Characterization and identification of selected metal-binding biomolecules from hepatic and gill cytosols of Vardar chub (Squalius vardarensis Karaman, 1928) by various techniques of liquid chromatography and mass spectrometry

Metals play crucial physiological roles, but they can also cause irreparable toxic effects through binding to important cellular biomolecules in aquatic organisms. The aim of this study was to determine the exact molecular masses and to identify several selected metal-binding biomolecules in hepatic and gill cytosols of Vardar chub (Squalius vardarensis Karaman, 1928). Methods applied for the achievement of this goal were SEC-AEC-HPLC for two-dimensional separation of cytosolic biomolecules, HR ICP-MS for metal measurements, and mass spectrometry (MALDI-TOF-MS and LC-MS/MS) for biomolecule mass determination and identification. Analyzed biomolecules reffered to: Fe-binding biomolecules, which were identified as hemoglobin subunits in the liver (molecular masses of 15 kDa), and hemoglobin subunits and in the gills (molecular masses of 11 kDa, 13 kDa and 15 kDa) ; heat-stable Cd-binding biomolecules, which were identified as MT isoforms MT-I and MT-II (molecular mass of 6.0 kDa in both liver and gills, and an additional 4.9 kDa isoform in the gills) ; heat-stable Mo-binding biomolecules of molecular masses equal to 3.3 kDa (in the gills) and 8.5 kDa (in the liver). An important finding of this study was obvious presence of different isoforms of the same biomolecules in the liver and gills. It was, among others, manifested through absence of Zn binding to MTs only in the gills, indicating that the same type of biomolecule can be responsible for different functions in different organs. Thus, for better understanding of metal behaviour in aquatic organisms, it is crucial to know cellular metal- binding biomolecules and their functions