アミノ酸,ペプチドの化学的性質を利用した生体高分子の選択的認識と検出(誌上シンポジウム)
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Recently, the fluorometric detection of biomacromolecules has attracted much attention. In this paper, we report the development of two new techniques utilizing the chemical properties of amino acids or peptides: 1) a fluorescence assay for serine/threonine kinase activity; and 2) "turn-on" fluorescent probes for protein labeling, which could be useful for bioimaging. To develop the novel kinase assay, we utilized the chemical reactivity of phosphorylated serine or threonine. Phosphorylated peptide on resin was successfully labeled fluorescently via base-mediated β-elimination, followed by Michael addition with novel coumarin derivatives. Protein kinase A and casein kinase I activities were detectable with our method. Also, this method was confirmed to be applicable for kinase inhibitor screening. For the development of the novel protein labeling technique, the selective interaction between "His-tag (His_6)" and "metal ion nitrilotriacetic acid (NTA) complex" was utilized. This interaction is useful for protein purification and immobilization. We designed fluorescent probes composed of a fluorophore and Ni^<2+> or Co^<2+>-NTA complex. These probes were found to be weakly fluorescent as expected. When His-tag peptide was added, these probes became brightly fluorescent. On the other hand, these probes remained non fluorescent with the addition of angiotensin I (H-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-OH). These probes will be powerful tools for the bioimaging of target proteins.
- 社団法人日本薬学会の論文
- 2007-12-01
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