Ca ~（2+）络合物形成的反应熵δ_0为+55.2 J × mol ~（-1）× K ~（-1），Mg ~（2+）络合物形成的反应熵δ_0为+54.8 × mol ~（-1）× K ~（-1）。因此，相应的金属结合过程由焓和熵结合驱动，并使人联想到Ca 2+与肌钙蛋白C的结合。在Mg 2 +-Ca 2+交换（0.4 J × mol-1 × k-1）过程中观察到的反应熵是可以忽略不计的，尽管Ca 2+和Mg 2+的水合熵显着不同。这表明小清蛋白的两种金属配合物不具有相同的构象熵。由于在金属交换后没有观察到内在蛋白质荧光的变化，因此构象差异必须限于金属结合位点的直接环境。
Microcalorimetry and equilibrium dialysis were used to determine the thermodynamic functions delta H0, delta G0 and delta S0 guiding the interaction of Ca2+ and Mg2+ with purified carp muscle isoparvalbumin of pI 4.25. The equilibrium dialysis studies indicate equal affinities of the two metal sites of parvalbumin for either Ca2+ or Mg2+ with equilibrium constants of KCa = 2.7 X 10(9) M-1, and KMg = 9.5 X 10(4) M-1. Binding of the two metal ions is fully competitive with no indication for cooperative effects. The apparent Ca2+ affinity constant K'Ca in the presence of 1 mM Mg2+ is 2.8 X 10(7) M-1, and the Mg2+-Ca2+ exchange equilibrium constant equals 2.8 X 10(4) M-1. Microcalorimetry shows that parvalbumin exhibits negative reaction enthalpies of -37.2 kJ/mol metal site for Ca2+ binding and -25.1 kJ/mol site for Mg2+-Ca2+ exchange, yielding delta H0 = -12.1 kJ/mol site for Mg2+ complex formation. Enthalpy changes are linearly dependent upon the amount of metal bound to the protein, thus corroborating the equal affinities of the two sites. Reaction entropies delta S0 are +55.2 J x mol-1 x K-1 for Ca2+ complex formation and +54.8 x mol-1 x K-1 for Mg2+ complex formation. Thus the respective metal binding processes are driven by both enthalpy and entropy conbinations, and are reminiscent of Ca2+ binding to troponin C. The reaction entropy observed during Mg2+-Ca2+ exchange (0.4 J x mol-1 x k-1) is negligible in spite of the markedly different hydration entropies for Ca2+ and Mg2+. This indicates that the two metal complexes of parvalbumin do not have the same conformation entropy. Since no variation in the intrinsic protein fluorescence was observed upon metal exchange, the conformation differences must be restricted to the immediate environment of the metal binding sites.