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

¹ Department of Biochemistry, Beckman Center, Stanford Medical Center, Stanford, CA 94305-5307, USA
² Centro de Biologia Molecular Estrutural, Laboratório Nacional de Luz Síncrotron, CP 6192, Campinas SP, 13084–971, Brazil

Reprint requests to: Carlos H.I. Ramos, Centro de Biologia Molecular Estrutural, Laboratório Nacional de Luz Síncrotron, CP 6192, Campinas SP, 13084–971, Brazil; e-mail: cramos{at}lnls.br; fax: (55)19-3287-7110.

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 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 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 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.

Keywords: Anion-specific binding; protein stabilization; net positive charge; ribonuclease A; thermal unfolding; Hofmeister effect

Abbreviations: Tm, temperature midpoint of thermal unfolding curve • G, unfolding free energy • Go, G at zero urea molarity • m, slope of curve of G versus urea molarity • Cm, urea molarity at midpoint of unfolding transition • GdmCl, guanidinium chloride • MES, 4-morpholinoethane sulfonic acid • MOPS, 3—(N-morpholino)propanesulfonic acid • TRIS, Tris(hydroxymethyl)aminomethane

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