Phosphorylation, one of the most common post-translational modifications of proteins, plays a critical role in many biological processes. We have previously developed several analytical methods for determining the phosphorylation status of certain proteins by using a phosphate-capturing binuclear metal complex known as Phos-tag. Here, we describe a novel method for the gel-based in vitro analysis of the phosphorylation status of a protein by a simple and rapid fluorometric staining method that uses a tetramethylrhodamine (TAMRA)-labeled Phos-tag derivative (TAMRA-Phos-tag). The entire staining protocol, which requires less than 2 h to complete, uses three buffer solutions for staining, washing, and dilution, respectively, at room temperature. The gel-based analysis of phosphoproteins in a polyacrylamide gel can be conducted by using a fluorescence imaging scanner with a 532-nm excitation laser and a 580-nm longpass emission filter. As a practical example of the use of the TAMRA-Phos-tag staining method, we examined the time course of dephosphorylation of ovalbumin by an alkaline phosphatase. In addition, inhibitor profiling of a tyrosine kinase Abl was performed by using an Abl-substrate (GST-Abltide) and an Abl-inhibitor (Imatinib).

In the bacterial signaling mechanisms known as two‐component systems (TCSs), signals are generally conveyed by means of a His–Asp phosphorelay. Each system consists of a histidine kinase (HK) and its cognate response regulator. Because of the labile nature of phosphorylated His and Asp residues, few approaches are available that permit a quantitative analysis of their phosphorylation status. Here, we show that the Phos‐tag dye technology is suitable for the fluorescent detection of His‐ and Asp‐phosphorylated proteins separated by SDS‐PAGE. The dynamics of the His–Asp phosphorelay of recombinant EnvZ‐OmpR, a TCS derived from Escherichia coli, were examined by SDS‐PAGE followed by simple rapid staining with Phos‐tag Magenta fluorescent dye. The technique permitted not only the quantitative monitoring of the autophosphorylation reactions of EnvZ and OmpR in the presence of adenosine triphosphate (ATP) or acetyl phosphate, respectively, but also that of the phosphotransfer reaction from EnvZ to OmpR, which occurs within 1 min in the presence of ATP. Furthermore, we demonstrate profiling of waldiomycin, an HK inhibitor, by using the Phos‐tag Cyan gel staining. We believe that the Phos‐tag dye technology provides a simple and convenient fluorometric approach for screening of HK inhibitors that have potential as new antimicrobial agents.

No 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) was determined to be 10 M 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 10 M. 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.