The separation and detection of complexes of aptamers and protein targets by capillary electrophoresis (CE) with laser-induced fluorescence was examined. Aptamer-thrombin and aptamer-immunoglobulin E (IgE) were used as model systems. Phosphate, 3-(N-morpholino)propanesulfonic acid with phosphate, and tris(hydroxyamino)methane-glycine-potassium (TGK) buffer at pH 8.4 were tested as electrophoresis media. Buffer had a large effect with TGK providing the most stable complexes for both protein-aptamer complexes. Conditions that suppressed electroosmotic flow, such as addition of hydroxypropylmethylcellulose to the media or modification of the capillary inner wall with polyacrylamide, were found to prevent detection of complexes. The effect of separation time and electric field were evaluated by monitoring complexes with electric field varied from 150-2850 V/cm and effective column lengths of 3.5 and 7.0 cm. As expected, shorter times on the column greatly increased peak heights for the complexes due to a combination of less dilution by diffusion and less dissociation on the column. High fields were found to have a detrimental effect on detection of complexes. It is concluded that the best conditions for detection of noncovalent complexes involve use of the minimal column length and electric field necessary to achieve separation. The results will be of interest in developing affinity probe CE assays wherein aptamers are used as affinity ligands.

Fluorescence anisotropy capillary electrophoresis (FACE) and affinity probe capillary electrophoresis (APCE) with laser-induced fluorescence detection were evaluated for analysis of peptide-protein interactions with rapid binding kinetics. The Src homology 2 domain of protein SH2-Bbeta (SH2-Bbeta (525-670)) and a tyrosine-phosphorylated peptide corresponding to the binding sequence of JAK2 were used as a model system. For peptide labeled with fluorescein, the K(d) = 82 +/- 7 nM as measured by fluorescence anisotropy (FA). APCE assays had a limit of detection (LOD) of 100 nM or 12 amol injected for SH2-Bbeta (525-670). The separation time of 4 s, achieved using an electric field of 2860 V/cm on 7-cm-long capillaries, was on the same time scale as complex dissociation allowing K(d) (101 +/- 12 nM in good agreement with FA measurements) and dissociation rate (k(off) = 0.95 +/- 0.02 s(-)(1) corresponding to a half-life of 0.73 s) to be determined. This measurement represents a 30-fold higher rate of complex dissociation than what had previously been measurable by nonequilibrium CE analysis of equilibrium mixtures. Using FACE, the protein was detected with an LOD of 300 nM or 7.5 fmol injected. FACE was not used for determining K(d) or k(off); however, this method provided better separation resolution for multiple forms of the protein than APCE. Both methods were found suitable for analysis of cell lysate. These results demonstrate that FACE and APCE may be useful complements to existing techniques for exploring binding interactions with rapid kinetics.

Hydrolysis of fluorescent GTP analogues BODIPY® FL guanosine 5-O-(thiotriphosphate) (BGTP␥S) and BODIPY® FL GTP (BGTP)by G␣ i1 and G␣ o was characterized using on-line capillary electrophoresis laser-induced fluorescence assays in order that changes in substrate, substrate-enzyme complex, and product could be monitored separately. Nanomolar RGS increased the rate of enzyme product formation (BODIPY® FL GDP (BGDP)) by 117-213% under steady-state conditions and accelerated the rate of G protein-BGTP complex decay by 199 -778% in pseudosingle-turnover assays. Stimulation of GTPase activity by RGS proteins was inhibited 38 -81% by 40 M YJ34, a previously reported peptide RGS inhibitor. Taken together, these results illustrate that G␣ subunits utilize BGTP as a substrate similarly to GTP, making BGTP a useful fluorescent indicator of G protein activity. The unexpected levels of BGTP␥S hydrolysis detected suggest that caution should be used when interpreting data from fluorescence assays with this probe. G proteins1 are involved in many important physiological processes. Ligands binding to cell surface receptors signaling through G protein pathways (like G protein-coupled receptors (GPCRs) or receptor tyrosine kinases (RTKs)) induce the G protein to exchange GDP for GTP, activating the protein. Activated G protein can transmit signals to downstream effectors such as adenylyl cyclase, phospholipases, ion channels, or cGMP phosphodiesterase, which ultimately result in cellular responses initiated by the extracellular ligand stimulus. Signaling is ceased by the hydrolysis of GTP to GDP via the intrinsic GTPase activity of the G protein (reviewed in Refs. 1 and 2). G protein deactivation can be accelerated by interactions with GTPase-activating proteins (GAPs) such as regulators of G protein signaling (RGS) (3, 4).Most studies of G protein-mediated signaling have relied on utilization of guanine nucleotide analogues that both behave similarly to the native species and have been modified such that they can be sensitively detected. The most common modification involves insertion of radiolabeled isotopes of phosphorous or sulfur to yield [␥-32 P]GTP and [␥-35 S]GTP␥S, which can then be used in filter-binding assays to measure GTPase activity and the extent of nucleotide binding, respectively. More recently, fluorescent guanine nucleotide derivatives N-methyl-3Ј-O-anthranoyl (MANT) and BODIPY® have been developed (5, 6), allowing for G protein assays with greater sensitivity than previous fluorescence assays that relied on detection of changes in intrinisic tryptophan fluorescence. A number of studies have reported on use of these fluorescent GTP derivatives with low molecular weight, Ras-like G proteins. In general, the binding affinities and rate constants reported for MANT analogues of GTP and GDP are within a factor of 2 of those for unmodified guanine nucleotides (7), rendering these probes useful for studies of basal and GAP-stimulated GTPase activity (8 -13) and interactions with guanine nucleotide exchange factors (14...

An affinity probe capillary electrophoresis (APCE) assay for guanine-nucleotide-binding proteins (G proteins) was developed using BODIPY FL GTPgammaS (BGTPgammaS), a fluorescently labeled GTP analogue, as the affinity probe. In the assay, BGTPgammaS was incubated with samples containing G proteins and the resulting mixtures of BGTPgammaS-G protein complexes and free BGTPgammaS were separated by capillary electrophoresis and detected with laser-induced fluorescence detection. Separations were completed in less than 30 s using 25 mM Tris, 192 mM glycine at pH 8.5 as the electrophoresis buffer and applying 555 V/cm over a 4-cm separation distance. BGTPgammaS-Galpha(o) peak heights increased linearly with Galpha(o) up to approximately 200 nM using a 50 nM BGTPgammaS probe. The detection limit for Galpha(o) was 2 nM, corresponding to a mass detection limit of 3 amol. The high speed of the APCE assays allowed reaction kinetics and the dissociation constant (Kd) to be determined. The on-rate and off-rate of BGTPgammaS to Galpha(o) were 0.0068 +/- 0.0004 and 0.000 23 +/- 0.000 01 s(-1), respectively. The half-life of the BGTPgammaS-Galpha(o) complex was 3060 +/- 240 s and Kd was 8.6 +/- 0.7 nM. The estimates of these parameters are in good agreement with those obtained using established techniques, indicating the suitability of this method for such measurements. Lowering the temperature of the separation improved the detection of the complex, allowing the assay to be performed on a commercial instrument with longer separation times. Additionally, the capability of the technique to detect several G proteins based on their binding to BGTPgammaS was demonstrated with assays for Galpha and Galpha(i1) and for Ras and Rab3A.

We describe a capillary electrophoresis (CE) assay to detect G protein-coupled receptor (GPCR)-stimulated G protein GTPase activity in cell membranes expressing alpha2A adrenoreceptor-Galphao1 wild-type (wt) or C351I mutant fusion proteins using a fluorescent, hydrolyzable GTP analogue. As no change in total fluorescence is observed by conversion of substrate to product, CE is used to separate the fluorescent substrate (*GTP) from the fluorescent product (*GDP). Using the assay, the alpha2a adrenoceptor agonist UK14,304 was shown to simulate specific production of *GDP in membranes from HEK293T cells expressing receptor-G protein fusion to 525% of basal levels with an EC50 of 0.48 +/- 0.20 microM. The EC50 increased to 9.4 +/- 5 muM with addition of the antagonist yohimbine. Nucleotide hydrolysis was increased further over agonist-stimulated levels with addition of the in vivo modulator protein RGS (regulator of G protein signaling). It is envisioned that this technique could be used for screening for novel GPCR ligands or other G protein signaling modifiers.

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