Refolding of Hemoglobin Under Macromolecular Confinement: Impersonating In Vivo Volume Exclusion

Biomacromolecules evolve and function inside the cell under crowded conditions. The effect of macromolecular crowding and confinement on nature and interactions of biomacromolecules cannot be ruled out. This study demonstrates the effect of volume exclusion due to macromolecular crowding on refolding rate of Gn-HCl induced unfolded hemoglobin. The in vivo like crowding milieu was created using dextran 70. Unfolding of Hb was followed by the absorbance at 280 nm and intrinsic fluorescence intensity along with a bathochromic shift that shows the destabilization of Hb in the presence of the denaturing agent. This was supported by a decrease in soret absorbance, increased hydrodynamic radii and loss in secondary structure, evidenced from dynamic light scattering and circular dichroism experiments respectively. Refolding process of Hb was followed by an increase in soret absorbance, decrease in intrinsic fluorescence intensity with a hypsochromic shift, decreased hydrodynamic radii and gain in secondary structural content. The results revealed that the effect of confinement and volume exclusion is insignificant on the process of Hb refolding.