A mechanistic analysis of the increase in the thermal stability of proteins in aqueous carboxylic acid salt solutions

Abstract

The stability of proteins is known to be affected significantly in the presence of high concentration of salts and is highly pH dependent. Extensive studies have been carried out on the stability of proteins in the presence of simple electrolytes and evaluated in terms of preferential interactions and increase in the surface tension of the medium. We have carried out an in-depth study of the effects of a series of carboxylic acid salts: ethylene diamine tetra acetate, butane tetra carboxylate, propane tricarballylate, citrate, succinate, tartarate, malonate, and gluconate on the thermal stability of five different proteins that vary in their physico-chemical properties: RNase A, cytochrome c, trypsin inhibitor, myoglobin, and lysozyme. Surface tension measurements of aqueous solutions of the salts indicate an increase in the surface tension of the medium that is very strongly correlated with the increase in the thermal stability of proteins. There is also a linear correlation of the increase in thermal stability with the number of carboxylic groups in the salt. Thermal stability has been found to increase by as much as 22 8C at 1 M concentration of salt. Such a high thermal stability at identical concentrations has not been reported before. The differences in the heat capacities of denaturation, DC p for RNase A, deduced from the transition curves obtained in the presence of varying concentrations of GdmCl and that of carboxylic acid salts as a function of pH, indicate that the nature of the solvent medium and its interactions with the two end states of the protein control the thermodynamics of protein denaturation. Among the physico-chemical properties of proteins, there seems to be an interplay of the hydrophobic and electrostatic interactions that lead to an overall stabilizing effect. Increase in surface free energy of the solvent medium upon addition of the carboxylic acid salts appears to be the dominant factor in governing the thermal stability of proteins.

Stabilization of Protein Structure in Freeze-Dried Amorphous Organic Acid Buffer Salts

“…In addition, the lower effective concentration of monosodium citrate compared to sucrose suggested the contribution of electrostatic (ion-ion, ion-dipole) interactions between the salt anion (hydroxycarboxylate ion) and basic amino acid residues on protein surfaces for the structural stabilization. 17,36,37) The high T g of the dried solids indicated mixing of the protein and salts required for the interaction. The topic of protein-stabilizing molecular interactions in the dried states, including the effect of differently ionized functional groups, will require further study for elucidation.…”

“…The citrate 3Ϫ and L-tartrate 2Ϫ ions are 'kosmotropic' anions that thermodynamically stabilize native protein structures by being preferentially excluded from the immediate surface of the protein molecules. 16,17,22,[38][39][40][41] In other words, the proteins are preferentially hydrated in the solute solutions.…”

“…Solution pH and anion structure are major factors that determine the thermal stability of proteins in aqueous carboxylic acid salt solutions. 17) Some di-and tricarboxylic acid salts protect proteins from thermal denaturation. The number of carboxyl and hydroxyl groups should is also likely to be important in protecting the secondary structure of BSA through the water-substituting hydrogen bonds and electrostatic interactions against the dehydration stress.…”

“…6) Some buffer components also favorably or adversely affect the protein stability through direct interactions and/or through changing the local environments in the dried state. For example, freezing of certain buffer systems (e.g., sodium phosphate) often induces crystallization of a component salt and resulting shift of the local pH surrounding the proteins.7-11) Freeze-drying from some buffer systems (e.g., L-histidine, citrate, or Tris) often leads to higher activity retention of proteins (e.g., coagulation factor VIII, recombinant human interleukin-1 receptor antagonist) relative to those from other buffers.12-15) Conformation of the proteins lyophilized in these buffer systems is of particular interest.Reported properties of some carboxylic acid salts, including stabilization of native protein conformation in aqueous solutions (e.g., antithrombin III) 16,17) and their propensity to form glass-state amorphous solids upon lyophilization, 18) suggest their ability to protect protein conformation against dehydration stress through mechanisms similar to disaccharides. Non-reducing disaccharides (e.g., sucrose, trehalose) are popular stabilizers in solution and freeze-dried protein formulations.…”

“…Reported properties of some carboxylic acid salts, including stabilization of native protein conformation in aqueous solutions (e.g., antithrombin III) 16,17) and their propensity to form glass-state amorphous solids upon lyophilization, 18) suggest their ability to protect protein conformation against dehydration stress through mechanisms similar to disaccharides. Non-reducing disaccharides (e.g., sucrose, trehalose) are popular stabilizers in solution and freeze-dried protein formulations.…”