Free energy changes in denaturation of ribonuclease A by mixed denaturants. Effects of combinations of guanidine hydrochloride and one of the denaturants lithium bromide, lithium chloride, and sodium bromide.

F Ahmad

doi:10.1016/s0021-9258(17)43027-4

Abstract

The denaturation of ribonuclease A by guanidine hydrochloride, lithium bromide, and lithium chloride and by mixed denaturants consisting of guanidine hydrochloride and one of the denaturants lithium chloride, lithium bromide, and sodium bromide was followed by difference spectral measurements at pH 4.8 and 25 degrees C. Both components of mixed denaturant systems enhance each other's effect in unfolding the protein. The effect of lithium bromide on the midpoint of guanidine hydrochloride denaturation transition is approximately the sum of the effects of the constituent ions. For all the mixed denaturants tested, the dependence of the free energy change on denaturation is linear. The conformational free energy associated with the guanidine hydrochloride denaturation transition in water is 7.5 +/- 0.1 kcal mol-1, and it is unchanged in the presence of low concentrations of lithium bromide, lithium chloride, and sodium bromide which by themselves are not concentrated enough to unfold the protein. The conformational free energy associated with the lithium bromide denaturation transition in water is 11.7 +/- 0.3 kcal mol-1, and it is not affected by the presence of low concentrations of guanidine hydrochloride which by themselves do not disrupt the structure of native ribonuclease A.

Highlights

Thedenaturation of ribonuclease A by guanidine We have reported elsewhere [3, 4] the results of the effect hydrochloride, lithium bromide, and lithium chloride of mixed denaturants
A t somethe presence olfow concentrations of lithium bromidew, hat higher concentrations of denaturant salts, we found that lithium chloride, and sodium bromide which by themt-he added denaturant cooperates withurea in unfolding, again selves are not concentrated enoughto unfold the pro- with no change in the nature of the end product of denaturtein
As we have suggested elsewhere [3], a possible explathe lithium bromide denaturation transitionin water is 11.7 f 0.3 kcal mol-’, and it is not affected by the presence of low concentrations of guanidine hydrochloride which bythemselvesdo not disruptthe structure of native ribonuclease A

Summary

Faizan Ahmad

From theDepartment of Chemistry, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2. Thedenaturation of ribonuclease A by guanidine We have reported elsewhere [3, 4] the results of the effect hydrochloride, lithium bromide, and lithium chloride of mixed denaturants (mixtures of urea and one of the denaand by mixed denaturantsconsisting of guanidinehy- turant salts LiCI,LiClO,, and CaCL). Guanidine hydrochloride, and GdnHCNS give the owing to the lack of a carbonyl oxygen in the molecule It is most extensively unfolded state which is devoid of all the expected that both components of the mixed denaelements of native structure andcause the moleculeto behave turants should enhance each other’s effect in denaturing as a cross-linked randomcoil [6, 7]. This finding seems to suggest that there is no "specific" binding site on the native RNase A for the denaturants used in thisstudy

MATERIALS AND METHODS

RESULTS

DISCUSSION