pH, ionic strength, and temperature dependences of ionization equilibria for the carboxyl groups in turkey ovomucoid...

Two-dimensional NMR spectroscopy has been used to monitor the pH dependences of proton chemical shifts for turkey ovomucoid third domain (OMTKY3). Sample pH was varied from 7.0 to 1.4 in order to determine the apparent pKa values of all six carboxyl groups in OMTKY3. At 35 degrees C and in the presence of 10 mM KCl, the pKa values for Asp 7, Glu 19, and Asp 27 (< 2.6, 3.2, and < 2.3, respectively) are more than 1 pH unit below those for model compounds. The pKa values for Glu 10 (4.1) and Glu 43 (4.7) show more modest deviations from model compound data. The low pKa for the alpha-carboxyl group of Cys 56 (< 2.5) is attributable, at least in part, to acidification by the disulfide group. Fitting the data to a modified Hill equation [Markley, J. L. (1975) Acc. Chem. Res. 8, 70-80] reveals little evidence for interactions between the acidic groups; most Hill coefficients fall between 0.8 and 1.2, with outlying values usually obtained with data that describe incomplete transitions. Most of the very low pKa values show increases in the presence of 1.0 M KCl but, with the exception of that for glutamate 19, remain well below model compound values. pH-dependent changes in amide proton chemical shifts permitted identification of hydrogen bonds involving the side chains of Asp 7, Glu 19, and Asp 27, which may partially explain the low pKa values for these groups. These hydrogen bonds, two of which involve side chains that are well exposed to solvent, were previously identified in high-resolution X-ray studies of turkey ovomucoid third domain [Fujinaga, M., Sielecki, A. R., Read, R. J., Ardelt, W., Laskowski, M., Jr., & James, M. N. G. (1987) J. Mol. Biol. 195, 397-418]. Results of additional experiments performed at 15, 25, and 40 degrees C suggest that apparent ionization enthalpies for all carboxyl groups in OMTKY3 are about 0 +/- 2 kcal/mol. In the accompanying paper [Swint, L., & Robertson, A. D. (1995) Biochemistry 34, 4724-4732], the pH dependence of OMTKY3 stability is described and compared to expectations based on the pKa values described herein.