HYDROGEN-BONDS AND THE PH-DEPENDENCE OF OVOMUCOID 3RD DOMAIN STABILITY

Thermal denaturation of turkey ovomucoid third domain (OMTKY3) has been monitored with differential scanning calorimetry (DSC) and circular dichroism in H2O and D2O pH 1.5 to 5 and ionic strength 0.01 to 0.71. Results from DSC experiments are in good agreement with spectroscopic studies [Swint, L., and Robertson, A.D. (1993) Protein Sci. 2, 2037-2049] and fit well to the two-state unfolding model. The average ratio of calorimetric to van't Hoff enthalpies is 0.99 +/- 0.03 (n = 16), and the average value of Delta C-p, is 620 +/- 20 cal/(mol K) (n = 7). The free energy of unfolding (Delta G(u)(0)) increases in the presence of salt at both pH 1.5 and 4.5. This stabilization is not due to ion binding and probably results from screened repulsive interactions between the cationic groups of OMTKY3. At very low ionic strength, the change in Delta G(u)(0) from pH 1.5 to 4.5, Delta Delta(pH)(0), is 3.5 +/- 0.2 kcal/mol. Few interactions between ionizing groups are affected by the addition of 200 mM KCl; Delta Delta(pH)(0), decreases by only 0.4 +/- 0.3 kcal/mol. Comparison of Delta Delta(pH)(0) with values calculated from the pK(a)s of all six carboxyl groups in OMTKY3 [Schaller, W.S., & Robertson, A.D. (1995) Biochemistry 34, 4714-4723] suggests that some pK(a)s in the denatured state may be lower than those of model compounds. Moreover, calculated values of Delta Delta(pH)(0) are very sensitive to modest changes in the cooperativity of proton binding. Aspartates 7 and 27, whose pK(a)s appear to be perturbed primarily by involvement in hydrogen bonds, are responsible for most of the pH dependence of OMTKY3 stability.