THEORETICAL AND EXPERIMENTAL-ANALYSIS OF IONIZATION EQUILIBRIA IN OVOMUCOID 3RD DOMAIN

2D-NMR experiments were used to determine the pK(a) values ranging fro m 8.0 to greater than or equal to 11.1 of seven basic residues in turk ey ovomucoid third domain (OMTKY3) and were compared to values predict ed as described by Antosiewicz et al. [(1996) Biochemistry 35, 7819-78 33]. Lys 13, 29, and 34 were previously attributed with increasing the acidity of numerous acidic residues [Schaller, W., and Robertson, A. D. (1995) Biochemistry, 34, 4714-4723]. These interactions were expect ed to raise the pK(a) values of those basic groups; however, the pK(a) values of Lys 13 and 34 are less than the model compound values. The pK(a) values of the other basic residues are greater than the model co mpound values and, unlike the acidic residues, all are surprisingly in sensitive to salt. While the calculations properly predict the directi on of most of the pK(a) shifts and provide valuable insight into the p ossible molecular origins of the interactions that perturb pK(a) value s, there is a tendency to overestimate the magnitude of the shifts and their salt dependence. Interestingly, the shapes of both the calculat ed and observed transitions are often more complex than expected for a simple titration, suggesting that pK(a) values at many sites are chan ging during the transition. Differences between predicted and experime ntal pK(a) values and titration profiles for some residues may be due to as yet uncharacterized structural changes at the extremes of pH.