COMPARISON OF SOLUTE-INDUCED PROTEIN STABILIZATION IN AQUEOUS-SOLUTION AND IN THE FROZEN AND DRIED STATES

A wide variety of solutes protect isolated proteins during freeze-thawing. These solutes come from chemically dissimilar classes, including sugars, polyols, amino acids, methylamines, and lyotropic salts. The only characteristic that these compounds have in common is that they have all been shown to be preferentially excluded from contact with the surface of proteins in aqueous solution. This interaction of solutes with proteins leads to the stabilization of proteins in nonfrozen aqueous systems. Conversely, those solutes, e.g., urea and guanidine HCl, that bind preferentially with proteins destabilize proteins in solution, and we found that they also enhance the inactivation of enzymes during freeze-thawing. Based on the results of freeze-thawing experiments with lactate dehydrogenase and phosphofructokinase and a review of the theory of protein stabilization in nonfrozen, aqueous solution, we demonstrated that the cryoprotection afforded to isolated proteins by solutes can be accounted for by the fact that these solutes are preferentially excluded from contact with the protein's surface. In contrast, carbohydrate-induced stabilization of labile enzymes during freeze-drying appears to be dependent on hydrogen bonding of the carbohydrate to the dried protein. Using Fourier transform infrared spectroscopy, we characterized this interaction and found that under conditions in which the carbohydrate did not hydrogen bond to the protein, no protein stabilization occurred. Thus, for freeze-dried systems, we conclude that certain carbohydrates provide protection to labile enzymes because these solutes serve as water substitutes for the dried protein by satisfying the hydrogen bonding requirements of polar groups on the protein's surface.