Reisler E, Liu J, Mercola M, Horwitz J
Biochim Biophys Acta. 1980 Jun 26;623(2):243-56. doi: 10.1016/0005-2795(80)90253-6.
Binding of troponin to Cibacron Blue F3GA-agarose column and its selective release from the gel in the presence of 0.5 M KCl provides the basis for a new purification method. The two-step procedure consists of isoelectric precipitation of tropomyosin and chromatography of the resultant crude troponin supernatant on Affi-Gel Blue column. Adsorption of troponin to the immobilized dye appears to occur through the troponin-T subunit. Troponin-I and troponin-C do not bind to the blue agarose column, whereas troponin-T binds to it very tightly. Binding of the dye to troponin-T prevents formation of troponin-T-troponin-C complex, but does not interfere with direct interaction of troponin-T with troponin-I. The activity of troponin in conferring calcium sensitivity on actomyosin ATPase is not affected by Cibacron Blue. Circular dichroism and difference absorption measurements of complexes of the blue dye with troponin and its subunits reveal the presence of a tight binding site on whole troponin and on troponin-T (KA greater than or equal to 10(6) M). The existence of weak binding sites for the dye on troponin and all of its subunits is deduced from difference absorption studies. Cibacron Blue appears to be a sensitive probe for subunit interactions in troponin.
肌钙蛋白与汽巴克隆蓝F3GA - 琼脂糖柱的结合以及在0.5 M KCl存在下从凝胶中的选择性释放为一种新的纯化方法提供了基础。两步法包括肌动蛋白调节蛋白的等电沉淀以及所得粗肌钙蛋白上清液在亲和蓝胶柱上的色谱分析。肌钙蛋白与固定化染料的吸附似乎是通过肌钙蛋白 - T亚基发生的。肌钙蛋白 - I和肌钙蛋白 - C不与蓝色琼脂糖柱结合，而肌钙蛋白 - T与之紧密结合。染料与肌钙蛋白 - T的结合阻止了肌钙蛋白 - T - 肌钙蛋白 - C复合物的形成，但不干扰肌钙蛋白 - T与肌钙蛋白 - I的直接相互作用。肌钙蛋白赋予肌动球蛋白ATP酶钙敏感性的活性不受汽巴克隆蓝的影响。蓝色染料与肌钙蛋白及其亚基复合物的圆二色性和差示吸收测量揭示了在整个肌钙蛋白和肌钙蛋白 - T上存在紧密结合位点（KA大于或等于10(6) M）。从差示吸收研究推断出染料在肌钙蛋白及其所有亚基上存在弱结合位点。汽巴克隆蓝似乎是肌钙蛋白中亚基相互作用的敏感探针。
