BIOPHYSICAL STUDIES OF STRUCTURAL ALTERATIONS INDUCED BY SDS IN THE HEMOCYANIN OF MACROBRACHIUM ACANTHURUS

Hemocyanins (Hcs) are high molecular weight, oligomeric, copper-containing proteins responsible for oxygen transport in arthropods. Beyond this classical role, Hcs exhibit immune-related functions and biotechnological potential. This study investigated the structural and functional effects of sodium dodecyl sulfate (SDS) on hemocyanin from Macrobrachium acanthurus (HcMac), focusing on conformational alterations, aggregation behavior, and enzymatic modulation. HcMac was isolated from hemolymph by ultracentrifugation and size exclusion chromatography. Spectroscopic analyses showed SDS-induced perturbations. Absorption spectra showed shifts in the Copper binding region and changes in aromatic residue environments. Fluorescence spectroscopy (λexc = 295 nm) indicated a concentration dependent increase in emission intensity, suggesting partial unfolding and exposure of tryptophan residues. Dynamic light scattering (DLS) analysis showed significant changes in HcMac hydrodynamic diameter with increasing SDS concentration. At low SDS concentrations (0.05–0.9 mmol·L⁻¹), particle size remained nearly constant (13.42 ± 0.20 nm). Between 1.0 and 3.5 mmol·L⁻¹ SDS, HcMac began to aggregate, forming particles larger than 200 nm and exhibiting high
polydispersity (PDI > 0.78), indicative of a heterogeneous supramolecular system. In contrast, increasing the temperature from 25 °C to 50 °C resulted in a gradual rise in particle size (from 13.35 to 13.98 nm), indicating that HcMac maintains moderate structural stability without undergoing major conformational transitions.Functionally, SDS modulated the phenoloxidase (PO) activity of HcMac, assessed dopachrome formation at 475 nm in the presence of dopamine. A progressive activation pattern was observed, reaching a maximum at 3.5 mmol.L-1 SDS, with PO activity increasing from an initial 0.0021 to values comparable to other crustacean Hcs reported in the literature.These findings suggest SDS facilitates exposure of the di-copper catalytic center, enhancing active site accessibility.
Overall, the results demonstrate that SDS promotes conformational changes, structural reorganization, and functional activation in HcMac. The protein showed notable thermal stability between 25 °C and 50 °C and remained structurally stable up to 0.9 mmol·L⁻¹ SDS, above the critical concentration required to phenoloxidase activity. These findings enhance the understanding of hemocyanin dynamics under surfactant influence and support their potential in catalysis,
immunogenic platforms, and protein engineering.