The Characteristics And Function Of Liver Ferritin Of Dasyatis Akajei

Ferritin from the liver of a salt water fish, Dasyatis akajei, herein called DALF, was isolated and purified by thermal denaturation, ammonium sulphate fractionation and ammonium reverse extraction, Sephacryl S-300 gel filtration and DEAE- cellulose anion exchange chromatography. Molecular mass analysis using Sephacryl 8-300 exclusion chromatography method revealed that the value was about 400kDa. Moreover, SDS-PAGE technology showed that DALF was a heteropolymer of two kinds of subunits of H and L, which molecular weight were 1 8kDa and 1 3kDa respectively, and the molecular sizes of both protein shell and iron core in diameter were measured by transmission electron microscope of 10-l4nm and 5-8nm respectively. Isoelectric focusing revealed heterogeneity with at least five bands in the gel slab of p1 values ranging from 5.4 to 6.2. Two kinds of DALF, DALFA and DALFB, was isolated in the DEAE-cellulose column using process elution method, SDS-PAGE analysis revealed the difference between the two eluates was the different ratio of H:L of the twenty four subunits. In vitro, DALF could store small organic molecular, such as dyes, in the protein shell and bind them on the outer surface, which showed that the quantity binding on the outer surface was quite difference but that stored in the shell was nearly the same. The kinetic characteristics of the two eluates, DALF storing and binding organic dyes, and DALF binding haem were studied, which further demonstrated the proposition that it was the self-regulation of the protein shell not the complex surface of the core played a key role in iron release and storage.In vitro, DALF could firmly keep most of the iron and phosphate in the core even in the high salinity or when dialyzing in the water for three days. And just as ferritin from the other sources, besides reconstituting iron and phosphate in the hole of the protein shell, it also could stored many kinds of heavy metal ions, which showed that the quantity of storage was in positive3relation to the concentration of metal ions in the medium and the time of storage, so the DALF had the possibility to monitor the heavy metal ions pollution in the inland sea. Further studies showed that the change of the salinity of the water and the content of iron in the core of DALF had no influence on the storage of metal ions, but the alteration of pH in the water and the content of phosphate in the core of DALF could much influence the storage. The cause was the phosphate in the core could provide more binding site for the metal ions, while the influence of pH might result from its promotion on the release of phosphate in the core. Accompanying by the metal ion storing, the iron in the core of DALF was released, which provided a new method to monitor the total metal that DALF stored. At the same time, the entire absorbance was decreased during the heavy metal ion stored, which also could be used as a new way for real time monitor.I tried to modify the DALF to construct a ferritin reactor using DALF, from our present research, we could not establish the kinetic equations for storing various metal ions with the reactor, there is a great deal work needed to be done in the future.