Salt Effects on Ionization Equilibria of Histidines in Myoglobin

Abstract

The salt dependence of histidine p K a values in sperm whale and horse myoglobin and in histidine-containing peptides was measured by 1H-NMR spectroscopy. Structure-based p K a calculations were performed with continuum methods to test their ability to capture the effects of solution conditions on p K a values. The measured p K a of most histidines, whether in the protein or in model compounds, increased by 0.3 pH units or more between 0.02 M and 1.5 M NaCl. In myoglobin two histidines (His 48 and His 36) exhibited a shallower dependence than the average, and one (His 113) showed a steeper dependence. The 1H-NMR data suggested that the salt dependence of histidine p K a values in the protein was determined primarily by the preferential stabilization of the charged form of histidine with increasing salt concentrations rather than by screening of electrostatic interactions. The magnitude and salt dependence of interactions between ionizable groups were exaggerated in p K a calculations with the finite-difference Poisson-Boltzmann method applied to a static structure, even when the protein interior was treated with arbitrarily high dielectric constants. Improvements in continuum methods for calculating salt effects on p K a values will require explicit consideration of the salt dependence of model compound p K a values used for reference in the calculations.