The aggregation behavior of two model proteins- i) bovine serum albumin (BSA) and ii) β-galactosidase (β-gal), was investigated by micro-flow imaging (MFI) during freeze-thaw cycling in phosphate buffered solutions. The pH shift was measured upon cooling the solutions from 20 to −25 °C. When the buffer concentration was 100 mM, cooling caused a pH decrease of 3.1 and 2.7 units (for BSA and β-gal, respectively) attributed to selective crystallization of disodium hydrogen phosphate as a dodecahydrate. The crystallizing solute phase was characterized by low temperature powder X-ray diffractometry. The pH shift resulted in protein aggregation, evident from the pronounced increase in particle count (by MFI). The addition of cellobiose attenuated the pH shift on cooling (pH decrease of ~1.0 unit), and no evidence of either buffer salt crystallization or protein aggregation was observed. Decreasing the buffer concentration to 10 mM, also prevented protein aggregation. The protein, by inhibiting buffer crystallization, prevented the pH shift and then the buffer, by maintaining the pH, enhanced protein stability.
Bibliographical noteFunding Information:
The project was partially supported by the William and Mildred Peters endowment fund . Parts of this work were carried out at the Characterization Facility, University of Minnesota , which received partial support from NSF through MRSEC program. Special thanks to Bio-Techne for providing access to their MFI 5200.
© 2020 Elsevier B.V.
Copyright 2020 Elsevier B.V., All rights reserved.
- pH shift
PubMed: MeSH publication types
- Journal Article
- Research Support, Non-U.S. Gov't
- Research Support, U.S. Gov't, Non-P.H.S.