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

Abstract

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, Na 2 SO 4 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 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 T m between 0.1 and 1.0 M Na 2 SO 4 , 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 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 Na 2 SO 4 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.