Selective Recognition and Detection of Biomacromolecules Utilizing Chemical Property of Amino Acid or Peptide

Abstract

Recently, the ‰uorometric 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 ‰uorescence assay for serine/threonine kinase activity; and 2)``turn-on'' ‰uorescent 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 ‰uorescently via base-mediated b-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 puriˆcation and immobilization. We designed ‰uorescent probes composed of a ‰uorophore and Ni 2＋ or Co 2＋ -NTA complex. These probes were found to be weakly ‰uorescent as expected. When His-tag peptide was added, these probes became brightly ‰uorescent. On the other hand, these probes remained non ‰uorescent with the addition of angiotensin I (H-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-HisLeu-OH). These probes will be powerful tools for the bioimaging of target proteins.