Dopamine hydrochloride promotes non-amyloid aggregation and secondary structural rearrangement of hemoglobin: Biophysical and spectroscopic evidence

Hemoglobin (Hb) is susceptible to oxidative and conformational perturbations that can promote protein aggregation and impair biological function. Dopamine hydrochloride (DA HCl), a redox-active catecholamine, undergoes autoxidation under physiological conditions, generating reactive intermediates that may influence protein structure and assembly. However, its effects on native Hb aggregation pathways remain poorly understood. Here, we investigated DA HCl-induced structural and aggregation-related changes in Hb using UV-visible absorption and intrinsic fluorescence spectroscopy, Fourier transform infrared (FTIR) spectroscopy, circular dichroism (CD), Thioflavin T (ThT), Congo Red (CR), and 8-anilino-1-naphthalenesulfonic acid (ANS) assays, dynamic light scattering (DLS), scanning electron microscopy (SEM), atomic force microscopy (AFM), fluorescence microscopy, and X-ray powder diffraction (XRPD). Exposure of Hb to DA HCl was associated with concentration-dependent changes in fluorescence and UV-visible spectral properties, while time-dependent UV-visible measurements confirmed progressive dopamine autoxidation. FTIR and CD analyses revealed reduced α-helical content accompanied by increased β-sheet and disordered structural elements, indicating modest conformational rearrangement rather than extensive unfolding. ThT, CR, and ANS assays showed enhanced aggregation and increased hydrophobic surface exposure; however, the responses were substantially weaker than those typically associated with mature amyloid fibrils. DLS demonstrated increased hydrodynamic diameters, whereas SEM, AFM, and fluorescence microscopy confirmed the formation of heterogeneous micron-scale aggregates. XRPD analysis showed no evidence of new crystalline phases or cross-β fibrillar ordering. Collectively, these findings indicate that DA HCl and/or its oxidation products are associated with concentration-dependent, non-fibrillar mesoscale aggregation of Hb, although the relative contributions of dopamine versus its oxidation products cannot be resolved from the present data.