engleski

Distribution of selected metals/metalloids among differently negatively charged cytosolic metal-binding biomolecules in the digestive gland of freshwater mussels

Cytosol is a metabolically active part of the cell that contains metalloproteins with isoelectric point (pI) values clustered between 4.0 and 6.0 [1]. Despite their responsibility for metabolic processes [2], metalloprotein characteristics (mass, charge, etc.) are poorly understood in many organisms, including mussel species. Therefore, the aim of this study was to analyse the distribution of 14 selected metals/metalloids among differently charged metal-binding biomolecules in the digestive gland cytosol of the mussel species Unio crassus (Philipson, 1788) using anion-exchange high-pressure liquid chromatography (AEX-HPLC) and mass spectrometric analysis (ICP-MS). Protein separation using AEX- HPLC was performed with a linear salt gradient (0- 500 mM NH4OAc ; 20 minutes) in 10 mM Tris buffer (pH 7.4). Metal-binding biomolecules with lower pI values tend to interact more strongly with AEX resins, requiring higher salt concentrations for elution [2]. In addition, the AEX- profiles of five protein standards with known pI values (alcohol dehydrogenase, carbonic anhydrase, metallothioneins 1 and 2, superoxide dismutase) were analysed. The results showed that the majority of biomolecules binding the elements Ag, As, Bi, Cd, Cs, Cu, Fe, Pb, Se, and Zn had elution times similar to those of the analysed protein standards (first 10 minutes ; pI higher or ≈ 4). Only a few elements (Ag, Co, Cd, Pb, and Mo) were bound to cytosolic biomolecules at the end of the salt gradient (15-20 min ; >375 mM NH4OAc), indicating the presence of more acidic proteins, i.e., proteins with very low pI values. Several elements (As, Co, Cs, Cu, Fe, Mn, Pb, Se, Zn) were eluted in the void volume indicating the presence of basic metal-binding proteins in the mussel digestive gland cytosol. Some elements (Ag, Co, and Mo ; Cd and Pb ; Mo and Zn ; Ag, Cu, and Cd) had overlapping peaks, suggesting that they are bound to the cytosolic biomolecules of the same charge in the digestive glands of this bivalve species. Further application of additional chromatographic separation and mass spectrometry techniques is needed to confirm the identity of these cytosolic biomolecules.

cytosol ; mussel ; AEX HPLC ; ICP-MS

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