A Chromatographic Approach For Protein-protein Interaction By Immobilized Dopamine

The physiological function of organisms is mainly participated 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. Hence, it is significant to elucidate protein-protein interactions for human life. Accroding to the previous research, the purpose of this study is to unceasingly explore the method for protein-protein interaction. To interact with proteins in porcine liver, such as monoamine Oxidase B, a dopamine molecule was immobilized on agarose beads using ethylenediamine and glutaraldehyde as spacer. The interactions between monoamine Oxidase B and its correlative proteins were investigated.The result of experiments indicated that: (1) It was found that the adsorption capabilities to monoamine Oxidase B were increased by promoting the density of dopamine on support, but the purity of monoamine Oxidase B went up first and then down. As far as higher enzymatic activeness and enzyme purity were concerned, the preferable density of dopamine for purifying monoamine Oxidase B was about 36μmol/mL gel. The activeness of monoamine Oxidase B was 3723 U/mg and its purity was 76% by Bandscan software analyse. (2) After the primary extraction of monoamine Oxidase B from liver crude sample by appropriate density dopamine support, gel filtration was used to progress farther purification. Based on optimization of operating conditions and the control of bovine serum albumin, a protein which may interact with monoamine Oxidase B was found, and its relative molecular weight was about 100000Da. (3) Compared with the purified enzyme eluate, enzymatic activeness of the eluate-cytoplasm mixture increased about eleven times. It primarily showed that some biomolecules in cytoplasm could interact with monoamine Oxidase B and participate in enzymatic catalyze.All these experiments indicated that it is feasible that a dopamine biomolecule is immobilized on support to interact with monoamine Oxidase B, and the interaction between monoamine Oxidase B and its correlative proteins in cells can be investigated. Therefore, it can be applied in the study of protein-protein interaction in functional proteomics as a new chromatographic method.