Reversible Self-Association in Lactate Dehydrogenase during Freeze-Thaw in Buffered Solutions using Neutron Scattering

Published

October 28, 2021

Author(s)

Jayesh Sonje, Seema Thakral, Susan T. Krueger, Raj Suryanarayanan

Abstract

The effect of freezing and thawing stresses on lactate dehydrogenase (LDH) stability were evaluated under three conditions: (i) in a solution buffered with sodium phosphate (NaP),where the selective crystallization of disodium hydrogen phosphate during freezing caused a pronounced pH shift, (ii) in a solution buffered with histidine, where there was no pH shift due to buffer salt crystallization, (iii) at different concentrations of LDH so as to determine the self-stabilizing ability of LDH. The change in LDH tetrameric conformation was measured by small-angle neutron scattering (SANS). The pH of the phosphate buffer solutions was monitored as a function of temperature to quantify the pH shift. Low temperature X-ray diffractometry of these solutions enabled us to identify the conditions of buffer component crystallization from solution. Finally, dynamic light scattering (DLS) enabled us to determine the effect of freeze-thawing on the protein aggregation behavior. LDH, at a high concentration (1000 µg/mL), has a pronounced self-stabilizing effect and did not aggregate after 5 freeze-thaw cycles. At lower LDH concentrations (10 and 100 µg/mL), irreversible aggregation could be avoided only with the selection of an appropriate buffer. While SANS provided evidence of the existence of aggregation, DLS provided quantitative information with respect to the size of the aggregates. SANS was the only technique which could characterize the protein both in the frozen and thawed states.

Citation

Molecular Pharmaceutics

Volume

18

Issue

12

Pub Type

Journals

Keywords

crystallizing buffer, freeze concentrate, freeze-thaw, lactate dehydrogenase, reversible self-association, protein interactions, reversible aggregation

Citation

Sonje, J. , Thakral, S. , Krueger, S. and Suryanarayanan, R. (2021), Reversible Self-Association in Lactate Dehydrogenase during Freeze-Thaw in Buffered Solutions using Neutron Scattering, Molecular Pharmaceutics (Accessed April 25, 2024)