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We have used rapid mix flow cytometry to analyze the early subsecond dynamics of the disassembly of ternary complexes of G-protein coupled receptors (GPCRs) immobilized on beads to examine individual steps associated with guanine nucleotide activation. Our earlier studies suggested that the slow dissociation of Gα and Gβγ subunits was unlikely to be an essential component of cell activation. However, these studies did not have adequate time resolution to define precisely the disassembly kinetics. Ternary complexes were assembled using three formyl peptide receptor constructs (wild type, FPR-Gαi2 fusion, and FPR-GFP fusion) and two isotypes of the α subunit (αi2 and αi3) and βγ dimer (β1γ2 and β4γ2). At saturating nucleotide levels, the disassembly of a significant fraction of ternary complexes occurred on a subsecond time frame for αi2 complexes and τ1/2 ≤ 4 sec for αi3 complexes, time scales which are compatible with cell activation. β1γ2 isotype complexes were generally more stable than β4γ2 associated complexes. The comparison of the three constructs however proved that the fast step was associated with the separation of receptor and G protein and that the dissociation of the ligand or of the α and βγ subunits was slower. These results are compatible with a cell activation model involving G protein conformational changes rather than disassembly of Gαβγ heterotrimer.

We describe a simple and rapid quantitative assay for biotin and biotin conjugates. The assay is based on the kinetic analysis of the enhancement of fluorescence of streptavidin/fluorescein biotin complexes in the presence of biotin. The kinetic response of fluorescence enhancement is proportional to the concentration of biotin. Standard calibration curves based on the kinetic response are obtained and detection limits of approximately 10(-9)M are established. Because the assay is amenable for use in small volumes of 5-50 microL or bead-based assays, the detection limits can be extended to the femtomole range. Since the assay depends on kinetic analysis, routine quantitation can be achieved without reference to standard curves. The dynamic aspects allow the assay to be extended to a broader range of applications including its use as an indicator of reagent mixing in laminar-flow assays carried out in microfluidic devices.

Epitope tagging of expressed proteins is a versatile tool for the detection and purification of the proteins. This approach has been used in protein-protein interaction studies, protein localization, and immunoprecipitation. Among the most popular tag systems is the FLAG epitope tag, which is recognized by three monoclonal antibodies M1, M2, and M5. We describe novel approaches to the detection of epitope-tagged proteins via fluorescence resonance energy transfer on beads. We have synthesized and characterized biotinylated and fluorescein-labeled FLAG peptides and examined the binding of FLAG peptides to commercial streptavidin beads using flow cytometric analysis. A requirement of assay development is the elucidation of parameters that characterize the binding interactions between component systems. We have thus compiled a set of Kd values determined from a series of equilibrium binding experiments with beads, peptides, and antibodies. We have defined conditions for binding biotinylated and fluoresceinated FLAG peptides to beads. Site occupancies of the peptides were determined to be on the order of several million sites per bead and Kd values in the 0.3-2.0 nM range. The affinity for antibody attachment to peptides was determined to be in the low nanomolar range (less than 10 nM) for measurements on beads and solution. We demonstrate the applicability of this methodology to assay development, by detecting femtomole amounts of N-terminal FLAG-bacteria alkaline phosphatase fusion protein. These characterizations form the basis of generalizable and high throughput assays for proteins with known epitopes, for research, proteomic, or clinical applications.

Multiple forms of glutathione S-transferase (EC 2.5.1.18), a family of proteins involved both with bilirubin transport and with the detoxification of electrophiles, have been purified from human liver using a scheme which employs an affinity chromatography column prepared by coupling glutathione to epoxy-activated Sepharose. This procedure offers a convenient method for obtaining highly purified transferases in good yields.