pH and temperature-dependent structural and functional stability of hemocyanin from Tachypleus gigas: A multispectroscopic, phenoloxidase activity and computational study.

Authors

Sudam Bhoi, K Sathya Sai Kiran, Bisnu Prasad Dash, Naresh Krishna Narasimha

Published in

Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy. Volume 363. Issue Pt 2. Pages 128466. Jul 17, 2026. Epub Jul 17, 2026.

Abstract

Hemocyanin, primarily an oxygen-transporting protein, has gained considerable attention due to its multifunctional role in therapeutic and biotechnological applications. A detailed assessment of structural and functional stability is crucial for its potential usage in various applications. Towards this, the stability of Tachypleus gigas hemocyanin (TgH) was evaluated across various temperatures and pH conditions. UV-visible spectroscopy confirmed stable oxygen binding at the di‑copper active site up to 50 °C between pH 5.0 and 8.0. Circular dichroism spectroscopy revealed a melting temperature of 77.98 °C and demonstrated that TgH successfully refolds into its native conformation from 70 °C upon removing thermal stress. Functional assay showed that TgH retains 80% phenoloxidase activity up to 70 °C, indicating remarkable functional stability and reversibility. Dynamic light scattering analysis revealed a decrease in particle diameter from 25.95 nm at pH 6.0 to 8.07 nm at pH 10.0, suggesting alkaline dissociation of oligomers into monomers, potentially due to electrostatic repulsion between subunits because of increased negative surface charge. Finally, molecular dynamics simulations corroborated these findings by demonstrating that the TgH subunit maintains its original conformation up to 70 °C, with notable deviations observed beyond this temperature, consistent with the experimental results. The structure remains stable without any notable deviation at pH 6.0 and 10.0, indicating structural stability of the monomer subunit despite alkaline dissociation. This comprehensive study highlights the thermostability and broad pH tolerance of TgH, emphasizing its potential for diverse therapeutic and biotechnological applications, especially in fluctuating environmental conditions.

PMID:
42485694
Bibliographic data and abstract were imported from PubMed on 23 Jul 2026.

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