Sulfate anion stabilization of native ribonuclease A both by anion binding and by the Hofmeister effect

Data are reported for T-m, the temperature midpoint of the thermal unfolding curve, of ribonuclease A, versus pH (range 2-9) and salt concentration (range 0-1 M) for two salts, Na2SO4 and NaCl. The results show stabilization by sulfate via anion-specific binding in the concentration range 0-0.1 M and via the Hofmeister effect in the concentration range 0.1-1.0 M. The increase in T-m caused by anion binding at 0.1 M sulfate is 20degrees at pH 2 but only 1degrees at pH 9, where the net proton charge on the protein is near 0. The 10degrees increase in T-m between 0.1 and 1.0 M Na2SO4, caused by the Hofmeister effect, is independent of pH. A striking property of the NaCl results is the absence of any significant stabilization by 0.1 M NaCl, which indicates that any Debye screening is small. pH-dependent stabilization is produced by 1 M NaCl: the increase in T-m between 0 and 1.0 M is 14degrees at pH 2 but only 1degrees at pH 9. The 14degrees increase at pH 2 may result from anion binding or from both binding and Debye screening. Taken together, the results for Na2SO4 and NaCl show that native ribonuclease A is stabilized at low pH in the same manner as molten globule forms of cytochrome c and apomyoglobin, which are stabilized at low pH by low concentrations of sulfate but only by high concentrations of chloride.