Maintenance of quaternary structure in the frozen state stabilizes lactatedehydrogenase during freeze-drying

Sugars inhibit protein unfolding during the drying step of lyophilization b y replacing hydrogen bonds to the protein lost upon removal of water. In ma ny cases, polymers fail to inhibit dehydration-induced damage to proteins b ecause steric hindrance prevents effective hydrogen bonding of the polymer to the protein's surface. However, in certain cases, polymers have been sho wn to stabilize multimeric enzymes during lyophilization. Here we test the hypothesis that this protection is due to inhibition of dissociation into s ubunits during freezing. To test this hypothesis, as a model system we used mixtures of lactate dehydrogenase isozymes that form electrophoretically d istinguishable hybrid tetramers during reversible dissociation. We examined hybridization and recovery of catalytic activity during freeze-thawing and freeze-drying in the presence of polymers (dextran, Ficoll, and polyethyle ne glycol), sugars (sucrose, trehalose, glucose), and surfactants (Tween 80 , Brij 35, hydroxypropyl p-cyclodextrin). The surfactants did not protect L DH during freeze-thawing or freeze-drying. Rather, in the presence of Brij 35, enhanced damage was seen during both freeze-thawing and freeze-drying, and the presence of Tween 80 exacerbated loss of active protein during free ze-drying. Polymers and sugars prevented dissociation of LDH during the fre ezing step of lyophilization, resulting in greater recovery of enzyme activ ity after lyophilization and rehydration. This beneficial effect was observ ed even in systems that do not form glassy solids during freezing and dryin g. We suggest that stabilization during drying results in part from greater inherent stability of the assembled holoenzyme relative to that of the dis sociated monomers. Polymers inhibit freezing- induced dissociation thermody namically because they are preferentially excluded from the surface of prot eins, which increases the free energy of dissociation and denaturation. (C) 2001 Academic Press.