Determination of Effective Protein Charge by Capillary Electrophoresis: Effects of Charge Regulation in the Analysis of Charge Ladders

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• 作者：Manoj K. Menon and Andrew L. Zydney
• 刊名：Analytical Chemistry
• 出版年：2000
• 出版时间：November 15, 2000
• 年：2000
• 卷：72
• 期：22
• 页码：5714 - 5717
• 全文大小：57K
• 年卷期：v.72,no.22(November 15, 2000)
• ISSN：1520-6882

文摘

Protein charge ladders are an effective tool for measuringprotein charge and studying electrostatic interactions.However, previous analyses have neglected the effects ofcharge regulation, the alteration in the extent of aminoacid ionization associated with differences between the pHat the protein surface and in the bulk solution. Experimental data were obtained with charge ladders constructed from bovine carbonic anhydrase. The proteincharge for each element in the ladder was calculated fromthe protein electrophoretic mobility as measured bycapillary electrophoresis using the hindrance factor for ahard sphere with equivalent hydrodynamic radius. Theprotein charge was also evaluated theoretically from theamino acid sequence by assuming a Boltzmann distribution in the hydrogen ion concentration. The calculationswere in excellent agreement with the data, demonstratingthe importance of charge regulation on the net proteincharge. These results have important implications for theuse of charge ladders to evaluate effective protein chargein solution.