ASSESSING THE STRUCTURAL INTEGRITY OF A LYOPHILIZED PROTEIN IN ORGANIC SOLVENTS

摘要

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 that are buried and strongly hydrogen bonded in aqueous solution are found to exchange with the solvent significantly within 24 h. In contrast, in water most of these protons do not exchange appreciably even after a week under otherwise similar conditions. The isotope exchange rates of the corresponding amide protons of BPTI are similar in these nonaqueous solvents, as well as in acetonitrile containing methanol or butanol (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 intensities of the amide protons vary greatly among the preparations. These data combined suggest that the structure of BPTI in the four aforementioned organic solvents is partially unfolded, but not more so than in lyophilized powder, i.e., that these solvents cause little additional protein denaturation beyond that brought about by lyophilization. Using the same methodology, the BPTI structure also has been studied in several protein-dissolving solvents containing 1% water. In dimethyl sulfoxide, dimethylformamide, or methanol, the same amide protons exchange almost completely within 24 h, while in glycerol (known to stabilize proteins and to function as a water mimic) they do not. These results demonstrate that some protein-dissolving organic solvents strongly denature 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 reduced (compared to water) enzymatic activity in nonaqueous media. [References: 56]