Byers D M, Kay C M
J Biol Chem. 1983 Mar 10;258(5):2951-4.
Bovine cardiac troponin-I (TN-I) and troponin-T (TN-T) have been examined in solution using ultracentrifugation, gel filtration, and viscosity. A new method of purifying TN-T, employing hydroxylapatite chromatography in 6 M urea, is reported. Cardiac TN-T (Mr = 36,000) undergoes a reversible, concentration-dependent association in nondenaturing buffers, probably to a tetramer. The Stokes radius (Rs) of aggregated TN-T, determined by sedimentation velocity and gel chromatography on Sephacryl S-300, is 80 A and the reduced viscosity of the subunit ranges from 20 to 25 ml/g at protein concentrations between 0.5 and 2.5 mg/ml. These data suggest that TN-T forms highly asymmetric aggregates in solution. Bovine cardiac TN-I also has a tendency toward self-association, but is essentially monomeric (Mr = 23,000) at protein concentrations below 1 mg/ml. The presence of reducing agent is necessary to avoid intermolecular disulfide bond formation. From gel filtration experiments, the value of Rs is 29 A indicating that TN-I is a moderately asymmetric protein (frictional ratio = 1.5). Similar properties are observed when both sulfhydryl groups of TN-I are modified by carboxamidomethylation.
已使用超速离心、凝胶过滤和粘度法对牛心肌肌钙蛋白-I（TN-I）和肌钙蛋白-T（TN-T）进行了溶液状态下的检测。本文报道了一种在6 M尿素中采用羟基磷灰石色谱法纯化TN-T的新方法。心脏TN-T（分子量=36,000）在非变性缓冲液中会发生可逆的、浓度依赖性缔合，可能形成四聚体。通过沉降速度和在Sephacryl S-300上的凝胶色谱法测定，聚集态TN-T的斯托克斯半径（Rs）为80 Å，在蛋白质浓度为0.5至2.5 mg/ml时，亚基的比浓粘度范围为20至25 ml/g。这些数据表明TN-T在溶液中形成高度不对称的聚集体。牛心肌TN-I也有自我缔合的倾向，但在蛋白质浓度低于1 mg/ml时基本为单体（分子量=23,000）。需要存在还原剂以避免分子间二硫键的形成。从凝胶过滤实验可知，Rs值为29 Å，表明TN-I是一种中等不对称的蛋白质（摩擦比=1.5）。当TN-I的两个巯基都通过羧甲基化进行修饰时，会观察到类似的性质。
