Protein Denaturation during Freezing and Thawing in Phosphate Buffer Systems: Monomeric and Tetrameric β-Galactosidase

Abstract

During freezing in sodium and potassium phosphate (NaP and KP) buffer solutions, changes in pH may impact the stability of proteins. Since the degradation pathways for the model proteins, monomeric and tetrameric β-galactosidase (β-gal), chosen for this study are governed by conformational changes (i.e., physical instability) as opposed to chemical transformations, we explored how the stresses of freezing and thawing alter the protein's native structure and if preservation of the native conformation during freeze–thawing is a requisite for optimal recovery of activity. During freezing in NaP buffer, a significant pH decrease from 7.0 to as low as 3.8 was observed due to the selective precipitation of the disodium phosphate; however, the pH during freezing in KP buffer only increased by at most 0.3 pH units. pH-induced inactivation was evident as seen by the lower recovery of activity when freeze–thawing in NaP buffer as compared to KP buffer for both sources of β-gal. In addition, we investigated the effects of cooling rate and warming rate on the recovery of activity for monomeric and tetrameric β-gal. Optimal recovery of activity for the NaP samples was obtained when the processing protocol involved a fast cool/fast warm combination, which minimizes exposure to acidic conditions and concentrated solutes. Alterations in the native secondary structure of monomeric β-gal as measured by infrared spectroscopy were more significant when freezing and thawing in NaP buffer as opposed to KP buffer. Conformational and activity analyses indicate that pH changes during freezing in NaP buffer contribute to denaturation of β-gal. These results suggest that proteins formulated in NaP buffer should be frozen and thawed rapidly to minimize exposure to low pH and high buffer salts.