A Novel Method For Biomolecule Interaction By Immobilized Small Molecule

The physiological functions of organisms, including humans, are mainly controlled and modulated by proteins in cells. Most of proteins firstly combine with ligand or form protein complex with other proteins, and then participate in the process of cell metabolism. Hence, protein-protein interactions are fundamental to understand biological processes. In this paper, a novel method for protein-protein interaction was founded. To form small molecule-target protein complex, a functional small molecule was immobilized on agarose beads and then interacted with target proteins. The complex and its correlative proteins in cells were investigated. This method was applied in the study of interaction between dopamine -monoamine oxidase B complex and its correlative proteins in liver cell cytoplasm, and the primary study of protein-protein interaction with dopamine in SH-SY5Y cell was also in progress.The result of experiments indicated that: (1) In virtue of the interaction between monoamine oxidase B and dopamine, low and high density dopamine immobilized on agarose beads were tandem used to separate and purify monoamine oxidase B in pig liver. Only the band of monoamine oxidase B was observed on sodium-dodecyl sulphate- polyacrylamide (SDS-PAGE) gel electrophoresis. Bandscan software analyse showed that its purity was about 95% and relative molecular weight was estimated to be approximately 60000 Da. The specific activity was 8921. 4U/mg and purification factor was 4.9. (2) Cytoplasm proteins of liver were adsorbed by the purified monoamine oxidase B and dopamine complex immobilized on agarose beads. Through the chromatographic process, three functional proteins that possibly participate in regulating monoamine oxidase B catalysis were obtained. Their relative molecular weight was from 50000Da to 80000Da. (3) Dopamine immobilized on agarose beads could interact with a functional protein in SH-SY5Y cells. MALDI-TOF-MS results showed that its molecular weight was 66652 and purity was over 96%.From the experiment it is concluded that: This method suggested is feasible that a small biomolecule is immobilized on agarose beads to interact with target proteins, creating small biomolecule-target protein complex,and then the interaction between small biomolecule-target protein complex and its correlative proteins in cells can be investigated.Hence, it can be applied in the study of functional protomics as a new method.