Protein denaturation during freezing and thawing in phosphate buffer systems: Monomeric and tetrameric beta-galactosidase

During freezing in sodium and potassium phosphate (NaP and KP) buffer solut ions, changes in pH may impact the stability of proteins. Since the degrada tion pathways for the model proteins, monomeric and tetrameric beta -galact osidase (beta -gal), chosen for this study are governed by conformational c hanges (i.e., physical instability) as opposed to chemical transformations, we explored how the stresses of freezing and thawing alter the protein's n ative structure and if preservation of the native conformation during freez e-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 obse rved 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 acti vity when freeze-thawing in NaP buffer as compared to KP buffer for both so urces of beta -gal. In addition, we investigated the effects of cooling rat e and warming rate on the recovery of activity for monomeric and tetrameric beta -gal. Optimal recovery of activity for the NaP samples was obtained w hen the processing protocol involved a fast cool/fast warm combination, whi ch minimizes exposure to acidic conditions and concentrated solutes. Altera tions in the native secondary structure of monomeric beta -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 i ndicate that pH changes during freezing in NaP buffer contribute to denatur ation of beta -gal. These results suggest that proteins formulated in NaP b uffer should be frozen and thawed rapidly to minimize exposure to low pH an d high buffer salts. (C) 2000 Academic Press.