Tůmová Tereza, Monincová Lenka, Čeřovský Václav, Kašička Václav
Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Prague, Czech Republic.
Faculty of Food and Biochemical Technology, University of Chemistry and Technology Prague, Prague, Czech Republic.
Electrophoresis. 2016 Dec;37(23-24):3186-3195. doi: 10.1002/elps.201600342. Epub 2016 Oct 19.
Capillary electrophoresis (CE) was employed for the determination of thermodynamic acidity constants (pK ) and actual ionic mobilities of polycationic antimicrobial peptides (AMPs). The effective electrophoretic mobilities of AMPs were measured by CE in a series of the background electrolytes within a wide pH range (2.00-12.25), at constant ionic strength (25 mM) and ambient temperature, using polybrene coated fused silica capillaries to suppress sorption of cationic AMPs to the capillary wall. Eventually, Haarhoff-Van der Linde peak fitting function was used for the determination of correct migration times of some AMPs peaks that were distorted by electromigration dispersion. The measured effective mobilities were corrected to 25°C. Mixed acidity constants, pKa,i mix , and actual ionic mobilities, m , of AMPs were determined by the nonlinear regression analysis of pH dependence of their effective mobilities. The pKa,i mix values were recalculated to thermodynamic pK s using the Debye-Hückel theory. Thermodynamic pK of imidazolium group of histidine residues was found to be in the range 3.72-4.98, pK of α-NH group was in the range 6.14-6.93, and pK of ε-NH group of lysine spanned the interval 7.26-9.84, depending on the particular amino acid sequence of the AMPs. Actual ionic mobilities of AMPs with positive charges from one to six elementary units achieved values (9.8 - 36.5) × 10 m V s .
采用毛细管电泳（CE）法测定聚阳离子抗菌肽（AMPs）的热力学酸度常数（pK）和实际离子迁移率。在宽pH范围（2.00 - 12.25）、恒定离子强度（25 mM）和环境温度下，使用涂有聚乙烯亚胺的熔融石英毛细管抑制阳离子AMPs在毛细管壁上的吸附，通过CE测量AMPs的有效电泳迁移率。最终，使用Haarhoff - Van der Linde峰拟合函数确定一些因电迁移扩散而扭曲的AMPs峰的正确迁移时间。将测量的有效迁移率校正到25°C。通过对AMPs有效迁移率的pH依赖性进行非线性回归分析，确定了AMPs的混合酸度常数pKa,i mix和实际离子迁移率m。使用德拜 - 休克尔理论将pKa,i mix值重新计算为热力学pK。发现组氨酸残基咪唑鎓基团的热力学pK在3.72 - 4.98范围内，α - NH基团的pK在6.14 - 6.93范围内，赖氨酸ε - NH基团的pK跨度为7.26 - 9.84，这取决于AMPs的特定氨基酸序列。带一到六个基本单位正电荷的AMPs的实际离子迁移率达到（9.8 - 36.5）×10 m V s的值。
