Thermodynamic and kinetic analysis on oligomeric protein dissociation using high-pressure native PAGE velocity method

Abstract

High pressure is known to dissociate several oligomeric proteins, and regarded as an important tool to shift the oligomerization equilibrium. Native polyacrylamide gel electrophoresis (native PAGE) at high pressure can characterize the dissociates and clearly discriminate the aggregates. However, a band smearing of migration profiles often hinders more detailed analyses (Miwa et al., High Pressure Res. (2019) 39, 218–224). In this paper, we focused on the band smearing dependent on the migration velocity so as to extract both thermodynamic and kinetic parameters. We systematically perturbed the migration velocity by changing the gel concentration and carried out numerical analysis for a series of the migration profiles based on a simple dissociation reaction scheme with limited thermodynamic and kinetic parameters. Then, complete volumetric properties on oligomerization process can be available. We term the present analysis method as a high-pressure native PAGE velocity method. We also report the application of this method to revisit the pressure dissociation of tetrameric lactate dehydrogenase (LDH) from pig heart.