Salt effects on ionization equilibria of histidines in myoglobin

The salt dependence of histidine pK(a) values in sperm whale and horse myog lobin and in histidine-containing peptides was measured by H-1-NMR spectros copy. Structure-based pK(a) calculations were performed with continuum meth ods to test their ability to capture the effects of solution conditions on pK(a) values. The measured pK(a) of most histidines, whether in the protein or in model compounds, increased by 0.3 pH units or more between 0.02 M an d 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 H-1-NMR data suggested that the salt dependence of histidi ne pK(a) values in the protein was determined primarily by the preferential stabilization of the charged form of histidine with increasing salt concen trations rather than by screening of electrostatic interactions. The magnit ude and salt dependence of interactions between ionizable groups were exagg erated in pK(a) calculations with the finite-difference Poisson-Boltzmann m ethod applied to a static structure, even when the protein interior was tre ated with arbitrarily high dielectric constants. Improvements in continuum methods for calculating salt effects on pK(a) values will require explicit consideration of the salt dependence of model compound pK(a) values used fo r reference in the calculations.