Purification Of Monoamine Oxidase B And The Chromatographic Approach For Analyzing Its Interactive Proteins In Porcine Liver

The physiological functions of organism, are mainly controlled and modulated by proteins in cells. Most proteins combined with ligand or form protein complexes with other proteins, and then execute the process of cell metabolism together. Enzymes as an important kind of protein, almost involve all life activities. Hense, it is significant to elucidate interactions between enzymes and their correlative protein in order to help people understand the physiological and pathological process of organisms. Accroding to the previous research, the purpose of this study is to continuously developed a chromatographic approach for analyzing interaction between Monoamine Oxidase B(MAOB) protein in procine liver:based on the specific interaction between substrate and enzyme, through method of computer autodocking, optimizing the density of ligands, and LC-ESI-MS/MS detection, we explored the feasibility of absorbing and purifying MAOB through immobized dopamine(DA) in order to lock the binding state of the substrate and enzyme.The result of experiments indicated that:(1)According to autodocking results, DADPA and glutaraldehyde spacer, DA ligand has high affinity with MAOB; Affinity material with high DA density can absorb MAOB and obtained a purification fold of 2.16. However, its selectivity to MAOB was very low, while affinity material with low DA density purifed a protein, which has no MAOB activity. Through LC-ESI-MS/MS Identification and activity measure, the obsorbed proteins were mainly catalase with serum albumin and carboxylase in it. (2)MAOB was purified from porcine liver by precipitation with ammonium sulfate, hydrophobic chromatography, and anion exchange chromatography. A purification of 18.2 fold was achieved, and the purified enzyme had a specific activity of 135 U/mg. The purified enzyme appeared homogeneous as shown by SDS-PAGE, and it had a molecular weight of about 60 kDa. The identity of the enzyme was confirmed by LC-ESI-MS/MS. (3) Immobilized DA and purifed MAOB didn't form stable complex which could be interrupted by 0.1M NaCl. So this immobilized substrate-enzyme complex can't be used to capture interaction proteins in cytoplasm. (4)Covalent immobilizing purifed MAOB using ethylenediamine and glutaraldehyde spacer with density of 5.75.7μmol/mL gel. The immobilized MAOB was 0.53 mg/mL gel and activity recovery is 40.98%. The optimal temperature, optimal pH and the stability of MAOB was increased after immobilization. (5) SDS-PAGE didn't detect any interaction protein with immobilized MAOB.All these experiments indicated that it is not feasible that using immobilizing DA to capture and purified MAOB and obtain stable DA-MAOB complex.