Biophysical Characterization of Shrimp Hemocyanins: Stability and Emerging Biotechnological Applications.

Abstract

Hemocyanins are oxygen-transporting proteins found in crustaceans and other arthropods, playing key roles in immune defense and metabolic regulation. Due to their stability and bioactive properties, Hcs have gained increasing interest in biotechnological and biomedical applications. However, detailed biophysical characterization is crucial to understanding their functional potential. In this study, the hemocyanin was extracted and purified from Macrobrachium acanthurus (HcMac) using ultracentrifugation and size-exclusion chromatography. The molecular mass of HcMac was determined by SDS-PAGE electrophoresis, MALDI-TOF mass spectrometry, and analytical ultracentrifugation. Spectroscopic analyses, including UV-Vis absorption, fluorescence emission, and light scattering intensity, were used to assess the structural stability of the compound under various pH conditions. HcMac was identified as a hexameric protein (~450 kDa) composed of monomeric subunits of 75 and 76 kDa. The protein maintained its oligomeric stability and oxygen-binding affinity in the pH range of 5.0-7.4. However, extreme pH conditions (below 4.4 and above 7.5) induced structural alterations, leading to dissociation and conformational changes, as evidenced by fluorescence emission and UV-Vis spectra. The isoelectric point was determined to be between pH 4.3 and 5.3, consistent with other crustacean HCs. These findings reinforce the structural robustness of HcMac and suggest its potential for biotechnological applications. The high stability of HcMac under physiological pH conditions indicates its suitability for biomedical research, including immunomodulatory and antimicrobial applications. Future studies integrating bioinformatics, proteomics, and immunological assays will be essential to explore the therapeutic potential of HcMac.