Preparation Of Ferritin From Salmon And The Effect Of Heat Treatment On Its Structure

As a kind of iron storage protein which exists widely in living organisms,ferritin plays a key role in the balance of iron metabolism.At present,many ferritin research institutions from both China and overseas have successively reported on the physicochemical properties and subunit cloning expression of ferritin in higher animals,but there are few reports on the structure and function of ferritin in the field of aquatic research.In addition,ferritin has special properties such as resistance to higher temperatures.By studying the effects of heat treatment on the structure-activity relationship and absorption characteristics of ferritin,it is possible to try to improve the loss of ferritin caused by digestion and absorption during processing.So that ferritin can play its biggest role.In this study,the ferritin was isolated and purified from salmon(Oncorhynchus kisutch)by the methods of salting out and column chromatography combined with membrane separation.By SDS-PAGE analysis,the tissues distribution of ferritin in salmon was investigated.The results show that the ferritin is highly expressed and distributed in the tissues of heart,liver and other visceral tissues.Then the molecular weight of ferritin and its subunit was analyzed using Native-PAGE and SDS-PAGE respectively,and the amino acid sequences of the ferritin subunit were determined using CE-MS.Ferritin heavy subunit of20.71 k Da was identified using BLAST analysis with genome sequence of O.kisutch from NCBI Gen Bank.Prokaryotic expression of ferritin was performed by the result of CE-MS.The primers were designed by the genome sequence.By constructing the recombinant expression plasmid p ET21a-fer H,E.coli BL21 was selected to induce the expression of ferritin heavy subunit.The induction lasted for 8h with 0.8m M IPTG at 37?.The expressed ferritin is present in the cells.According to cell fragmentation and SDS-PAGE analysis,the expression product exists in both insoluble inclusion bodies and soluble proteins.The soluble protein was selected for further purification.The protein was further purified in a DEAE-Sepharose Fast Flow column.The purified ferritin was subjected to crystal diffraction and its crystal structure was investigated.The analysis results in the molecular structure of the fer H subunit and the surface charge map of the active center,wherein the subunit contains an iron ion as a ligand.The axis of symmetry of ferritin is F 4 3 2,and a sub-base aggregates to form a cage structure.Glu at position 23,Glu at position 58 and His at position 61 form an active center that interacts with iron ions.The denaturation temperature of ferritin was determined by DSC to be 76 ? and the heat treatment of the salmon ferritin was carried out at five temperatures of 65 ° C,75?,85?,95?,and 100? for 20 min.The structural changes of ferritin after different temperature treatments were analyzed by SDS-PAGE and Native-PAGE.There was no change in the molecular weight of the subunits,indicating that the heat treatment had no effect on the primary structure of ferritin,while Native-PAGE showed that high partial depolymerization occurs with temperature increased and complete depolymerization at100 °C.The secondary structure changes of ferritin were analyzed by circular dichroism spectroscopy,particle size analyzer and fluorescence spectroscopy.As the heat treatment temperature increased,the ?-helix content gradually decreased,and the irregular curl content increased gradually.The particle size analysis showed that the macromolecular substance was observed.Aggregation occurs and the particle size gradually increases.The fluorescence intensity of the sample at 340 nm decreases with the increase of heat treatment temperature.After heating at 100 °C,the fluorescence peak is red-shifted.At this time,the tryptophan group inside ferritin has been exposed to protein molecules.Externally,it indicates that the horsefish ferritin has been depolymerized after 20 minutes of heat treatment.