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

This study investigates how different cryoprotective agents (compounds that protect biological tissue from freezing damage) affect the thermodynamic and kinetic properties of both horse and human hemoglobin molecules. The findings suggest that two out of three tested cryoprotectants reduce the thermal stability of hemoglobin, with implications for the process of cryopreservation.

Objectives and Methods

• The research aims to better understand the mechanism of damage and protection during the cryopreservation process. Specifically, it looks at how conformational transitions of biopolymers occur at low temperatures when in the presence of various cryoprotective agents. Glycerol, 1.2-propanediol (1.2-PD), and dimethyl sulfoxide (DMSO) were studied.
• The researchers used a differential scanning adiabatic microcalorimeter, a device that measures heat capacity, to record temperature changes and thermal reactions of the hemoglobin molecules. This method allowed for accurate monitoring of biopolymer denaturation kinetics under varied environmental conditions.

Findings and Results

• The temperatures and enthalpy (a measure of a system’s thermal energy) changes under denaturation, with the addition of cryoprotective agents from 0 to 40% concentration, were determined.
• It was found that within this entire concentration range, glycerol barely changed the denaturation temperature of the hemoglobin, whereas DMSO and 1.2-PD caused a significant decrease.
• Adding cryoprotectants to the hemoglobin solutions induced complex behavior in the enthalpy concentration dependencies of denaturation.
• This behavior is attributed to intra- and intermolecular processes such as hydration of the porphyrin cycle, heme cleavage from heme-containing proteins, and disorder in hydrophobic contacts with globin, among others. These factors can reduce thermal stability by loosening hemoglobin molecules and spatially disrupting protein fragments.
• The researchers also estimated the activation energy needed for the irreversible unfolding of equine hemoglobin during heating and found that the presence of DMSO and 1.2-PD decreased these energy values.

Conclusion

• Overall, this study demonstrates that the presence of certain cryoprotectants, DMSO and 1.2-PD, can reduce the thermal stability of hemoglobin. On the other hand, glycerol increases the thermal stability of hemoglobin within the studied concentration range.
• The results have implications for the cryopreservation of biological objects, potentially influencing the choice of cryoprotective agents used in these processes.