Comparative analysis of cryoprotective agents influence on thermodynamic and kinetic parameters of equine and human hemoglobin molecules.

BACKGROUND: Critical to the understanding the mechanism of destruction and protection during cryopreservation of biological objects is the knowledge of the conformational transitions of biopolymers experiencing low temperatures in the presence of cryoprotective agents. This information may be derived from the kinetic and thermodynamic parameters of macromolecular thermal denaturation kinetics under different environmental conditions. OBJECTIVE: The study deals with the influence of cryoprotective agents (glycerol, 1.2-propanediol (1.2-PD), and dimethyl sulfoxide (DMSO)) on thermodynamic and kinetic parameters of equine and human hemoglobin. METHODS: Thermograms were recorded with differential scanning adiabatic microcalorimeter (DASM-4, Biopribor, Russia). RESULTS: Temperatures and enthalpy changes in the denaturation of hemoglobins (ΔHcal) in the presence of cryoprotective agents from 0-40% (w/w) were determined. Within the whole concentration range glycerol slightly alters the hemoglobin denaturation temperature while DMSO and 1.2-PD lead to a significant decrease. The addition of cryoprotectants to hemoglobin solutions induces a complex behavior of ΔHcal concentration dependences of denaturation caused by intra- and intermolecular processes such as hydration of the porphyrin cycle, heme cleavage from heme-containing proteins, disorder in hydrophobic contacts with globin etc. These factors may decrease thermal stability by loosening hemoglobin molecules and spatial disruption of fragments of the protein. Activation energy of irreversible unfolding of equine hemoglobin at heating was estimated using the approach of Sanchez-Ruiz et al. DMSO and 1.2-PD decreased activation energy values. CONCLUSIONS: The data indicate the reduction of protein thermal stability by DMSO and 1.2-PD. Glycerol slightly increases hemoglobin thermal stability within the studied range of concentrations.