POLYMERS PROTECT LACTATE-DEHYDROGENASE DURING FREEZE-DRYING BY INHIBITING DISSOCIATION IN THE FROZEN STATE

Enzymes subjected to freeze-thawing are known to be protected by polym ers that are preferentially excluded from the hydrated surface of prot eins [reviewed in Carpenter et al. (1994) ACS Symp. Ser. 567, 134-147] . Preferentially excluded solutes are also known to stabilize quaterna ry structure, which enhances the thermostability of multimeric protein s in aqueous systems. Also, it has been suggested that retention of qu aternary structure may play a role in the protection of multimeric pro teins by polymers during freeze-drying (lyophilization). Although pref erential solute exclusion cannot occur in the absence of water, we rea soned that polymers could protect multimeric proteins during freeze-dr ying by stabilizing quaternary structure in the frozen state. Our resu lts are consistent with this hypothesis and demonstrate that bovine se rum albumin and polyvinylpyrrolidone stabilize lactate dehydrogenase b y inhibiting dissociation in the frozen solution, during the initial p hase of the sublimation step of lyophilization. Dissociation at this c ritical step correlated directly with decreased recovery of enzyme act ivity after rehydration. The damage to the protein, under conditions w here dissociation was studied, was due to a large decrease in pH in th e frozen state (e.g., from pH 7.5 to 4.5), which was attenuated by pro tective levels of polymers. Thus, inhibition of freezing-induced pH sh ifts, in addition to stabilization by the preferential exclusion mecha nism, plays an important role in the protection conferred by polymers. Furthermore, high concentrations of these polymers were capable of ma intaining quaternary structure during subsequent drying and rehydratio n. We suggest that the proximate cause for increased recovery of activ e, native protein after lyophilization is that the holoenzyme is more resistant to the stresses of drying/rehydration than unassociated mono mers. (C) 1996 Academic Press, Inc.