Purification, Characterization And Preliminary Study On Iron Release Kinetics From The Liver Ferritins Of Acipenser Baeri Brandt

Ferritin is a protein that is widely present in living organisms and it could regulate the balance of iron metabolism in the body.In the processing of aquatic products,a large number of by-products,such as liver,pancreas,and intestines,were often produced.If they were comprehensively utilized,they would have important significance for the aquatic industry.In this paper,ferritin was prepared from Acipenser baeri Brandt liver through single factor experiment combined with response surface method.The ferritin of Acipenser baeri Brandt liver（AbBLF）was separated and isolated by a series of purification technology,including ammonium sulfate precipitation,anion exchange chromatography and gel filtration chromatography.The preliminary characterization of ferritin constructure was performed by Native-PAGE gel electrophoresis,SDS-PAGE gel electrophoresis,2-dimensional electrophoresis,FT-IR and LC-MS/MS.Finally,the iron release kinetics of AbBLF was analyzed.The results provided theoretical guidance and technical support for the comprehensive utilization of by-products of aquatic product processing as well as the basic research of ferritin.Based on single factor experiment,factors including solid-liquid ratio,ammonium sulfate saturation and pH were selected as independent variables,and AbBLF extraction rate was selected as the response value.According to the principle of Box-Behnken.The result showed the optimum extraction condition for AbBLF was as follows:solid-liquid of 1:3.8,ammonium sulfate saturation of 57%,pH of 8.12.Through verification test,the yield of AbBLF was 32.95 mg·kg^-1,which was close to the theoretical value(34mg·kg^-1).The AbBLF was separated and isolated by a series of purification technology,including ammonium sulfate precipitation,DEAE Cellulose 52 anion exchange and Sephacryl S-300gel chromatography.By Native-PAGE gel electrophoresis,SDS-PAGE gel electrophoresis and two-dimensional electrophoresis,the molecular weight of the AbBLF is about 400 kDa,which contains two different subunits（Light-chain subunit and Heavy-chain subunit）,and their molecular weights were 20.5 kDa and 21.1 kDa,and the isoelectric points were 6.1and 6.6,respectively.The AbBLF subunits were identified by LC-MS/MS,and 9 peptide sequences were matched in the Unipot database.The homology of the two subunits to the ferritin subunit of Acipenser sinensis liver was 69%and 68%,and the homology to ferritin of Anoplopoma fimbria liver was 60%and 59%,respectively,which proved that the purified ferritin was AbBLF.By FT-IR analysis,the percentages ofβ-sheet,α-helix,β-turn,random coil of the AbBLF was 40.61%,35.86%,7.12%and 16.41%respectively.The ascorbic acid and Ferrozine were used as reducing agent and ferrous ion chelating agent,respectively,to analyze the iron release kinetics of the AbBLF in different pH values and solution systems.The results showed that the rate of iron release from AbBLF in the acidic environment was significantly higher than that in an alkaline environment;and the rate of iron release in Tris-HCl buffer is higher than that in phosphate buffer,demonstrated that H⁺,OH^-,and PO₄^3-in the solution affected iron release from the intact iron core of ferritin.