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single-labeled peptide probe for measuring peptide phosphorylation status was developed by using a phosphate sensitive terbium chelate. The activity of Abl protein tyrosine kinase and T-cell protein Tyrosine phosphatase (TC PTP) was monitored in real time. To study the probe design in detail, variable substrate peptide sequences, where the enzyme target site was located from two to five amino acids apart from the nearest tyrosine residue, were synthesized. The maximum change observed in fluorescence intensity after phosphorylation was up to 320%, when the phosphorylated tyrosine was located two amino acids from the lysine coupled to the phosphate sensitive terbium chelate, demonstrating an excellent performance for a homogeneous assay. Also the longer distance of five amino acids between the phosphorylated tyrosine residue and terbium chelate resulted up to 260% change in fluorescence intensity. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

A homogeneous high-throughput screening method based on time-resolved fluorescence resonance energy transfer (TR-FRET) for the measurement of calcium-dependent multimerization of an EF-hand protein, sorcin, is described. The assay is based on a specific sorcin binding peptide conjugated either with an intrinsically highly fluorescent europium chelate (donor) or an Alexa Fluor 700 fluorophore (acceptor). Addition of calcium results in multimerization of sorcin, allowing several peptides to bind simultaneously to the epitopes of the multimeric protein complex, and the proximity of peptides labeled either with donor or acceptor label results in fluorescence resonance energy transfer between the 2 labels. When no calcium is present, the protein remains in a monomer form, and thus no FRET can take place. In the optimized assay construct, the assay was performed in 45 min, and a more than 20-fold signal-to-background ratio was achieved. The reversibility of sorcin multimerization was shown by chelating free calcium with ethylenediamine tetraacetic acid (EDTA). The developed homogeneous assay can be used in screening molecules that either inhibit or enhance multimerization of sorcin, and the assay format is applicable to various noncompetitive high-throughput screening assays detecting protein multimerization reactions. (Journal of Biomolecular Screening 2007:842-848) [ABSTRACT FROM AUTHOR]

Abstract: The control of cell death is an intricate process involving a multitude of intracellular modulators. Among these molecules, the caspases have a central role and have become an interesting group of enzymes in the current pharmaceutical industry. We have developed a novel dual-step fluorescence energy transfer-based separation-free assay method for the primary screening of caspase-3 inhibitors in vitro. This method relies on fluorescent europium(III)-chelate-doped nanoparticle donors coated with streptavidin in conjunction with a dual-labeled (N-terminal Alexa Fluor 680 fluorescent acceptor and C-terminal BlackBerry Quencher 650) caspase-3-specific peptide substrate modified with a biotinyl moiety. In the assay, the nanoparticle donor excites the fluorescent acceptor, whose emission is monitored with time-resolved measurements. The intensity of the acceptor reflects the activity of the enzyme because the intensity is controlled by the proximity of the quencher. Owing to the dual-step fluorescence resonance energy transfer, this method enables a sensitized fluorescence signal directly proportional to the extent of enzymatic activity with relatively background fluorescence-free measurements in the event of complete enzyme inhibition. The generic nanoparticle donors further promote versatility and cost-efficiency of the method. The performance evaluated as the inhibitor (Z-DEVD-FMK) dose–response curve (IC50 value of ∼ 12 nM) was in good agreement with that of the recent methods found in literature. This assay serves as a model application proving the feasibility of the europium-chelate-doped nanoparticle labels in a homogeneous assay for proteolytic activity. [Copyright &y& Elsevier]