The preferential interactions of bovine serum albumin, lysozyme, chymotrypsinogen, ribonuclease A, and 6-lactoglobulin with polyethylene glycols (PEGS) of molecular weight 200-6,OOO have been measured by dialysis equilibrium coupled with high precision densimetry. All the proteins were found to be preferentially hydrated in all the PEGs, and the magnitude of the preferential hydration increased with increasing PEG size for each protein.The change in the chemical potentials of the proteins with the addition of the PEGs had highly positive values, indicating a strong thermodynamic destabilization of the system by the PEGs. A viscosity study of the PEGs showed them to be randomly coiled polymers, as their radii of gyration were related to the molecular weight by R, = a M 0 , 5 s . The thickness of the effective shell impenetrable to PEG around protein molecules, calculated from the preferential hydration, was found to vary with PEG molecular weight in similar fashion as the PEG radius of gyration, supporting the proposal (Arakawa, T. & Timasheff, S.N., 1985a, Biochemistry 24,6756-6762) that the preferential exclusion of PEGs from proteins is due principally to the steric exclusion of PEG from the protein domain, although favorable interactions with protein surface residues, in particular nonpolar ones, may compete with the exclusion. These thermodynamically unfavorable preferential exclusion interactions lead to the action of PEGs as precipitants, although they may destabilize protein structure at higher temperatures.

Trehalose, a naturally occurring osmolyte, is known to be an exceptional stabilizer of proteins and helps retain the activity of enzymes in solution as well as in the freeze-dried state. To understand the mechanism of action of trehalose in detail, we have conducted a thorough investigation of its effect on the thermal stability in aqueous solutions of five well characterized proteins differing in their various physico-chemical properties. Among them, RNase A has been used as a model enzyme to investigate the effect of trehalose on the retention of enzymatic activity upon incubation at high temperatures. 2 M trehalose was observed to raise the transition temperature, T m of RNase A by as much as 18°C and Gibbs free energy by 4.8 kcal mol ؊1 at pH 2.5. There is a decrease in the heat capacity of protein denaturation (⌬C p ) in trehalose solutions for all the studied proteins. An increase in the ⌬G and a decrease in the ⌬C p values for all the proteins points toward a general mechanism of stabilization due to the elevation and broadening of the stability curve (⌬G versus T). A direct correlation of the surface tension of trehalose solutions and the thermal stability of various proteins has been observed. Wyman linkage analysis indicates that at 1.5 M concentration 4 -7 molecules of trehalose are excluded from the vicinity of protein molecules upon denaturation. We further show that an increase in the stability of proteins in the presence of trehalose depends upon the length of the polypeptide chain. The pH dependence data suggest that even though the charge status of a protein contributes significantly, trehalose can be expected to work as a universal stabilizer of protein conformation due to its exceptional effect on the structure and properties of solvent water compared with other sugars and polyols.Sugars have been known to protect proteins against loss of activity (1, 2), chemical (3, 4), and thermal denaturation (5-9). Among several sugars, ␣,␣-trehalose (␣-D-glucopyranosyl(131)-␣-D-glucopyranoside) has been known to be a superior stabilizer in providing protection to biological materials against dehydration and desiccation (10, 11). It is a compatible osmolyte that gets accumulated in organisms under stress conditions (12, 13). Because of this unique property, tremendous interest has been generated in understanding the molecular basis of stress management through induction of trehalose biosynthesis (13, 14).Trehalose has also been found to be very effective in the stabilization of labile proteins during lyophilization (15, 16) and exposure to high temperatures in solution (2,8,9). Sugars in general protect proteins against dehydration by hydrogen bonding to the dried protein by serving as water substitute (15, 17). Several studies carried out by Timasheff and coworkers (9,18) show that sugars and polyols stabilize the folded structure of proteins in solution as a result of greater preferential hydration of the unfolded state compared with the native state. The mechanism is fundamentally different from stabil...

The correlation between protein solubility and the preferential interactions of proteins with solvent components was critically examined with aqueous MgCl2 as the solvent system. Preferential interaction and solubility measurements with three proteins, beta-lactoglobulin, bovine serum albumin, and lysozyme, resulted in similar patterns of interaction. At acid pH (pH 2-3) and lower salt concentrations (less than 2 M), the proteins were preferentially hydrated, while at higher salt concentrations, the interaction was either that of preferential salt binding or low salt exclusion. At pH 4.5-5, all three proteins exhibited either very low preferential hydration or preferential binding of MgCl2. These results were analyzed in terms of the balance between salt binding and salt exclusion attributed to the increase in the surface tension of water by salts, which is invariant with conditions. It was shown that the increase in salt binding at high salt concentration is a reflection of mass action, while its decrease at acid pH is due to the electrostatic repulsion between Mg2+ ions and the high net positive charge on the protein. The preferential interaction pattern was paralleled by the variation of protein solubility with solvent conditions. Calculation of the transfer free energies from water to the salt solutions for proteins in solution and in the precipitate showed dependencies on salt concentration. This indicates that the nature of interactions between proteins and solvent components is the same in solution and in the solid state, which implies no change in protein structure during precipitation. Analysis of the transfer free energies and preferential interaction parameter in terms of the salting-in, salting-out, and weak ion binding contributions has led to the conclusions that, when the weak ion binding contribution is small, the predominant protein-salt interaction must be that of preferential salt exclusion most probably caused by the increase of the surface tension of water by addition of the salt. A necessary consequence of this is salting-out of the protein, if the protein structure is to remain unaltered.

BackgroundAggregation of unfolded proteins occurs mainly through the exposed hydrophobic surfaces. Any mechanism of inhibition of this aggregation should explain the prevention of these hydrophobic interactions. Though arginine is prevalently used as an aggregation suppressor, its mechanism of action is not clearly understood. We propose a mechanism based on the hydrophobic interactions of arginine.MethodologyWe have analyzed arginine solution for its hydrotropic effect by pyrene solubility and the presence of hydrophobic environment by 1-anilino-8-naphthalene sulfonic acid fluorescence. Mass spectroscopic analyses show that arginine forms molecular clusters in the gas phase and the cluster composition is dependent on the solution conditions. Light scattering studies indicate that arginine exists as clusters in solution. In the presence of arginine, the reverse phase chromatographic elution profile of Alzheimer's amyloid beta 1-42 (Aβ1-42) peptide is modified. Changes in the hydrodynamic volume of Aβ1-42 in the presence of arginine measured by size exclusion chromatography show that arginine binds to Aβ1-42. Arginine increases the solubility of Aβ1-42 peptide in aqueous medium. It decreases the aggregation of Aβ1-42 as observed by atomic force microscopy.ConclusionsBased on our experimental results we propose that molecular clusters of arginine in aqueous solutions display a hydrophobic surface by the alignment of its three methylene groups. The hydrophobic surfaces present on the proteins interact with the hydrophobic surface presented by the arginine clusters. The masking of hydrophobic surface inhibits protein-protein aggregation. This mechanism is also responsible for the hydrotropic effect of arginine on various compounds. It is also explained why other amino acids fail to inhibit the protein aggregation.

The observed preferential hydration of proteins in aqueous MgCl2 solutions at low pH and low salt concentration (Arakawa et al., 1990) prompted a scrutiny of possible protein stabilization by MgCl2 under the same conditions, in view of earlier observations in aqueous solutions of sugars, amino acids, and a number of salts that preferential hydration is usually accompanied by the stabilization of the native structure of globular proteins. The results of thermal transition experiments on five proteins (ribonuclease A, lysozyme, beta-lactoglobulin, chymotrypsinogen, and bovine serum albumin) revealed neither significant stabilization nor destabilization of the protein structures by MgCl2 both at acid conditions (except for ribonuclease A, which was stabilized, but to a much smaller extent than by MgSO4) and at higher pH at which MgCl2 displayed little preferential hydration. This was in contrast to the great stabilizing action of MgSO4 at the same conditions. 2-Methyl-2,4-pentanediol (MPD), which gives a very large preferential hydration of native ribonuclease A at pH 5.8 [Pittz & Timasheff (1978) Biochemistry 17, 615-623], was found to be a strong destabilizer of that protein at the same conditions. Analysis of the preferentially hydrating solvent systems led to their classification into two categories: those in which the preferential hydration is independent of solution conditions and those in which it varies with conditions. The first always stabilize protein structure, while the second do not. In the first category the predominant interaction is that of cosolvent exclusion, determined by solvent properties, with the protein being essentially inert. In the second category interactions are determined to a major extent by the chemical nature of the protein surface.(ABSTRACT TRUNCATED AT 250 WORDS)