The Pathway Of Iron Deposition In Soybean Seed Ferritin And Research On Ferritin Uptake By Caco-2Cells In Vitro

Ferritin is a ubiquitous intracellular iron storage protein in all living organisms. It serves the dual function of iron detoxification and iron metabolism. In contrast to animal ferritin, relatively little information is available on phytoferritin. So far, about90%iron from legumes are stored inside the phytoferritin. Therefore, legume seed ferritins are considered as an ideal research model to study phytoferritin. Compared to animal ferritin, phytoferritins exhibit3distinctive features in structure. First, at the N-terminal of each phytoferritin subunit, there is an extension peptide (EP) located at the surface of phytoferritin and taking part in iron binding and oxidation, while animal ferritin lacks. Second, only H-type subunit has been identified in phytoferritin which is usually a heteropolymer consisting of two different subunits, H-1and H-2, sharing about80%amino acid sequence identity. The subunit composition of phytoferritins varied depending on the source of them. Third, the3-fold channels and4-fold channels of phytoferritin are hydrophilic. However, the3-fold channels of animal ferritin are hydrophilic and the4-fold channels are hydrophobic. It has been reported that all the channels of ferritin are responsible for substance exchanging. In this paper, phytoferritin has been further studied. From the viewpoint of phytoferritin structure, the function of3-fold channels and4-fold channels has been investigated, elucidating the reasons for the hydrophilic channels. On the other hand, the digestive stability of ferritin in vivo and in vitro was widely studied. Meanwhile, the bioavailability of ferritin from different sources has been identified to provide useful information for developing ferritin as potential iron supplements. All the results are as follows:1. The rH-1, rH-2and the variants of rH-2(H193AH197A, E165IE167AE171A, E165IE167AE171AH193AH197A) were prepared successfully by virtue of molecular cloning, site-specific mutagenesis, vector construction and expression and purification. We also successfully got purified rH-1, rH-2and the variants of rH-2by means of Sephacryl S-300gel filtration column after the DEAE-Sepharose Fast Flow column. Therefore, it will provide materials for further research.2. It has been demonstrated that the hydrophilic3-fold channels and4-fold channels are both responsible for iron diffusion and oxidative deposition into phytoferritins by comparing the iron oxidative activities, the association properties and the iron release activities of rH-2and its variants (H193AH197A, E165IE167AE171A,E165IE167AE171AH193AH197A).3. The digestive stabilities of phytoferritin with different subunit compositions and animal ferritin, and the effects of cooking and skim milk on the in vitro and in vivo digestive stability of ferritin were extensively studied, indicating that most of the phytoferritin and animal ferritin can pass through the gastrointestinal digestion and reach the small intestine, which can be further absorbed by epithelial cells. In addition, skim milk plays a protective role during in vivo digestion.4. Phytoferritins with different subunit compositions and animal ferritin were labeled with59Fe to further study the iron bioavailability of them and the interactions between ferritin and putative receptors at Caco-2cell surface. In this paper, it was found that the bioavailability of soybean seed ferritin (rH-1: rH-2=1:1) is the highest among all of the studied ferritins, and the binding affinity of soybean seed ferritin is the highest either. Therefore, soybean seed ferritin can be a novel and effective iron supplement in the future.