Purification, Characterization And Biochemical Properties Of Ferritin From Chick Pea Seeds

Ferritin is a ubiquitous intracellular iron storage protein in all living organisms. It serves the dual function of iron metabolism and iron detoxification. In contrast to animal ferritin, relatively little information is available on phytoferritin. Former reports say that in plants, especially in legume seeds, more than 90% of iron is stored in the form of ferritin in amyloplast. Therefore, the legume seed ferritins are considered as the ideal model to study phytoferritin. In this paper, taking the chickpea as research object,we succeed in exploring and realizing chickpea ferritin preparation, and have studied the structure and properties of chickpea ferritin. Chick pea seed ferritin (CSF) and PSF share very high identity in amino acid sequence through N-terminal sequence and MALDI-TOF-MS analyses. However, CSF exhibit the high activities in iron uptaking and releasing. All the details and results are as follows:1. With chickpea as raw materials, we have got get electrophoresis pure chickpea seed ferritin.In this aera, the extraction temperature, pH and heating conditions were consideried, using gradient screening of ammonium sulfate and column chromatography purification system.2.The protein we got was analysised that it has a 27.0 kDa subunit and the molecule is 560 kDa. N-terminal sequences, MALDI-TOF-MS, and Western-blot analyses indicate that the purified protein was chick pae seed ferritin (CSF) and it shares very high identity in amino acid sequence with PSF. However, the two phytoferritin exhibit the different activities in iron uptake and release. The reason may be due to the two proteins have different subunit compositions.3. The prodcuting mechanism of apoferritin was established, and the spectra variation of apoferritin was compared with holoferritin. Sodium Hyposulfite is strong reducing agents, therefore, which was applied to deoxidize holoferritin to release iron ion, and connection of iron of buffer was measured by the 2,2-Dipyridyl. Apoferritin was detected by ICP-MS. Holoferritin has no absorption compared with apoferritin by UV analysis, and Holoferritin has no fluorescence emission spectra in contrast with apoferritin by fluorescence analysis.4. By comparing the aggregate and iron oxidative deposition activities of the PSF and CSF, demonstrating that CSF exhibit the high activities in iron uptaking and releasing in whatever conditions.Phytoferritin was not stable enough at 25℃to be against at 4℃tempreture.The degradation start from 39 days at 4℃construct to 15 days at 25℃.