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, 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 degrees at pH 2 but only 1 degree at pH 9, where the net proton charge on the protein is near 0. The 10 degrees 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 degrees at pH 2 but only 1 degree at pH 9. The 14 degree 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.
Ramos
Carlos H I
CH
Department of Biochemistry, Beckman Center, Stanford Medical Center, Stanford, CA 94305-5307, USA. cramos@lnls.br
Baldwin
Robert L
RL
D000838:Anions / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D011485:Protein Binding; D011489:Protein Denaturation; D012259:Ribonuclease, Pancreatic / Q000378:metabolism*; D012492:Salts / Q000378:metabolism; D013431:Sulfates / Q000378:metabolism*; D013816:Thermodynamics; D014508:Urea
D000838:Anions; D002712:Chlorides; D012492:Salts; D013431:Sulfates; D014508:Urea; D012259:Ribonuclease, Pancreatic