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

Abstract

The aims of this work were to evaluate the effect of freezing and thawing stresses on lactate dehydrogenase (LDH) stability under three conditions. (i) In a solution buffered with sodium phosphate (NaP; 10 and 100 mM). 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. The conditions of buffer component crystallization from solution were identified using low-temperature X-ray diffractometry. 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; buffer concentration 10 mM), has a pronounced self-stabilizing effect and did not aggregate after five freeze–thaw cycles. At lower LDH concentrations (10 and 100 μg/mL), only with the selection of an appropriate buffer, irreversible aggregation could be avoided. While SANS provided qualitative information with respect to protein conformation, the insights from DLS were quantitative with respect to the particle size of the aggregates. SANS is the only technique which can characterize the protein both in the frozen and thawed states.

Role of Poloxamer 188 in Preventing Ice-Surface-Induced Protein Destabilization during Freeze–Thawing

“…Dynamic light scattering (DLS) was used to determine the Z -average diameter before and after five FT cycles. The LDH diameter in the freshly prepared LDH solution was ∼10 nm, revealing its tetrameric native state . After five FT cycles, in a system without a surfactant, the diameter substantially increased to >1000 nm (the upper detection limit of the DLS instrument), reflecting aggregation (Table S1).…”

“…In the FT experiments, LDH at a low concentration of 10 μg/mL was used to avoid its self-stabilization effect . Dynamic light scattering (DLS) was used to determine the Z -average diameter before and after five FT cycles.…”

“…The LDH diameter in the freshly prepared LDH solution was ∼10 nm, revealing its tetrameric native state. 30 After five FT cycles, in a system without a surfactant, the diameter substantially increased to >1000 nm (the upper detection limit of the DLS instrument), reflecting aggregation (Table S1). The addition of sucrose or trehalose (1.0%, w/v) could not inhibit LDH aggregation.…”

“…In the FT experiments, LDH at a low concentration of 10 μg/mL was used to avoid its selfstabilization effect. 30 Dynamic light scattering (DLS) was used to determine the Z-average diameter before and after five FT cycles. The LDH diameter in the freshly prepared LDH solution was ∼10 nm, revealing its tetrameric native state.…”

Poly(l-alanine-co-l-threonine succinate) as a Biomimetic Cryoprotectant

“…6 Because LDH is a representative protein that is particularly unstable and prone to lose its activity during freezing, it was used as a model protein to assay the protein stabilizing activity of the PATs during the repeated freeze− thaw cycles. 6,24 ■ EXPERIMENTAL SECTION Materials. N-Carboxy anhydrides of L-Ala (KPX Life, Korea) were used as received.…”