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

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Abstract
Data are reported for Tm, 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 Tm caused by anion binding at 0.1 M sulfate is 20° at pH 2 but only 1° at pH 9, where the net proton charge on the protein is near 0. The 10° increase in Tm 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 Tm between 0 and 1.0 M is 14° at pH 2 but only 1° at pH 9. The 14° 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.