PROTEIN-STRUCTURE IN THE LYOPHILIZED STATE - A HYDROGEN ISOTOPE-EXCHANGE NMR-STUDY WITH BOVINE PANCREATIC TRYPSIN-INHIBITOR

The structure of a stable model protein, bovine pancreatic trypsin inh ibitor (BPTI), in the lyophilized form has been investigated using the hydrogen isotope exchange/high-resolution NMR methodology. Six amide protons of BPTI that are buried in the protein interior and strongly h ydrogen-bonded in aqueous solution are found to exchange with water va pors within hours in the lyophilized state; in aqueous solution, most of these protons do not exchange appreciably even after a week under o therwise identical conditions. When BPTI is lyophilized in the presenc e of the lyoprotectant sorbitol, no significant hydrogen isotope excha nge of these protons in the solid state is detected. On the basis of t hese and other observations it is concluded that the structure of BPTI is partially (and reversibly) denatured on lyophilization. This concl usion, if true for other proteins, may explain the drastically reduced enzymatic activity in nonaqueous media compared to that in water.