In Vitro Elucidation of the Folding Intermediates and Aggregate Formation of Hemoglobin Induced by Acetonitrile: A Multispectroscopic Approach.

Acetonitrile is a mild solvent, which induces β-sheet conformation in proteins. The global conformational changes in Hb in the presence of ACN were studied using intrinsic fluorescence experiments, acrylamide quenching, ANS fluorescence measurements, soret absorbance spectroscopy, fourier transform infrared spectroscopy, circular dichroism, thioflavin T and congo red assay. Molecular docking showed the binding of hydrophobic residues of Hb to ACN. Hb exists as a partially unfolded intermediate state at 30% v/v ACN. Hb aggregates were obtained at 60% v/v ACN concentration, which were further confirmed by transmission electron microscopy.