SDS-Induced Structural Transitions of Hemoglobin Revealed by Spectroscopic Techniques and Small-Angle Neutron Scattering

Abstract

Hemoglobin A₀ (HbA₀) is a physiologically important tetrameric protein, consisting of two alpha (α) and two beta (β) subunits, resides inside the red blood cell and carries oxygen to the cell. In this study, the interaction of HbA₀ protein with anionic sodium dodecyl sulfate (SDS) has been investigated by small-angle neutron scattering (SANS), UV-visible spectroscopy, fluorescence spectroscopy, and circular dichroism (CD) spectroscopy. The previous studies on the HbA₀-surfactant system by DLS and spectroscopic techniques only confirmed the unfolding of HbA₀ without deciphering the exact process and differentiating between the subunit dissociation and extended structure formation. Our spectroscopic results reveal disruption of the heme pocket, loss of Tyr→Trp energy transfer, and spectral shifts indicative of methemoglobin formation. Utilizing the advantages of SANS, we have explored the subunit dissociation of tetramer units of HbA₀ and their different forms (monomer, dimer, etc.) with the sequential addition of SDS. The results reveal that the addition of SDS at low concentrations leads to the dissociation of tetramer structure into dimers and monomers, driven by molecular-level binding of SDS monomers to hydrophobic and electrostatic patches at the subunit interfaces rather than by micellization. Above higher SDS concentrations, a fraction of nonbound SDS forms free micelles that stabilize the dissociated monomers without promoting further unfolding. In the presence of electrolytes, electrostatic screening enhances protein–surfactant interactions and promotes the formation of extended rod-like complexes. These findings establish a detailed structural pathway for SDS-induced remodelling of HbA₀ and highlight the distinct behaviour of multimeric proteins compared to single-chain globular proteins.