On the stabilizing action of protein denaturants: acetonitrile effect on stability of lysozyme in aqueous solutions.

Stability of hen lysozyme in the presence of acetonitrile (MeCN) at different pH values of the medium was studied by scanning microcalorimetry with a special emphasis on determination of reliable values of the denaturational heat capacity change. It was found that the temperature of denaturation decreases on addition of MeCN. However, the free energy extrapolation showed that below room temperature the thermodynamic stability increases at low concentrations of MeCN in spite of the general destabilizing effect at higher concentrations and temperatures. Charge-induced contribution to this stabilization was shown to be negligible (no pH-dependence was found); therefore, the most probable cause for the phenomenon is an increase of hydrophobic interactions at low temperatures in aqueous solutions containing small amounts of the organic additive. The difference in preferential solvation of native and denatured states of lysozyme was calculated from the stabilization free energy data. It was found that the change in preferential solvation strongly depends on the temperature in the water-rich region. At the higher MeCN content this dependence decreases until, at 0.06 mole fractions of MeCN, the difference in the preferential solvation between native and denatured lysozyme becomes independent of the temperature over a range of 60 K. The importance of taking into account non-ideality of a mixed solution, when analyzing preferential solvation phenomena was emphasized.

[Influence of kinetic factors on heat denaturation and renaturation of biopolymers]. / Vliianie kineticheskikh faktorov na teplovuiu denaturatsiiu i renaturatsiiu biopolimerov.

Possible influence of experimental nonequilibrium conditions on heat denaturation and renaturation of biopolymers has been studied. The analysis has been made using a standard kinetic model of transition between two states. Regularities are revealed which determine the position and shape of the heat absorption peak in calorimetric experiments on direct and reverse scanning (heating and cooling). The evaluation formulas for obtaining kinetic information from such experiments are given and their application is discussed. The results obtained can be useful in analysis of real calorimetric curves. It is shown that upon heating and cooling in nonequilibrium conditions the behavior of the system can differ qualitatively. The above difference takes place when the renaturation rate decreases on lowering the temperature. In this case, heating under nonequilibrium conditions leads only to a shift of denaturation transition to the higher temperatures while nonequilibrium cooling may result in complete or partial irreversibility.

Folding under inequilibrium conditions as a possible reason for partial irreversibility of heat-denatured proteins: computer simulation study.

Using computer simulations we have studied possible effects of heating and cooling at different scan rates on unfolding and refolding of macromolecules. We have shown that even the simplest two-state reversible transition can behave irreversibly when an unfavorable combination of cooling rate, relaxation time and activation energy of refolding occurs. On the basis of this finding we suppose that apparent irreversibility of some proteins denatured by heat may result from slow relaxation on cooling rather than thermodynamic instability and/or irreversible alterations of the polypeptide chain. Using this kinetic reversible two-state model, we estimated the effects of the scan rate and kinetic parameters of the macromolecule on its unfolding-refolding process. A few recommendations are suggested on how to reach maximal possible recovery after denaturation if refolding appears to be under kinetic control.

Preferential solvation changes upon lysozyme heat denaturation in mixed solvents.

On the basis of scanning microcalorimetry data from literature and our own measurements, we have calculated the changes in preferential solvation of lysozyme upon heat denaturation in six solvent systems: water + methanol, ethanol, propanol [data from Velicelebi, G., & Sturtevant, J. M. (1979) Biochemistry 18, 1180], acetone, p-dioxane [data from Fujita, Y., & Noda, Y. (1983) Bull. Chem. Soc.Jpn. 56, 233], and dimethylsulfoxide [our data Kovrigin, E. L., Kirkitadze, M. D., & Potekhin, S. A. (1996) Biofizika 41, 549-553; Kovrigin, E. L., & Potekhin, S. A. (1996) Biofizika 41, 1201-1206]. These preferential solvation changes are (in effect) the numbers of cosolvent molecules entering or leaving the solvation shell of the protein upon denaturation. It has been shown that for a group of five substances in the initial activity range (approximately up to 0. 3) the denaturational changes of preferential solvation of lysozyme do not depend on the nature of the solvent and depend only on its activity. This suggests that lysozyme does not distinguish these substances in the initial activity range and preferential solvation has a nonspecific character. It has been shown also that preferential solvation DeltaGamma23 does not depend on the pH value at least for dimethylsulfoxide-water solutions. This indicates that the chargeable groups exposed on denaturation do not contribute significantly to preferential interaction of the protein surface with the solution components.

[Microcalorimetric study of the effect of dimethylsulfoxide on the heat denaturation of lysozyme]. / Mikrokalorimetricheskoe issledovanie vliianiia dimetilsul'foksida na teplovuiu denaturatsiiu lizotsima.

The effect of dimethylsulfoxide on heat denaturation of lysozyme has been studied by scanning microcalorimetry. Measurements have been performed in a wide range of apparent pH values and organic component concentrations and have revealed a destabilizing effect of dimethylsulfoxide on the structure of lysozyme. The whole range of dimethylsulfoxide concentrations can be subdivided into three regions according to the effect exerted on the enthalpy of denaturation: in one region the enthalpy increases, in the second falls and in the third the denaturational transition is not revealed. In a narrow range of dimethylsulfoxide concentrations (55-60% of the volume) at pH 2.5 the so-called "anomalous heat denaturation" has been revealed. This phenomenon is characterized by a sharp decrease of denaturation enthalpy and a considerable excess of effective enthalpy over calorimetric one while denaturation remains absolutely reversible. It has been shown that this is not connected with oligomerization of the protein; it has been suggested that this phenomenon is of kinetic origin.

[Enthalpy stabilization of chicken egg lysozyme in aqueous dimethylsulfoxide solutions]. / Ental'piia stabilizatsii struktury lizotsima kurinogo iaitsa v vodnykh rastvorakh dimetilsul'foksida.

Scanning microcalorimetry data have been used to plot the dependences of the denaturation enthalpy of hen egg lysozyme on dimethylsulfoxide concentration at fixed temperatures. It has been shown that at dimethylsulfoxide concentrations below 40% (v/v) the enthalpy does not depend on pH of the medium. An increase of dimethylsulfoxide concentrations in this range leads to a linear growth of enthalpy. The rate of enthalpy growth decreases with the temperature increase. The denaturation enthalpy begins to considerably depend on pH at dimethylsulfoxide concentrations more than 40%. Spectroscopy data indicate that conformational changes occur in the protein in this range of concentrations already at room temperature, whereas according to scanning microcalorimetry, they take place at much higher temperatures. This difference is probably due to a decrease of the real temperature of protein melting below room temperature and a very inhibited character of the denaturational transition. This results in a decrease of calorimetric enthalpy at acidic pH owing to incomplete protein renaturation upon calorimeter cooling to the starting point.