MODULATION OF THE CONFORMATIONAL STABILITY OF Macrobrachium acanthurus HEMOCYANIN BY pH AND UREA: EVIDENCE FROM INTRINSIC FLUORESCENCE AND DYNAMIC LIGHT SCATTERING

Hemocyanins are large copper-containing respiratory proteins whose biological functions depend on the maintenance of their native oligomeric organization. The present study investigated the effects of pH and urea-induced denaturation on the conformational stability of Macrobrachium acanthurus hemocyanin (HcMac) using UV-Vis spectroscopy, intrinsic fluorescence, and dynamic light scattering (DLS). HcMac samples were incubated for 2 h in 20 mmol·L⁻¹ acetate-phosphate buffer at pH 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0 in the presence of increasing urea concentrations (0–7 mol·L⁻¹).DLS analyses demonstrated that HcMac remained predominantly monodisperse over a broad pH range, exhibiting Z-average values of approximately (17.9, 15.0, 16.4, 14.0 and 15.7 nm) at pH 4.0, 5.0, 6.0, 7.0 and 8.0, respectively. The lowest polydispersity index was observed at pH 7.0 (PDI ≈ 0.13), indicating maximum colloidal stability. In contrast, samples at pH 9.0 displayed increased heterogeneity (PDI ≈ 0.43) and the emergence of larger particle populations, suggesting structural rearrangements and partial destabilization. Intrinsic fluorescence measurements revealed significant changes in both emission intensity and wavelength maximum as a function of urea concentration. A progressive red shift from approximately 327 to 342 nm was observed at higher denaturant concentrations, indicating increased exposure of aromatic residues to the aqueous environment. Simultaneously, fluorescence intensity profiles suggested the presence of intermediate conformational states during the unfolding process. UV-Vis spectroscopy revealed pH-dependent alterations in the absorption band associated with the dicopper active center. At pH 4.0, absorbance increased up to 2 mol·L⁻¹ urea before decreasing at higher concentrations, whereas at pH 9.0 a progressive reduction was observed throughout the denaturation process, indicating disruption of the local environment surrounding the copper-binding sites. Overall, the results demonstrate that HcMac exhibits remarkable structural stability between pH 5.0 and 8.0, while alkaline conditions and increasing urea concentrations promote conformational destabilization, changes in tertiary structure, and alterations in supramolecular organization. These findings provide new insights into the physicochemical stability of crustacean hemocyanins and their potential biotechnological applications.
This work was supported by Conselho Nac. Des. Cient. Tecnologico (CNPq) and by the and by the Institute for Advanced Marine Studies (IEAMar/UNESP).