Doi Y, Kim F, Kido S
Department of Food Science, Kyoto Women's University, Japan.
Biochemistry. 1990 Feb 13;29(6):1392-7. doi: 10.1021/bi00458a008.
Calcium binding of swine plasma gelsolin was examined. When applied to ion-exchange chromatography, its elution volume was drastically altered depending on the free Ca2+ concentration of the medium. The presence of two classes of Ca2+ binding sites, high-affinity sites (Kd = 7 microM) and low-affinity sites (Kd = 1 mM), was suggested from the concentration dependence of the elution volume. The tight binding sites were specific for Ca2+. The weakly bound Ca2+ could be replaced by Mg2+ once the tight binding sites were occupied with Ca2+. The binding of metal ions was totally reversible. Circular dichroism measurement of plasma gelsolin indicated that most change in secondary structure was associated with Ca2+ binding to the high-affinity sites. Binding of Mg2+ to the low-affinity sites caused a secondary structural change different from that caused by Ca2+ bound to the high-affinity sites. Gel permeation chromatography exhibited a small change in Stokes radius with and without Ca2+. Microheterogeneity revealed by isoelectric focusing did not relate to the presence of two classes of Ca2+ binding sites. These results indicated that plasma gelsolin drastically altered its surface charge property due to binding of Ca2+ or Ca2+, Mg2+ with a concomitant conformational change.
对猪血浆凝溶胶蛋白的钙结合情况进行了检测。当应用于离子交换色谱时，其洗脱体积会根据介质中游离Ca2+浓度而发生显著变化。从洗脱体积的浓度依赖性推测存在两类Ca2+结合位点，即高亲和力位点（Kd = 7 microM）和低亲和力位点（Kd = 1 mM）。紧密结合位点对Ca2+具有特异性。一旦紧密结合位点被Ca2+占据，弱结合的Ca2+可被Mg2+取代。金属离子的结合是完全可逆的。血浆凝溶胶蛋白的圆二色性测量表明，二级结构的大多数变化与Ca2+结合到高亲和力位点有关。Mg2+结合到低亲和力位点引起的二级结构变化与Ca2+结合到高亲和力位点引起的不同。凝胶渗透色谱显示，有或没有Ca2+时，斯托克斯半径有微小变化。等电聚焦揭示的微不均一性与两类Ca2+结合位点的存在无关。这些结果表明，血浆凝溶胶蛋白由于Ca2+或Ca2+、Mg2+的结合以及伴随的构象变化而使其表面电荷性质发生显著改变。
