Behlke J, Ristau O
Max Delbrück Center for Molecular Medicine, Berlin, Germany.
Biophys Chem. 1999 Jan 11;76(1):13-23. doi: 10.1016/s0301-4622(98)00212-9.
This paper presents a modified method to determine experimentally the second virial coefficient of protein solutions by sedimentation equilibrium experiments. The improvement is based on the possibility of fitting simultaneously up to seven radial concentration distribution curves of solutions with different loading concentrations. The possibility of precise determination of the second virial coefficient allows estimation of the net charge and the excluded volume of a monomeric protein. Application of the method is demonstrated for lysozyme and ovalbumin. In 0.1 M sodium acetate buffer, pH 4.5, the second virial coefficient of hen egg white lysozyme amounts to 24 +/- 1 ml/g. Analysis based on spherical particle theory yield an excluded volume of 3.5 ml/g and a charge dependent value of 20.5 ml/g which is induced by a net charge number of 14.1 +/- 1. Under low salt conditions self-association processes on lysozyme are unfavorable due to electrostatic repulsion. To overcome these repulsive contributions, either a shift to neutral pH or addition of at least 2% NaCl is necessary. In this way the charge dependent contribution decreases below the value responsible for the excluded volume and allows crystallization of the protein. Similar effects can be observed with ovalbumin. The high virial coefficient observed at pH 8.5 is induced by the high net charge number of 27 +/- 1.
本文提出了一种改进方法，通过沉降平衡实验以实验方式测定蛋白质溶液的第二维里系数。改进基于同时拟合多达七种不同加载浓度溶液的径向浓度分布曲线的可能性。精确测定第二维里系数的可能性使得能够估算单体蛋白质的净电荷和排阻体积。该方法在溶菌酶和卵清蛋白上的应用得到了证明。在pH 4.5的0.1 M醋酸钠缓冲液中，鸡蛋清溶菌酶的第二维里系数为24±1 ml/g。基于球形颗粒理论的分析得出排阻体积为3.5 ml/g，由净电荷数14.1±1引起的电荷相关值为20.5 ml/g。在低盐条件下，由于静电排斥，溶菌酶上的自缔合过程不利。为了克服这些排斥作用，要么将pH值调至中性，要么加入至少2%的NaCl。通过这种方式，电荷相关贡献降低到低于负责排阻体积的值，并允许蛋白质结晶。在卵清蛋白上也能观察到类似的效果。在pH 8.5时观察到的高维里系数是由27±1的高净电荷数引起的。
