Roman V. Rariy

Protein refolding in predominantly organic media markedly enhanced by common salts

The refolding/reoxidation of unfolded/reduced hen egg-white lysozyme was investigated in a variety of predominantly nonaqueous media consisting of protein-dissolving organic solvents and water. It was discovered that LiCl and other common salts dramatically (up to more than 100-fold) increased the refolding yield of lysozyme in such nonaqueous systems, while reducing it in water. The mechanism of this surprising phenomenon appears to involve salt-induced suppression of nonspecific lysozyme aggregation during refolding due to an enhanced protein solubility.

Calorimetric evidence for a native-like conformation of hen egg-white lysozyme dissolved in glycerol.

Hen egg-white lysozyme, lyophilized from aqueous solutions of different pH (from pH 2.5 to 10.0) and then dissolved in water and in anhydrous glycerol, has been studied by high-sensitivity differential scanning microcalorimetry over the temperature range from 10 to 150 degrees C. All lysozyme samples exhibit a cooperative conformational transition in both solvents occurring between 10 and 100 degrees C. The transition temperatures in glycerol are similar to those in water at the corresponding pHs. The transition enthalpies in glycerol are substantially lower than in water but follow similar pH dependences. The transition heat capacity increment in glycerol does not depend on the pH and is 1.25+/-0.31 kJ mol(-1) K(-1), which is less than one fifth of that in water (6. 72+/-0.23 kJ mol(-1) K(-1)). The thermal transition in glycerol is reversible and equilibrium, as demonstrated for the pH 8.0 sample, and follows the classical two-state mechanism. In contrast to lysozyme in water, the protein dissolved in glycerol undergoes an additional, irreversible cooperative transition with a marginal endothermic heat effect at temperatures of 120-130 degrees C. The transition temperature of this second transition increases with the heating rate which is characteristic of kinetically controlled processes. Thermodynamic analysis of the calorimetric data reveals that the stability of the folded conformation of lysozyme in glycerol is similar to that in water at 20-80 degrees C but exceeds it at lower and higher temperatures. It is hypothesized that the thermal unfolding in glycerol follows the scheme: N ifho-MG-->U, where N is a native-like conformation, ho-MG is a highly ordered molten globule state, and U is the unfolded state of the protein.

On the Relationship Between Enzymatic Enantioselectivity in Organic Solvents and Enzyme Flexibility

The enantioselectivities of subtilisin Carlsberg and α-chymotrypsin suspended in seven anhydrous solvents were determined in the acylation of sec-phenethyl alcohol with vinyl butyrate. In every case, both a dry lyophilized enzyme and one hydrated by a prior exposure to a humid environment were used as asymmetric catalysts in a given solvent. This hydration was found to boost the water content of the enzyme which, in turn, is known to enhance the enzymes conformational flexibility. These data together resulted in the following correlation: as enzyme flexibility increases, the enantioselectivity decreases or remains the same. This relationship is opposite to that recently claimed for subtilisin by Broos et al. (Broos, J., Visser, A.J.W.G., Engbersen, J.F.J., Verboom, M., van Hock, A. and Reinhoudt, D.N. (1995) J. Am. Chem. Soc., 117, 12657–12663). The basis for their conclusion is critically analyzed herein and found to be questionable.

COMPUTATIONAL AND EXPERIMENTAL EXAMINATION OF THE EFFECT OF INORGANIC SALTS ON CHYMOTRYPTIC ENANTIOSELECTIVITY IN WATER

Enantioselectivity (E) of α-chymotrypsin in the hydrolysis of tropic (3-hydroxy-2-phenylpro-pionic) acid methyl ester in water was found to be 31 in favor of the R enantiomer. Addition of 1.5 M ammonium or sodium sulfate resulted in a nearly 3-fold drop in the E value. In contrast, thermodynamic calculations predict a 30-40% increase in E. Importantly, these calculations are based on the assumption that the solvent-dependent differences in the desolvation energetics of substrate enantiomers upon formation of their enzyme-bound transition states is the dominant factor by which the solvent affects enzymatic enantioselectivity. Therefore this assumption, validated previously for enzymatic transesterifications in organic solvents and herein for enzymatic hydrolysis in water, does not apply to concentrated aqueous salt solutions. It is hypothesized instead that the sulfate salts may alter the conformation of chymotrypsin. However, the fluorescence and UV absorbance spectra of the enzyme in water and in 1.5 M a...