Resurrecting A Desiccation-Inactivated Enzyme

For decades, the osmolyte and disaccharide trehalose has been FDA-approved as an excipient to stabilize pharmaceuticals, including biologic drugs. The protein human serum albumin is also a useful excipient. However, their mechanisms of action remain controversial. We show that trehalose and the sucrose polymer Ficoll 70™, which both contain non-reducing linkages, protect the enzyme lactate dehydrogenase against desiccation-induced inactivation, but sugars containing reducing linkages are not protective. Trimethylamine N-oxide, but not the other osmolytes tested, shows protective properties similar to those of trehalose. We also identified both globular and disordered proteins with robust protective properties against desiccation-induced inactivation. Unlike osmolytes, these protective proteins have no known biological function in desiccation tolerance. Polyethylene glycol 3350 lacks a protective effect, suggesting that protection does not stem from simple hard-core repulsions. Of the molecules that protect lactate dehydrogenase against desiccation-induced inactivation, the proteins were more effective at protecting the desiccated enzyme against heat-induced inactivation. In addition, desiccation-inactivated lactate dehydrogenase was resurrected by re-suspension in dilute protein solutions, a phenomenon not observed with osmolytes. This finding suggests that properly folded and disordered protein crowders may facilitate refolding of unfolded or misfolded lactate dehydrogenase upon rehydration rather than protect the enzyme against desiccation-induced inactivation. The fact that proteins with no known link to desiccation tolerance protect and resurrect lactate dehydrogenase activity also suggests that these phenomena result from non-specific protein-protein interactions.