Vapor Pressure Osmometry Studies of Osmolyte−Protein Interactions: Implications for the Action of Osmoprotectants in Vivo and for the Interpretation of “Osmotic Stress” Experiments in Vitro

Top Cited Papers
Publisher Website
Google Scholar
Abstract
To interpret or to predict the responses of biopolymer processes in vivo and in vitro to changes in solute concentration and to coupled changes in water activity (osmotic stress), a quantitative understanding of the thermodynamic consequences of interactions of solutes and water with biopolymer surfaces is required. To this end, we report isoosmolal preferential interaction coefficients (Γμ1) determined by vapor pressure osmometry (VPO) over a wide range of concentrations for interactions between native bovine serum albumin (BSA) and six small solutes. These include Escherichia coli cytoplasmic osmolytes [potassium glutamate (K+Glu-), trehalose], E. coli osmoprotectants (proline, glycine betaine), and also glycerol and trimethylamine N-oxide (TMAO). For all six solutes, Γμ1 and the corresponding dialysis preferential interaction coefficient Γμ1,μ3 (both calculated from the VPO data) are negative; Γμ1,μ3 is proportional to bulk solute molality ( ) at least up to 1 m (molal). Negative values of Γμ1,μ3 indicate preferential exclusion of these solutes from a BSA solution at dialysis equilibrium and correspond to local concentrations of these solutes in the vicinity of BSA which are lower than their bulk concentrations. Of the solutes investigated, betaine is the most excluded (Γμ1,μ3/ = −49 ± 1 m-1); glycerol is the least excluded (Γμ1,μ3/ = −10 ± 1 m-1). Between these extremes, the magnitude of Γμ1,μ3/ decreases in the order glycine betaine ≫ proline >TMAO > trehalose ≈ K+Glu- > glycerol. The order of exclusion of E. coli osmolytes from BSA surface correlates with their effectiveness as osmoprotectants, which increase the growth rate of E. coli at high external osmolality. For the most excluded solute (betaine), Γμ1,μ3 provides a minimum estimate of the hydration of native BSA of approximately 2.8 × 103 H2O/BSA, which corresponds to slightly less than a monolayer (estimated to be ∼3.2 × 103 H2O). Consequently, of the solutes investigated here, only betaine might be suitable for use in osmotic stress experiments in vitro as a direct probe to quantify changes in hydration of protein surface in biopolymer processes. More generally, however, our results and analysis lead to the proposal that any of these solutes can be used to quantify changes in water-accessible surface area (ASA) in biopolymer processes once preferential interactions of the solute with biopolymer surface are properly taken into account.