Ho C H, Hlady V, Nyquist G, Andrade J D, Caldwell K D
Department of Materials Science & Engineering, University of Utah, Salt Lake City 84112.
J Biomed Mater Res. 1991 Apr;25(4):423-41. doi: 10.1002/jbm.820250402.
In order to further the understanding of protein-surface interactions in the coagulation system, we have chosen to study plasma protein adsorption onto heparin-immobilized surfaces. Heparin-binding proteins are abundant in plasma: a search of amino acid sequences revealed that many plasma proteins have possible heparin binding sites. Plasma protein adsorption to the heparinized surfaces is monitored by a novel technique in which the solution depletion of proteins is analytically determined using quantitative two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). This method enables simultaneous, quantitative detection of the majority of plasma proteins before, during, and after their adsorption onto high surface area adsorbents. Using computerized densitometry of silver-stained 2-D PAGE gels, the amount of each protein can be determined from the integrated optical density of each protein "spot." Kinetics of adsorption and adsorption isotherms of four important heparin binding proteins, antithrombin III (ATIII), complement factor C3 (C3), apolipoprotein AI (Apo-AI) and apolipoprotein AIV (Apo-AIV) are reported in this paper. From the adsorption isotherms, the apparent binding constants of each protein-immobilized heparin complex, Ka, were calculated. The surface binding constants were of the same order of magnitude as the respective solution binding constants in the literature. The surface binding constants followed the same order as the respective solution binding constants: Ka (ATIII) greater than Ka (Apo-AIV) greater than Ka (C3) greater than Ka (Apo-AI), indicating that protein binding to the immobilized heparin used is not essentially different from solution binding.
为了进一步了解凝血系统中蛋白质与表面的相互作用，我们选择研究血浆蛋白在固定化肝素表面的吸附情况。肝素结合蛋白在血浆中含量丰富：对氨基酸序列的搜索表明，许多血浆蛋白都可能具有肝素结合位点。血浆蛋白在肝素化表面的吸附情况通过一种新技术进行监测，该技术利用定量二维聚丙烯酰胺凝胶电泳（2-D PAGE）分析测定蛋白质溶液的消耗情况。这种方法能够同时定量检测大多数血浆蛋白在吸附到高比表面积吸附剂之前、期间和之后的情况。通过对银染2-D PAGE凝胶进行计算机密度测定，可以根据每个蛋白质“斑点”的积分光密度确定每种蛋白质的含量。本文报道了四种重要的肝素结合蛋白，抗凝血酶III（ATIII）、补体因子C3（C3）、载脂蛋白AI（Apo-AI）和载脂蛋白AIV（Apo-AIV）的吸附动力学和吸附等温线。根据吸附等温线，计算了每种蛋白质 - 固定化肝素复合物的表观结合常数Ka。表面结合常数与文献中各自的溶液结合常数处于相同的数量级。表面结合常数与各自的溶液结合常数遵循相同的顺序：Ka（ATIII）＞Ka（Apo-AIV）＞Ka（C3）＞Ka（Apo-AI），表明蛋白质与所用固定化肝素的结合与溶液结合本质上没有差异。
