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.Keywords: Anion-specific binding; protein stabilization; net positive charge; ribonuclease A; thermal unfolding; Hofmeister effect Specific anions induce the formation of molten globule forms from acid-unfolded cytochrome c and apomyoglobin (Goto et al. 1990). When various anions are ranked by their effectiveness in inducing molten globules according to the anion concentration needed, the same anion series is found for both cytochrome c and apomyoglobin. Today, specific anion stabilization of molten globules at low pH is considered to be a characteristic property of molten globules. The question then arises whether specific anion stabilization is a property only of molten globules or applies also to native proteins (see also Makhatadze et al. 1998;Bedell et al. 2000;Sakurai et al. 2001).The mechanism by which specific anions stabilize molten globules is reduction of the net positive charge on the protein through anion binding. At pH 2, the basic residues (Lys, His, Arg) of molten globules are fully ionized whereas most of the acidic groups (Asp, Glu) are protonated. The resulting net positive charge on the protein is strongly destabilizing, according to unfolding experiments in which the net charge is varied. Goto and Nishikiori (1991) carried out partial acetylation of the lysine amino groups of cytochrome c and separated the partially reacted species by net charge, using electrofocusing. Thermal unfolding experiments on the separated species at pH 1.8 revealed a strong dependence of molten globule stability on net charge. Later work showed a linear relation between unfolding free energy and the Reprint requests to: Carlos H.I. Ramos, Centro de Biologia Molecular Estrutural, Laboratório Nacional de Luz Síncrotron, CP 6192, Campin...

Antibody Solubility Behavior in Monovalent Salt Solutions Reveals Specific Anion Effects at Low Ionic Strength

“…The reason is that Debye screening simply treats different monovalent ions equally as a single point charge. 9 Therefore, our observation suggests that the monovalent anions display specific interactions with the antibody. Namely, SCN − has the strongest binding with the antibody and most effectively decreases the Q * , weakens the repulsive electrostatic interactions, and reduces the antibody solubility, followed by Cl − and F − .…”

“…Although the general trend of antibody solubility drop by salts may be explained by Debye screening 9 (salt ions screen the repulsive electrostatic interactions), it does not explain the observed anion specificity for reducing the antibody solubility at the ionic strength below approximately 50 mM. The reason is that Debye screening simply treats different monovalent ions equally as a single point charge.…”

“…The Hofmeister series effects are typically used for explaining protein solution behavior at moderate-to-high (>0.3 M) salt concentration when the repulsive electrostatic intermolecular interactions are completely screened. 4,9,10 We chose the above three anions because they follow the order of F − > Cl − > SCN − for precipitating proteins according to the (direct) Hofmeister series. [4][5][6][7][8] Although the exact interaction mechanisms of the electrolyte ions with proteins remain unclear, [11][12][13][14] the above three monovalent anions should perturb solubility of the antibody as a function of pH and ionic strength because they rank from the strongly hydrated F − to the weakly hydrated SCN − .…”

Stability of Protein Pharmaceuticals: An Update

“…For example, sulfate appears to bind to endostatin (429) and ribonuclease (430). Ribonuclease has also been shown to bind two moles of chloride ion, resulting in significant structural stabilization (∼2-3 kJ/mol) (431).…”

Ion‐specific modulation of protein interactions: Anion‐induced, reversible oligomerization of a fusion protein

“…Ions have been observed to impact protein stability, 3 fibril formation, [4][5][6] reversible self-association, [7][8][9] and even protein solution rheology. 10,11 An ion-specific dependence is often discussed in terms of the Hofmeister effect or the electroselectivity series; [3][4][5][12][13][14] however, the exact nature of ion-protein interactions remains unresolved. The classical view holds that such interactions are mediated indirectly through the alteration of bulk water structure, 15 although there is increasing recognition that ions interact directly with proteins.…”