Purification and characterisation of ferritin from the Baltic blue mussel Mytilus trossulus

Abstract

Baltic blue mussels Mytilus trossulus were collected from the Gulf of Gdansk (southern Baltic Sea) in order to isolate ferritin from its soft tissues, as well as to purify and characterise this protein. Proteins were isolated from the inner organs of M. trossulus (hepatopancreas, gills and soft tissue residue) by thermal denaturation (70◦C) and acidification (pH 4.5) of the homogenates, followed by ammonium sulphate ((NH4)2SO4) fractionation. The ferritin was then separated by ultracentrifugation (100 000× g, 120 min.). The protein content in the purified homogenates was determined by the Lowry method using bovine serum albumin (BSA) and horse spleen ferritin (HSF) as standards. PAGE-SDS and Western blotting analysis permitted identification of ferritin in the purified preparations. Additionally, the purified homogenates and mussel soft tissue were analysed for their heavy metal contents (especially cadmium and iron) in a Video 11 E atomic absorption spectrophotometer, following wet digestion of the samples (HNO3/HClO4). * This work was supported financially by the Committee for Scientific Research, grant No. 3/P04E/035/22, and the Institute of Oceanology statutory research grant. The complete text of the paper is available at http://www.iopan.gda.pl/oceanologia/ 526 J. Potrykus, A. Kosakowska The electrophoregrams showed that the inner organs of M. trossulus contained ferritin, which, like plant ferritin, is characterised by the presence of subunits in the electrophoregram in the 26.6–28.0 kDa range. The highest ferritin content was recorded in the hepatopancreas, followed by the gills and the soft tissue residue. With regard to the sampling stations, the highest content of ferritin was noted in the animals sampled off Sopot (station D3), and in those collected by a diver off Jastarnia (W1) and Gdynia (W4). Ferritin isolated from the inner organs of mussels collected from these stations also contained the largest quantities of heavy metals (Cd and Fe). Ferritin isolated from the inner organs of mussels collected by a diver from wrecks – sites where the concentrations of iron and other trace metals in the sea water are high – contained higher quantities of heavy metals (Cd and Fe) than the ferritin isolated from the inner organs of mussels collected with the drag. This confirms that ferritin is a protein able to store and transport not only iron, but also, though to a lesser extent, some other heavy metals, including cadmium.