Yun J H, Meyerhoff M E, Yang V C
College of Pharmacy, University of Michigan, Ann Arbor 48109-1065.
Anal Biochem. 1995 Jan 1;224(1):212-20. doi: 10.1006/abio.1995.1032.
A polymeric membrane electrode that exhibits significant and analytically useful potentiometric response to submicromolar levels of the heparin antagonist, protamine, is reported. The sensor is prepared by incorporating a lipophilic cation exchanger, potassium tetrakis(4-chlorophenyl)borate (KTpClPB) (at 1 wt%), within a specially formulated polymer membrane composed of 33 wt% 2-nitrophenyl octyl ether (2-NPOE), and 66 wt% poly(vinyl chloride) (PVC). When the polymer film is mounted in an appropriate electrode body, the resulting membrane electrode responds reproducibly to protamine via a nonequilibrium quasi-steady-state change in the phase boundary potential at the membrane/sample interface. Such response can be used to directly monitor, via classical potentiometric titrations, the binding between protamine and a variety of native (porcine and beef) as well as low-molecular-weight heparins. Scatchard analysis of the EMF titration data provides binding constants and stoichiometries for protamine-heparin interactions. The electrode can be further used to follow the enzymatic digestion of protamine by trypsin. In the presence of a given level of protamine, initial rates of potential decrease (-dE/dt) are shown to be linearly related to trypsin activity in solution over the range of 0-130 units/ml. The speed and simplicity of the protamine sensor make it an attractive alternative to classical methods for studying the interaction of protamine with other biologically important macromolecules as well as the proteolytic activity and reaction kinetics of trypsin.
据报道，一种聚合物膜电极对亚微摩尔水平的肝素拮抗剂鱼精蛋白表现出显著且具有分析用途的电位响应。该传感器通过将亲脂性阳离子交换剂四（4-氯苯基）硼酸钾（KTpClPB）（1 wt%）掺入由33 wt% 2-硝基苯基辛醚（2-NPOE）和66 wt%聚氯乙烯（PVC）组成的特殊配方聚合物膜中制备而成。当将聚合物膜安装在合适的电极体中时，所得的膜电极通过膜/样品界面处相界电位的非平衡准稳态变化对鱼精蛋白产生可重复的响应。这种响应可用于通过经典电位滴定法直接监测鱼精蛋白与多种天然（猪和牛）以及低分子量肝素之间的结合。对电动势滴定数据进行Scatchard分析可提供鱼精蛋白-肝素相互作用的结合常数和化学计量比。该电极还可进一步用于跟踪胰蛋白酶对鱼精蛋白的酶促消化。在给定水平的鱼精蛋白存在下，在0 - 130单位/毫升的范围内，电位下降的初始速率（-dE/dt）与溶液中的胰蛋白酶活性呈线性相关。鱼精蛋白传感器的快速性和简便性使其成为研究鱼精蛋白与其他重要生物大分子相互作用以及胰蛋白酶的蛋白水解活性和反应动力学的经典方法的有吸引力的替代方法。
