Maintenance of Quaternary Structure in the Frozen State Stabilizes Lactate Dehydrogenase during Freeze?Drying

Maintenance of Quaternary Structure in the Frozen State Stabilizes Lactate Dehydrogenase during Freeze?Drying
Received 19 September 2000; revised 5 March 2001. Available online 12 March 2002.
Thomas J. Anchordoquya, Ken-Ichi Izutsub, a, Theodore W. Randolphc and John F. Carpentera, 1
Archives of Biochemistry and Biophysics
Volume 390, Issue 1 , 1 June 2001
a Department of Pharmaceutical Sciences, University of Colorado Health Sciences Center, Denver, Colorado, 80262
b National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagayaku, Tokyo, 158, Japan
c Department of Chemical Engineering, University of Colorado, Boulder, Colorado, 80309
Abstract
Sugars inhibit protein unfolding during the drying step of lyophilization by replacing hydrogen bonds to the protein lost upon removal of water. In many cases, polymers fail to inhibit dehydration-induced damage to proteins because steric hindrance prevents effective hydrogen bonding of the polymer to the protein's surface. However, in certain cases, polymers have been shown to stabilize multimeric enzymes during lyophilization. Here we test the hypothesis that this protection is due to inhibition of dissociation into subunits during freezing. To test this hypothesis, as a model system we used mixtures of lactate dehydrogenase isozymes that form electrophoretically distinguishable 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 polyethylene glycol), sugars (sucrose, trehalose, glucose), and surfactants (Tween 80, Brij 35, hydroxy-propyl ?-cyclodextrin). The surfactants did not protect LDH 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 freeze?drying. Polymers and sugars prevented dissociation of LDH during the freezing step of lyophilization, resulting in greater recovery of enzyme activity after lyophilization and rehydration. This beneficial effect was observed even in systems that do not form glassy solids during freezing and drying. We suggest that stabilization during drying results in part from greater inherent stability of the assembled holoenzyme relative to that of the dissociatedmonomers. Polymers inhibit freezing-induced dissociation thermodynamically because they are preferentially excluded from the surface of proteins, which increases the free energy of dissociation and denaturation.
Author Keywords: lyophilization; freeze?thawing; freeze?drying; protein stabilization; dissociation
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