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

Mol Pharm. 2021 Dec 6;18(12):4459-4474. doi: 10.1021/acs.molpharmaceut.1c00666. Epub 2021 Oct 28.


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.

Keywords: LDH conformation; aggregation; dynamic light scattering; pH shift; self-stabilization; small-angle neutron scattering; sodium phosphate buffer.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Buffers
  • Enzyme Stability
  • Freezing*
  • Hydrogen-Ion Concentration
  • L-Lactate Dehydrogenase / chemistry*
  • Neutron Diffraction*
  • Protein Aggregates
  • Protein Conformation
  • Protein Multimerization
  • Scattering, Small Angle*
  • Solutions


  • Buffers
  • Protein Aggregates
  • Solutions
  • L-Lactate Dehydrogenase