Calorimetric study of interaction of ovalbumin with vanillin

The effect of vanillin on thermal denaturation of ovalbumin was studied using high-sensitivity differential scanning calorimetry. At neutral and acidic (6.7 and 3.0) pH values thermal denaturation of ovalbumin depends on the heating rate and is irreversible. The experimental denaturation thermograms of ovalbumin were compared with thermograms simulated according to the Lumry–Eyring model of irreversible protein denaturation (N↔D→A). It was shown that general tendencies of changes in the calorimetric parameters of ovalbumin denaturation as a function of the heating rate are in accordance with those predicted by this model. It was concluded that the heating rate could be considered as a factor shifting the denaturation equilibrium of ovalbumin (N↔D) similarly to its shifts caused by such physicochemical factors as pH, ionic strength or ligand concentration. Both the temperature and the enthalpy of ovalbumin denaturation decrease with an increase in the vanillin concentration within the pH range from 3.0 to 9.0. This effect of vanillin binding does not depend on the heating rate. The equilibrium parameters of ovalbumin denaturation have been determined by an extrapolation procedure at different vanillin concentrations. The equilibrium denaturation parameters have been used for calculation of the excess free energy of denaturation as a function of vanillin concentration. The vanillin binding constant Kb equals 5 M−1 at pH 6.7 and 33 M−1 at pH 3.0. The denaturation increment of the numbers of binding sites is Δdν=20 at pH 6.7 and Δdν=3 at pH 3.0. Possible mechanism of the vanillin binding to ovalbumin and its applied aspects are discussed.