Anderot Maria, Nilsson Mikael, Végvári Akos, Moeller Eva Horn, van de Weert Marco, Isaksson Roland
Chemistry and Biomedical Sciences, University of Kalmar, SE-391 82 Kalmar, Sweden.
J Chromatogr B Analyt Technol Biomed Life Sci. 2009 Apr 1;877(10):892-6. doi: 10.1016/j.jchromb.2009.02.021. Epub 2009 Feb 11.
In this manuscript we report the binding affinity between two model proteins, human serum albumin (HSA) and ribonuclease A (RNase A), and negatively charged polyelectrolytes, two different heparin fractions and dextran sulfate, by means of partial filling and affinity capillary electrophoresis. The apparent dissociation constants, K(d), obtained by use of the partial-filling method, between HSA and heparin (17kDa), heparin (3kDa) and dextran sulfate (8kDa) were 33 and 307microM, respectively. A new method was developed to determine affinities that take in account different migration directions between the protein and the polyelectrolyte, which was required to study RNase A. By use of this affinity capillary electrophoresis two K(d) values were observed for the interaction between RNase A and heparin 17kDa, yielding a high affinity binding with K(d1) 0.0075microM, and a lower affinity binding with K(d2) 8.7microM. For dextran sulfate 8kDa these K(d) values were 0.027 and 10.4microM, respectively. Heparin 3kDa only showed a single K(d) value of 0.52microM. The results show that the magnitude of the binding affinity depends on the type of polyelectrolyte and its molecular weight.
在本论文中，我们通过部分填充亲和毛细管电泳法，报道了两种模型蛋白——人血清白蛋白（HSA）和核糖核酸酶A（RNase A）与带负电荷的聚电解质（两种不同的肝素级分和硫酸葡聚糖）之间的结合亲和力。使用部分填充法测得HSA与17 kDa肝素、3 kDa肝素和8 kDa硫酸葡聚糖之间的表观解离常数K(d)分别为33 μM和307 μM。我们开发了一种新方法来测定亲和力，该方法考虑了蛋白质和聚电解质之间不同的迁移方向，这是研究RNase A所必需的。通过这种亲和毛细管电泳法，观察到RNase A与17 kDa肝素相互作用时有两个K(d)值，高亲和力结合的K(d1)为0.0075 μM，低亲和力结合的K(d2)为8.7 μM。对于8 kDa硫酸葡聚糖，这些K(d)值分别为0.027 μM和10.4 μM。3 kDa肝素仅显示单一的K(d)值为0.52 μM。结果表明，结合亲和力的大小取决于聚电解质的类型及其分子量。
