ASSESSING THE STRUCTURAL INTEGRITY OF A LYOPHILIZED PROTEIN IN ORGANIC-SOLVENTS

The structure of a model. protein, bovine pancreatic trypsin inhibitor (BPTI), in organic solvents has been examined using hydrogen isotope exchange/high-resolution NMR methodology. When lyophilized deuterated BPTI is suspended in acetonitrile, tetrahydrofuran, ethyl acetate, or butanol, each containing 1% (H2O)-H-1, several protein amide protons t hat are buried and strongly hydrogen bonded in aqueous solution are fo und to exchange with the solvent significantly within 24 h. In contras t, in water most of these protons do not exchange appreciably even aft er a week under otherwise similar conditions. The isotope exchange rat es of the corresponding amide protons of BPTI are similar in these non aqueous solvents, as well as in acetonitrile containing methanol or bu tanol (instead of water) as a H donor. When solid BPTI is prepared by different methods, such as rotary evaporation, acetone precipitation, or lyophilization from a dimethyl sulfoxide solution, and subsequently suspended in acetonitrile containing 1% water, the exchange intensiti es of the amide protons vary greatly among the preparations. These dat a combined suggest that the structure of BPTI in the four aforemention ed organic solvents is partially unfolded, but not more so than in lyo philized powder, i.e., that these solvents cause little additional pro tein denaturation beyond that brought about by lyophilization. Using t he same methodology, the BPTI structure also has been studied in sever al protein-dissolving solvents containing 1% water. In dimethyl sulfox ide, dimethylformamide, or methanol, the same amide protons exchange a lmost completely within 24 h, while in glycerol (known to stabilize pr oteins and to function as a water mimic) they do not. These results de monstrate that some protein-dissolving organic solvents strongly denat ure BPTI and that intermolecular contacts in the suspended protein are important in maintaining the protein conformation in organic solvents . These findings, if general, explain the considerable but much reduce d (compared to water) enzymatic activity in nonaqueous media.