Hydrogen-ion titrations of amino acids and proteins in solutions containing concentrated electrolyte

摘要

This report describes a first attempt to quantify the net charge as a function of solution pH for lysozyme and (alpha)-chymotrypsin at 0.1 M, 1.0 M and 3.0 M ionic strength, (IS). The calculations are based on the residue (titratable group) pK(sub a)'s in the amino-acid sequence of the protein. To determine these pK(sub a)'s, a simple theory was used which assumes that the pK(sub a)'s are independent from each other in the protein and are equal to their pK(sub a) values in free amino-acid solution (Independent-Site Theory, IST). Residue pK(sub a)'s were obtained from amino-acid hydrogen-ion titrations at three different KCl concentrations corresponding to 0.1M, 1.0M and 3.0M ionic strength. After construction of a suitable apparatus, the experimental procedure and data reduction were computerized to perform a large number of titrations. Most measured pK(sub a)'s showed high reproducibility (the difference of pK(sub a) values observed between two experiments was less than 0.05). For IS = 0.1M, observed pK(sub a)'s agreed with literature values to within a few hundredths of a pH unit. Furthermore, the ionic-strength dependence of the pK(sub a)'s followed the trends reported in the literature, viz. pK(sub a) values decrease with increasing ionic strength until they reach a minimum at about IS = 0.5M. At still higher IS, pK(sub a)'s increase as the ionic strength rises to 3M. The known pK(sub a)'s of all titratable groups in a protein were used with the IST to give a first approximation of how the protein net charge varies with pH at high ionic strength. A comparison of the titration curves based on the IST with experimental lysozyme and (alpha)-chymotrypsin titration data indicates acceptable agreement at IS = 0.1M. However, comparison of measured and calculated titration curves at IS = 1M and IS = 3M indicates only quantitative agreement.