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A simple method for determining the ligand affinity toward a zinc-enzyme model by using a TAMRA/TAMRA interaction

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Abstract

Thiolate coordination to zinc(II) ions occurs widely in such functional biomolecules as zinc enzymes or zinc finger proteins. Here, we introduce a simple method for determining the affinity of ligands toward the zinc-enzyme active-center model tetramethylrhodamine (TAMRA)-labeled 1,4,7,10-tetraazacyclododecane (cyclen)–zinc(II) complex (TAMRA–ZnL). The 1 : 1 complexation of TAMRA-labeled cysteine (TAMRA–Cys) with TAMRA–ZnL (each at 2.5 μM), in which the TAMRA moieties approach one another closely, induces remarkable changes in the visible absorption and fluorescence spectra at pH 7.4 and 25 °C. The 1 : 1 complex formation constant (K = [thiolate-bound zinc(II) complex]/[uncomplexed TAMRA–ZnL][uncomplexed TAMRA–Cys], M−1) was determined to be 106.7 M−1 from a Job's plot of the absorbances at 552 nm. By a ligand-competition method with the 1 : 1 complexation equilibrium, analogous K values for thiol-containing ligands, such as N-acetyl-L-cysteine, L-glutathione, and N-acetyl-L-cysteinamide, were evaluated to have similar values of about 104 M−1. As a result of the ligand affinities to TAMRA–ZnL, nonlabeled zinc(II)–cyclen induced remarkable stabilization of the reduced form of L-glutathione and a cysteine-containing enolase peptide to aerial oxidation in aqueous solution at pH 7.4 and 25 °C.

Graphical abstract: A simple method for determining the ligand affinity toward a zinc-enzyme model by using a TAMRA/TAMRA interaction

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Publication details

The article was received on 20 Nov 2017, accepted on 08 Jan 2018 and first published on 08 Jan 2018


Article type: Paper
DOI: 10.1039/C7DT04364C
Citation: Dalton Trans., 2018, Advance Article
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    A simple method for determining the ligand affinity toward a zinc-enzyme model by using a TAMRA/TAMRA interaction

    H. Kusamoto, A. Shiba, M. Tsunehiro, H. Fujioka, E. Kinoshita-Kikuta, E. Kinoshita and T. Koike, Dalton Trans., 2018, Advance Article , DOI: 10.1039/C7DT04364C

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