Peroxynitrite‐induced structural remodeling and aggregation of hemoglobin under nitroxidative stress

Abstract

Peroxynitrite (PN), generated by the reaction of nitric oxide with superoxide, is a potent reactive nitrogen species (RNS) implicated in nitroxidative protein damage in cardiovascular and neurodegenerative diseases. Hemoglobin (Hb), the principal oxygen‐transport protein of erythrocytes, is a key intravascular scavenger and target of PN; however, how controlled PN exposure translates into structural remodeling and aggregation of Hb remains poorly understood. Purified human Hb was exposed to PN across sub‐stoichiometric to moderate oxidant excess and characterized using intrinsic fluorescence, UV–visible (UV–vis) spectroscopy, 2,4‐dinitrophenylhydrazine (DNPH) carbonyl assay, Thioflavin T (ThT), 8‐anilino‐1‐naphthalenesulfonic acid (ANS), Congo Red (CR), Fourier‐transform infrared (FTIR) spectroscopy with Amide I deconvolution, dynamic light scattering (DLS) with zeta potential, x‐ray powder diffraction (XRPD), scanning electron microscopy (SEM), atomic force microscopy (AFM), and fluorescence microscopy. At low oxidant loads, PN caused concentration‐dependent fluorescence quenching, carbonyl accumulation, and graded UV–visible absorbance increases, consistent with heme‐centered oxidation and limited aromatic residue modification. At higher concentrations, Amide I deconvolution revealed collapse of α ‐helical content from 66.41% to 8.81% and β ‐sheet enrichment to 71.18%, accompanied by surface charge neutralization, increased hydrodynamic diameter, and enhanced nanoscale roughness. Despite β ‐sheet accumulation and ThT/CR‐positive aggregate formation, XRPD, AFM, SEM, and fluorescence microscopy showed no long‐range crystalline order or fibrillar morphology, demonstrating that PN drives Hb toward amorphous and oligomeric assemblies rather than canonical amyloid fibrils. Time‐dependent assays at 37°C confirmed that PN modification lowers the nucleation barrier for thermally driven aggregation. These findings define physicochemical thresholds separating protective scavenging from structural destabilization and offer a framework for distinguishing nitroxidative protein aggregation from classical amyloid fibrillation.