Separation of freezing- and drying-induced denaturation of lyophilized proteins using stress-specific stabilization. II. Structural studies using infrared spectroscopy

Abstract

The conformation of two labile enzymes, lactate dehydrogenase and phosphofructokinase, has been examined in the aqueous and lyophilized states, using infrared spectroscopy. In the preceding paper it was demonstrated that a stress-specific stabilization scheme, which employs a combination of a cryoprotectant (polyethylene glycol) and a compound which protects the dried protein (sugars or mannitol), can be used to optimize recovery of activity of these enzymes upon freeze-drying and rehydration. The purpose of the present study is to determine the effects of these additives on the conformation of these enzymes during lyophilization. Lyophilization in the absence of stabilizers was observed to induce significant conformational changes in both enzymes. Addition of 10 mM mannitol, lactose, or trehalose or 1% polyethylene glycol to the enzyme solutions attenuated the unfolding, but significant spectral differences for the enzymes in the dried state are still observed when compared to the aqueous conformation. Addition of any one of these stabilizers does not improve recovery of activity. However, when a combination of 1% PEG and either 10 mM mannitol, lactose, or trehalose is added, the native structure is preserved during lyophilization and essentially full enzymatic activity is recovered upon reconstitution. The ability of the stabilizers to preserve the native structure during lyophilization correlates directly with the recovery of enzymatic activity upon reconstitution. It appears that for labile proteins, preservation of the native structure during lyophilization is requisite for recovery of activity following rehydration. This study demonstrates that the infrared spectroscopic technique is a rapid and useful method for studying protein conformation in the dried state and can aid in determining the optimal conditions for stabilization of proteins during lyophilization.