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Analysis of Fe distribution among cytosolic biomolecules and identification of Fe-binding compounds in the liver and gills of Vardar chub (Squalius vardarensis Karaman)

Many proteins contain essential metal Fe in ionic form, either within their own structures or bound to their active sites (del Castillo Busto et al., 2010). Iron distribution profiles among cytosolic biomolecules were determined for hepatic and gill cytosols of Vardar chub (Squalius vardarensis Karaman) from Macedonian rivers. Application of size-exclusion high performance liquid chromatography (SEC-HPLC) coupled offline with high resolution inductively coupled plasma mass spectrometry (HR ICP-MS) resulted with Fe separation in two peaks, the first one corresponding to biomolecules of 230-630 kDa, and the second one corresponding to biomolecules of 24-51 kDa. The first peak most likely corresponds to ferritin, the major Fe storage protein (450 kDa), and was more prominent in liver than in gills, which is in accordance with more significant role of liver in Fe storage. To obtain more information about biomolecules comprised by the second peak, which was observed in both studied organs, we have used anion exchange chromatography (AEX-HPLC) coupled offline with HR ICP-MS, as a second step of separation. Cytosolic biomolecules of 24-51 kDa from both organs were further separated according to their ionic strength, and clearly distinguishable Fe peaks were observed at elution times from 10th to 14th minute. The last step was application of two mass spectrometry techniques for the identification of Fe-binding biomolecules of 24-51 kDa which were purified by two-dimensional chromatographic separation. By use of MALDI-TOF, three protein peaks were obtained, corresponding to molecular masses of 15 kDa, 32 kDa and 47 kDa in the liver, whereas in the gills we have obtained two peaks corresponding to 11 kDa and 14 kDa. Application of liquid chromatography- mass spectrometry system (LC-MS) enabled identification of α and β subunits of haemoglobin (Hb, 15-16 kDa) in both organs, whereas hepatic peaks of higher molecular masses probably referred to Hb dimers and trimers.

iron ; Vardar chub ; liver ; gills ; chromatography ; mass spectrometry ; protein identification

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