Pinkerton T C, Koeplinger K A
Control Division, Upjohn Company, Kalamazoo, Michigan 49001.
Anal Chem. 1990 Oct 1;62(19):2114-22. doi: 10.1021/ac00218a013.
The Hummel-Dreyer size-exclusion high-performance liquid chromatographic method for the determination of protein binding parameters has been improved and automated by use of an internal surface reversed-phase (ISRP) column (5 cm X 4.6 mm) and a computer-controlled mobile-phase delivery system with low volume syringe mixing. The high-efficiency ISRP columns, which are nonadsorptive and exclusionary to serum proteins but allow partitioning of small molecules with an internal peptide bonded phase, maintain high performance after many injections of human serum albumin (HSA), enable the use of short columns, and provide for the resolution of primary ligand from protein binding displacers. The modified Hummel-Dreyer high-performance liquid chromatographic method was demonstrated by the determination of binding parameters for warfarin-HSA in phosphate buffer, which were found to be n1 = 1.0, n2 = 2.1, K1 = 3.30 X 10(5) M-1, and K2 = 2.03 X 10(4) M-1. The necessary sequence of chromatographic experiments was repeated 4 times at 18 separate warfarin mobile phase concentrations. Each automated sequence required 8 h to complete. The parameters were measured with a precision of less than 10% relative standard deviation.
用于测定蛋白质结合参数的胡默尔 - 德雷尔尺寸排阻高效液相色谱法已通过使用内表面反相（ISRP）柱（5 cm×4.6 mm）和带有小体积注射器混合的计算机控制流动相输送系统得到改进并实现自动化。这种高效的ISRP柱对血清蛋白无吸附且具有排阻作用，但允许小分子与内部肽键合相进行分配，在多次注射人血清白蛋白（HSA）后仍能保持高性能，可使用短柱，并能分离主要配体与蛋白质结合置换剂。通过测定磷酸盐缓冲液中华法林 - HSA的结合参数，证明了改进后的胡默尔 - 德雷尔高效液相色谱法，结果发现n1 = 1.0，n2 = 2.1，K1 = 3.30×10⁵ M⁻¹，K2 = 2.03×10⁴ M⁻¹。在18种不同的华法林流动相浓度下，将色谱实验的必要序列重复了4次。每个自动化序列需要8小时完成。测量参数的相对标准偏差精度小于10%。
