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

yophilization (freeze-drying) is a common method for preparing protein samples for long-term storage. However, the freezing and drying processes themselves can cause protein denaturation. Preservation of protein structure and function during lyophilization requires stabilization against both freezing and drying with the appropriate additives, which protect proteins during these stresses by distinctly different mechanisms. Protection during freezing occurs by the same mechanism as that for thermodynamic stabilization of the native protein structure in unfrozen solution, i.e., preferential solute exclusion. As described by Timasheff and his colleagues, additives that are preferentially excluded from the protein surface increase the chemical potential of the protein; the magnitudes of both effects are directly proportional to the protein surface area. Because protein unfolding increases protein surface area, in the presence of preferentially excluded solutes the free energy of unfolding is increased. Preferentially excluded solutes can also stabilize quaternary structure because the combined surface area of the dissociated subunits is greater than that for the fully assembled protein. Further, considering that holoenzymes are known to be more stable than dissociated subunits, preferentially excluded solutes could potentially provide additional stabilization of multimeric proteins by maintaining the fully assembled state.
The preferential solute exclusion mechanism does not apply to protection of proteins during dehydration. Rather, effective stabilizers such as sugars inhibit dehydration- induced protein unfolding by replacing water and hydrogen bonding directly to the dried protein. In contrast, polymeric additives (e.g., polyvinylpyrrolidone, dextran, polyethylene glycol) are sterically hindered from effectively hydrogen bonding with the surface of the dried protein and are unable to inhibit directly dehydration-induced protein unfolding. Furthermore, surfactants are a class of stabilizers that do not protect via the preferential exclusion mechanism or hydrogen bonding. In an apparent contradiction to this observation, Hellman et al. reported that polyvinylpyrrolidone did protect multimeric as paraginase during lyophilization. This result suggested that polymers protect proteins during drying by a method other than water replacement. Hellman et al. employed a sodium phosphate buffer system that is known to acidify dramatically during freezing due to the precipitation of Na2HPO4. More recently, employing lactate dehydrogenase (LDH) 2 as a model multimeric protein, we demonstrated that this acidification promotes enzyme dissociation in the frozen state, which results in further irreversible denaturation during subsequent drying. Furthermore, our previous work shows that polymers (e.g., polyvinylpyrrolidone) form glasses that prevent buffer acidification and the consequent dissociation during freezing. Therefore, the observed increase in recovery after lyophilization resulted from the ability of polymers to prevent acidification and preserve quaternary structure in the frozen state .In addition, we hypothesized that preferential exclusion of polymers from the surface of LDHs also fosters maintenance of quaternary structure in the frozen state. Accordingly, preferentially excluded solutes (e.g., polymers, sugars) should prevent freezing-induced dissociation, which occurs in the absence of buffer acidification; and as a result, protein stability during subsequent drying should be increased. To test this hypothesis, in the current study we employ two electrophoretically distinct isoforms of LDH dissolved in a potassium phosphate–KI buffer system that fosters dissociation but does not acidify during freezing .Our experiments monitor quaternary structure and enzyme activity during different stages of the freezing– drying process to assess the ability of a range of additives (sugars, polymers, and surfactants) to prevent enzyme dissociation and preserve LDH activity.