Investigation of cosolute-protein preferential interaction coefficients: new insight into the mechanism by which arginine inhibits aggregation

Abstract

The relatively new technique of vapor pressure osmometry was utilized to determine the preferential interaction of five common solution additives (arginine HCl, guanidine HCl, glycerol, glucose, and urea) using three different model proteins (BSA, lysozyme, and alpha-chymotrypsinogen). Results for guanidine, glycerol, glucose, and urea are comparable to literature values, which utilized the dialysis/densimetry technique. However, values for arginine differ greatly from literature values, making it unclear what is the nature of arginine-protein interactions. A repeat of the dialysis/densimetry measurements reported in the literature supports the vapor pressure osmometry measurements and reveals a never before seen trend in the interaction of arginine with proteins as a function of concentration. This trend is dependent on the protein size and shows arginine to be unique among solution additives. For concentrations below 0.5 M, arginine has a preferential interaction coefficient near zero (slightly greater than zero for small proteins but decreases as the size of the protein increases), which indicates that arginine is neither strongly bound nor excluded from the protein surface. This trend differs greatly from cosolutes that influence the protein folding equilibrium. However, as the concentration of arginine increases beyond 0.5 M, arginine becomes increasingly excluded. Such behavior might be indicative of the protein surface becoming saturated with arginine, thus causing any additional arginine added to the solution to be excluded from interacting with the surface. All of this behavior is most likely the result of a balance between the affinity arginine has for the peptide backbone and certain amino acids and the repulsion generated by surface tension increment and volume exclusion effects [Arakawa et al. Biophysical Chemistry 2007, 127, 1]. In addition, such behavior may explain why arginine has little effect on protein folding equilibrium but is an effective aggregation suppressor.

Figure 1

Comparison of Arginine Γ_μ3 values from VPO measurements (▲), dialysis/densimetry measurements (△), and literature values obtained from Reference (■) for (A) BSA and (B) lysozyme.

Figure 2

Comparison of arginine preferential interaction coefficient values (□) to that for urea (○) (a preferentially bound and denaturing cosolute) and glycerol (△) (a preferentially excluded and stabilizing cosolute) for all three proteins tested, (A) lysozyme, (B) aCgn, and (C) BSA.

Figure 3

Comparison of arginine preferential interaction coefficient values for all three proteins tested demonstrating the relationship with arginine concentration and protein size; lysozyme, 14.3 kDa (●), aCgn, 25.7 kDa (■), and BSA, 66.4 kDa (▲).

Figure 4

Comparison of arginine binding (assuming A₁=0.3 g/g) for all three proteins tested demonstrating that arginine possibly saturates the surface of the protein at high concentrations leading to preferential exclusion; lysozyme, 14.3 kDa (●), aCgn, 25.7 kDa (■), and BSA, 66.4 kDa (▲).