PH, IONIC-STRENGTH, AND TEMPERATURE DEPENDENCES OF IONIZATION EQUILIBRIA FOR THE CARBOXYL GROUPS IN TURKEY OVOMUCOID 3RD DOMAIN

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 pK(a), values of all six carboxyl groups in OMTKY3. At 35 degrees C and in the presence of 10 mM KCl, the pK, 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 pK, values for Glu 10 (4.1) and Glu 43 (4.7) show more modest deviations from model compound data. The low pK, for the a-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) Ace, 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 pK(a) 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 pK, 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., and 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., and Robertson, A.D. (1995) Biochemistry 34, 4724-4732], the pH dependence of OMTKY3 stability is described and compared to expectations based on the pK(a) values described herein.