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Cytosolic Cd-, Cs-, Pb-, and Tl-binding metalloproteins of the digestive gland of the bivalve Unio crassus analysed by anion-/cation-exchange high-performance liquid chromatography and inductively coupled plasma mass spectrometry

The isoelectric point (pI) is a fundamental parameter intrinsic to any protein, representing the pH at which the protein has an overall neutral charge. This attribute is useful in characterizing the acidic or basic nature of complex proteins. Depending on the pH of the solution, proteins may carry a net positive or negative charge relative to their pI [1]. Consequently, ion exchange chromatography represents a powerful technique to achieve the separation of ionizable proteins based on their charge properties [2]. The aim of this study was to analyse the distribution of toxic elements Cd, Cs, Pb, and Tl among negatively and positively charged biomolecules in the digestive gland cytosol of the bivalve species Unio crassus using anion- and cation-exchange liquid chromatography (AEX-HPLC ; CEX-HPLC) coupled with inductively coupled plasma mass spectrometry (ICP- MS). Protein separation using both HPLC methods was performed with a liner salt gradient (0-500 mM ammonium acetate ; 20 minutes) in appropriate buffers at pH 7.4 (10 mM Tris buffer for AEX-ICP- MS ; 15 mM phosphate buffer for CEX-ICP-MS). In addition, AEX-ICP-MS profiles of six protein standards (alcohol dehydrogenase, carbonic anhydrase, metallothioneins 1 and 2, superoxide dismutase, and transferrin) and CEX-ICP-MS profiles of two protein standards (cytochrome-C and lysozyme) with known pI values were analysed. The results of AEX-ICP-MS showed that biomolecules that bind Cd, Pb and Tl were mainly eluted later than the measured protein standards. Only the first peak of the Pb-binding biomolecules had an elution time similar to that of transferrin (last eluted protein standard, 12.1 minutes, pI~6). Other Pb peaks, in addition to the peaks of Cd- binding biomolecules, were found in the last third of the salt gradient (375 mM – 500 mM ammonium acetate), indicating the more acidic nature of these metalloproteins. Moreover, Cd and Pb were eluted in overlapping peaks, revealing that they are bound to the biomolecules of the comparable negative charge in the digestive gland of U. crassus. This was confirmed by separation on CEX- column, where biomolecules that bind Cd and Pb, due to their appearant negative charge at neutral pH, were completely eluted in the void volume (in the first minute). Conversely, biomolecules that bind Cs were entirely eluted in the void volume (in the first minute) when AEX-column was used, but their elution was evident on the CEX-ICP-MS profile, with the main Cs peak appearing at 7.7 minutes and indicating that proteins that bind this metal are mainly positively charged at neutral pH. A peak of Tl-binding biomolecules was also observed on the CEX-ICP-MS profile (at ~15 minutes), indicating the presence of both acidic and basic cytosolic proteins responsible for Tl binding in U. crassus digestive gland. Protein standards (cytochrome-C with a pI value of 9.6 and lysozyme with a pI value of 11.1) had CEX-ICP-MS elution times at 19 and 24 minutes, respectively. This suggested that basic biomolecules that bind Cs and Tl have lower pI values than standards, probably closer to the pH of the buffer solution. Additional chromatographic separations and analyses by different techniques of mass spectrometry are needed to further identify and characterize these biomolecules.

cytosol; mussel; AEX HPLC; CEX-HPLC; ICP-MS

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