: 10384 : 200026037 UDC MALDI-TOF 2003 10 18 2003 11 01 2003 2003 10
i
1 3..5 1 5 1.1..5 1.2..8 1.3...10 1.4...14 1.5....16 1.5.1 16 1.5.2....17 1.5.3...18 1.5.4....20 1.5.5....21 1.6..24 1.7 MALDI-TOF.. 25 1.8 27 2.. 29 2.1...29 2.2...29 2.3....30 2.4...30 2.4.1....30 2.4.2....31 2.4.3....31 2.4.4....32 2.4.5....32 2.4.6....33 ii
2.4.7....35 2.4.8 36 2.4.9 36 2.4.10 ( ).......36 2.4.11......38 2.4.12......38 3.. 39 3.1 (DALF)... 39 3.2 DALF.....40 3.3 DALF.43 3.4 HPLC.....45 3.5....48 3.6 MALDI-TOF SDS- ( )...51 3.7 SDS- ( ).. 55 3.8 SDS-PAGE....59 3.9 DALF. 61 3.10......62 MALDI-TOF. 64 1.. 64 1.1....64 1.2...65 1.3...68 1.4...69 2.....70 2.1........... 70 2.2 70 2.3 70 2.4 71 iii
2.4.1 MALDI-TOF.......71 2.4.2 71 2.4.3 INS.. 71 2.4.4 INS..... 72 2.4.5 INS-...... 72 2.4.6 INS-...... 72 2.4.7 INS-...... 72 2.4.8 INS-...... 72 2.4.9 INS-. 72 2.4.10 INS-......... 73 3.. 74 3.1... 74 3.2... 76 3.3... 78 3.4... 81 3.5... 82 3.6... 84 3.7... 86 3.8 INS- 88 3.9 INS- 89 3.10 INS- INS.. 91 93 94 105 106 iv
Liver feritin of Dasyatis akajei DALF DEAE-52 G-25 Sephacryl S-300 DALF SDS-PAGE DALF 2 H L 20kDa H 19kDa L L H R-HPLC Matrix-assisted laser desorption/ ionization time-of-flight MALDI-TOF DALF H L MALDI-TOF DALF MALDI DALF H 20550.53Da SDS- PAGE H SDS SDS-PAGE DALF MALDI-TOF H 20534.18Da MALDI-TOF DALF H H 1
MALDI-TOF - - MALDI-TOF ph2-3 A B Vc A B : MALDI-TOF, 2
Abstract Ferritin is the most common hydrophilic iron-storage protein. Ferritin not only plays very important functions in organisms, but also has very direct relations with many kinds of diseases. So studies on the functions and structure of ferritin can lead to diagnosis and treatment of human diseases and development of ferritin to be used in medicine, environment science, engineering and material science. Ferritin was isolated from the liver of fish named Dasyatis akajei. On the basis of thermostable property of ferritin, we used the thermal denaturation method to discard most of impurities. Dasyatis akajei liver ferritin (DALF) was then purified by DEAE-cellulose anion exchange chromatography and gel filtration chromatography. We also studied the molecular mass and polymer forms of ferritin by using the methods like Sephacryl S-300 exclusion chromatography, native PAGE and electron microscope. The information may help us to understand the advanced structure and the acting forces between subunits. DALF was also studied by SDS-PAGE and the result showed us that DALF is heteropolymer and composed of two kinds of subunits, H subunit and L subunit. Their molecular weight is 20kDa (H subunit) and 19kDa(L subunit) respectively. It is obvious that the concentration of H chain is more than that of L chain. We also used R-HPLC and MALDI-TOF to prove the composition and proportion of DALF subunits. This phenomenon is very unusual because the liver ferritin is rich of L chain to store the iron in the most case of animals. The result may be attributed to the difference between fish and advanced vertebrates in land. We used Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry as one new technique to study ferritin. The MALDI-TOF mass spectrum of full molecular of ferritin showed mass spectrum of H chain whose molecular mass is 20550.53 Da, which is bigger than the result from SDS-PAGE. As both SDS-PAGE and MALDI-TOF technique have them own advantages and shortages, we coupled both methods to analyze property of ferrtin subunits. DALF H chain was extracted from negative stained gel of SDS-PAGE by using organic solution, and then the subunit was introduced to MALDI-TOF to make sure its 3
molecular mass. The result indicated that the MALDI-TOF mass spectrum of DALF H chain is similar to that of DALF, and proved that DALF H chain has 20534.18 Da of molecular mass. We also used trypsin to digest the extracted H chain and found the DALF H chain has high homology with bacteriferritin and drosophila H chain. Ferritin is one kind of ideal nanomaterials and can be developed as carrier for nanoparticles. As the model polypeptide, insulin was analyzed by MALDI-TOF under different environment. The results can establish foundations for further research on ferritin as nanoparticles carriers. It was also declared that MALDI-TOF is very suitable for research on nanopartilces study as the fast, high-throughout advantages it has. The stability and activation of peptide is always the obstacles for its applying in medicine. As one of the earliest known polypeptide medicine, insulin is very important medicine for diabetes treatment. MALDI-TOF study showed that insulin was stable under weak acid environment and not inclined to form polymer. We also could learn the property and structure changes when insulin was treated by different ph gradient. Under the effect of sulfhydryl reducer, insulin was reduced to form A chain and B chain. We could observe their mass spectra peaks and adducts respectively in the mass spectrum. When we use Vc to reduce insulin, we could not observe any adducts. The results may reflect the mechanism difference between the two reduce process. In conclusion, MALDI-TOF is the very convenient and easy method for inspecting the stability and structure changes of insulin and polysaccharide nanopaticles. Keyword: ferritin, structure and function, MALDI-TOF, insulin MS and electrophoresis, polysaccharide nanopaticles 4
1 1.1 [1-5] 24 [1] 7-8nm [2] 2~2.5nm 24 [3,4] ; four-fold channel [2] [5] X-ray Horse spleen ferritin, HSF 0.4-.05nm 0.4nm ( :1,10-phenanthroline ),Yang and Chasteen (Electron paramagnetic reasonance) (nitroxide spin), HSF H (human H-chain ferritin), 0.7nm [2] 1974 Harrison HSF 4500 [1,3] 0.25M HSF 50% Apoferritin PAGE 440kDa 440kDa PAGE 5
(Fe 3+ ) [2] [FeO(OH)] 8 [FeO(H 2 PO 4 )] [3], 879.7 g/mol, 18.48 0.10 nm,,, [4] [5] ph DNA [6] [7] 92 [8] [9] (Bacterioferritin) Iron protein Fe 2+ Fe 3+ [4] (DNA and mrna) 2000 4500 6
H L [10,11] HSF Dasyatis akajei 100 7
1.2 H L H 182 21.1kDa L 53 L 83-95 174 19.9 kda [12,13] 15 24 4 3 2 6 8 8 4 A-D E B C L L N C D E A B AB C D CD BC L L 8 19 kda 21 kda [14] 8
3D 3D L B C C- E Fe 2+ Fe 2+ [8] 24 H 12 Listeria innocua Dps DNA 30 12 23 4 2 ferritin like Dps Dps like Dps [15,16] 9
1.3 Listeria innocua 12 24 H L Fe 2+ H L H Fe 2+ L 1000 H C L L H H/L L H 25 H/L [17,18,19] H L pi four-helix-bundle H L [20] H H dinuclear, E27 E61 E62 H62 E107 Q141 L L, H E57, E60, E64 L L H/L H H/L 10
L [21] L H H H L H L Ferritin cdna [22] H (heavy) M (middle) L (light) L E60, E57, E64 H M H M L M L 4 Mg 2+ Glu23 Glu58 His61 Glu103 Gln137 Asp140 H [23] [24,25,26], (bacterioferritin, BFR) Escherichia coli Azotobacter vinelandii Pseudomonas aeruginosa Nitrobacter winogradskyi ;, ferritin FTN Bacteroides fragilis, Helicobacter pylori Escherichia coli FTN BFR 20 BFR b1 b2 0.5 0.2, 1, [27] 11
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