METHOD FOR QUANTITATIVE ANALYSIS OF INTERACTIONS BETWEEN HIF-1ALPHA C-TERMINAL PEPTIDES AND CBP OR P300 PROTEINS AND METHOD OF SCREENING INHIBITORS AGAINST FORMATION OF PROTEIN COMPLEX USING THE SAME

摘要

A method for quantitative analysis of interactions between HIF-1alpha C-terminal peptides and CBP(cyclic AMP-response element-binding protein) or p300 proteins is provided to reduce the analysis costs by removal of separation for a protein complex of HIF-1alpha C-terminal peptides and CBP or p300 proteins and automation with well plate, screen a large quantity of inhibitors against the protein complex, and analyze efficacy of post-translational modification of HIF-1alpha. A method for quantitative analysis of interactions between HIF-1alpha C-terminal peptides and CBP or p300 proteins comprises the steps of: (1) attaching a fluorescent material to the HIF-1alpha C-terminal peptide to prepare a fluorescent probe; (2) contacting the fluorescence-labeled HIF-1alpha C-terminal peptide with CBP or p300 protein to react the fluorescent material with the CBP or p300; and (3) measuring fluorescence polarization of the reaction product, and comparing it with the fluorescence polarization value of fluorescent probe itself. A method for screening inhibitors against formation of a protein complex comprises the steps of: (1) reacting the HIF-1alpha C-terminal peptides with CBP or p300 protein and measuring the fluorescence polarization of the reaction product; (2) adding a candidate inhibitor into the mixture and measuring the fluorescence polarization of the mixture; and (3) comparing the fluorescence polarization values of steps (1) and (2), and deciding that the candidate material as an inhibitor against the protein complex when the fluorescence polarization value of step (2) is smaller than that of step (1).