Fluorescence Screens

The fluorophores are either intrinsic or extrinsic fluorophores. Several biological molecules contain naturally occurring fluorophores (intrinsic fluorophores). Tryp-tophan is the most highly fluorescent amino acid in proteins and contributes more than 90% of the fluorescence of a protein. Proteins absorb at 280 nm, and the emission maximum ranges between 320 and 350 nm. Though tyrosine is fluorescent in solution, its emission in a protein is weaker. Nucleotides and nucleic acids are not fluorescent except yeast t-RNAphe, which contains a highly fluorescent Y base. NADH is highly fluorescent, with absorption and emission maxima at 340 nm and 450 nm, respectively, but NAD+ is nonfluorescent. The quantum yield of NADH increases 4-fold when bound to a protein. FMN and FAD are fluorescent with absorption and emission maxima at 450 nm and riboflavin at 515 nm [2].

When the intrinsic fluorescence of a macromolecule is not adequate, external fluorophores are conjugated to them to improve spectral properties. In most of the biological assays extrinsic fluorophores conjugated with biomolecules have been used. Fluorescein, rhodamine, Texas Red, and 4,4-difluro-4-bora-3a,4a-diazo-s-indacene (BODIPY) dyes have been widely used for labeling proteins and nucleic acids. These fluorescent dyes have longer wavelengths of excitation and emission that minimizes the background fluorescence of biological samples. Some fluorophores such as coumarin derivatives, and dansyl chloride are excited with shorter wavelengths. Dansyl chloride is a very widely used fluorophore to label proteins and amines. Various derivatives of these fluorescence dyes are available that react with a primary amino group, the e-amino group of lysine, —SH group of cysteine, —OH, or —COOH groups of amino acids, and —OH or phosphate group of sugar moiety in nucleotides. ATP derivatives with etheno bridge (e-ATP derivatives) or lin-benzo AMP are highly fluorescent and retain hydrogen bonding properties and can be used for labeling nucleotides. 1-anilino-

8-naphthalenesulfonic acid (1,8-ANS), to-anilino-8-naphthalenesulfonic acid (bis-ANS), and 2-p-toluidinyl-naphthalene-6-sulfonic acid (2,6-TNS) are non-fluorescent in water but highly fluorescent in nonpolar solvents and when bound to proteins at hydrophobic pockets. Lipids in the membranes can be labeled with

9-vinyl anthracene, 1,6-diphenylhexatriene, or perylene. The probes are insoluble in water and partition into the lipid layer of membranes [2].

A. Fluorescence Intensity Screens

The readout of these assays is either an increase or a decrease in the fluorescence intensity. Fluorogenic assays and fluorescence quench relaxation assays are manifested in the increase of fluorescence intensity, whereas fluorescence quench assays show a decrease [3].

1. Fluorogenic Assays

The reactants (such as methylumbelliferyl derivatives, ANS, bisANS, nucleic acid specific dyes) of the assay are not fluorescent, but the products generated are fluorescent and measured as an increase in fluorescence intensity. P-Glucuron-idase, nuclease, and polymerase assays are discussed here.

Instrumentation. Several fluorescence plate readers from different manufacturers that can read fluorescence of 96- and 384-well microtiter plates with stackers are available. A few being used in the author's lab are: Victor 1420 (Wallac, Gaithersburg, MD), Analyst and Acqueyst (LJL BioSystems, Sunnyvale, CA), CytoFluor 4000 (PerSeptive Biosystems, Framingham, MA), Titer-tek (Flow Laboratories, AL), Spectramax Gemini (Molecular Devices), and HTS 7000 (Perkin-Elmer Corp., Norwalk, CT). The fluorescence assay can be fully automated using a robot, a liquid handler system, and a plate reader, and the throughput can be increased to 50,000 to 100,000 compounds per day.

P-Glucuronidase Assay. In the homogeneous fluorogenic assay for human P-glucuronidase the fluorogenic substrate, 4-methylumbelliferyl-d-glucuro-nide (MUG) is hydrolyzed to fluorescent 4-methyl umbelliferone (4-MeU) (Fig. 1) [4,5]. The product 4-MeU is fluorescent only when the hydroxyl group is ionized (the pKa of this hydroxyl group is 8-9), and maximal fluorescence is obtained at pH > 10. The enzyme activity is proportional to the fluorescence signal and is measured in a fluorescence plate reader with excitation at 355 nm and emission at 465 nm. This fluorogenic assay was found to be 100-fold more sensitive than the colorimetric methods. The P-glucuronidase assay can be minia-

Figure 1 Fluorogenic P-glucuronidase assay. The substrate 4-methylumbelliferyl-D-glucuronide is nonfluorescent. The product 4-methylumbelliferone formed is also non-fluorescent but becomes fluorescent in alkaline solution. Fluorescence can be read in a fluorescence plate reader with excitation at 355 nm and emission at 465 nm.

Figure 1 Fluorogenic P-glucuronidase assay. The substrate 4-methylumbelliferyl-D-glucuronide is nonfluorescent. The product 4-methylumbelliferone formed is also non-fluorescent but becomes fluorescent in alkaline solution. Fluorescence can be read in a fluorescence plate reader with excitation at 355 nm and emission at 465 nm.

turized into 384-well plate, and the signal is comparable to that of 96-well microplate (Fig. 2). The fluorimetric P-glucuronidase assay is a simple, homogeneous, robust assay that does not involve any separations and washes. The assay is amenable for miniaturization in high density plates (384- and 1536-well plates).

Nuclease and Polymerase Assays. Molecular Probes developed several dyes (nonfluorescent or low intrinsic fluorescence) that specifically interact with double strand DNA (dsDNA), single strand DNA (ssDNA) or RNA and, upon binding to the nucleic acids, exhibit several-fold fluorescence enhancement and quantum yield increases. PicoGreen interacts with dsDNA and produces a fluorescence signal while ssDNA and RNA in the sample do not contribute to this fluorescence signal. PicoGreen is used for detection of picogram level of dsDNA in solution [6]. OliGreen specifically binds to ssDNA and oligonucleotides and produces a fluorescence signal. RiboGreen binds specifically to RNA and has

Figure 2 Comparison of P-glucuronidase screen in (A) 384-well and (B) 96-well format. Increasing concentrations of MUG were incubated with 3 or 10 ng P-glucuronidase in acetate buffer, pH 4.8, at room temperature in a total volume of 15 or 50 ||L in a 384-or 96-well micro plate, respectively. The reaction was terminated with the addition of 15 or 50 |L stop buffer (0.4 M Na2CO3, pH 10) to each well in a 384- or 96-well micro plate respectively. The 96-well plate was read in Titertek fluorescence reader and the 384-well plate was read in Victor 1420 (Wallac). The saturation binding curves were similar

Figure 2 Comparison of P-glucuronidase screen in (A) 384-well and (B) 96-well format. Increasing concentrations of MUG were incubated with 3 or 10 ng P-glucuronidase in acetate buffer, pH 4.8, at room temperature in a total volume of 15 or 50 ||L in a 384-or 96-well micro plate, respectively. The reaction was terminated with the addition of 15 or 50 |L stop buffer (0.4 M Na2CO3, pH 10) to each well in a 384- or 96-well micro plate respectively. The 96-well plate was read in Titertek fluorescence reader and the 384-well plate was read in Victor 1420 (Wallac). The saturation binding curves were similar and the Kms obtained were also similar.

been used for quantitation of RNA in solution [7]. Nuclease or polymerase assays using the specific nucleic acid binding dyes can be developed.

RNase H hydrolyzes the RNA strand from the RNA-DNA hybrid substrate. A homogeneous fluorescence RNase H assay can be developed with PicoGreen dye. In this assay, the substrate poly r(A)-d(T)12-i8 is incubated with the RNase H enzyme at 37°C for 60 min. The reaction is terminated with the addition of PicoGreen dye solution and read in a fluorescence plate reader. The dye binds to DNA in the intact DNA-RNA duplex, resulting in a fluorescence signal. As the RNA strand is hydrolyzed with the enzyme action, the double strand hybrid is depleted, resulting in a decrease in the dye bound to the substrate. Thus with the RNase H activity the fluorescence intensity is decreased.

A DNA polymerase assay can be developed wherein the fluorescence signal increases with enzyme activity. In this assay, the dsDNA synthesized in the enzyme reaction binds the dye and increases the fluorescence signal, whereas the ssDNA substrate does not bind. These assays can be performed in 96- or 384-well plate format or even in higher density plates. A limitation of these assays is the identification of false positives with compounds that interact with the DNA-RNA by intercalation and interference from colored compounds. Nevertheless, this nonradioactive, simple, homogeneous assay can be used for rapid primary screening, and the false negatives can be eliminated in the secondary screening.

2. Fluorescence Quench Assays

When a fluorescent molecule is constrained by covalent modification of the reactive groups (substrate), the fluorescence is quenched. During the assay reaction, if the covalent bond of the substrate is cleaved, free fluorescent dye is released, and the fluorescence intensity increases due to relaxation of quenching.

Peptidase Assay. Several amine containing dyes, 7-amino-4-methylcou-marin, 7-amino-4-chloromethylcoumarin, 6-aminoquinoline, rhodamine 110 (R-110), N-(4-chloromethyl)benzoyl rhodamine 110, 5-(and 6-)chloromethylrho-damine 110, and 6-amino-6-deoxyluciferin (Molecular Probes), when covalently linked to amino acids or peptides, change the spectral properties and cause the fluorescence to be quenched [6].

Rhodamine 110 exhibits spectral properties similar to fluorescein with excitation and emission X of 496 and 520 nm, respectively. Several bisamide derivatives of rhodamine 110 have been used as specific substrates for protease activity in solution and living cells [6]. These substrates contain peptides covalently linked to the two amino groups of rhodamine, thereby suppressing its absorption and fluorescence; thus the fluorescence of R-110 is quenched in these substrates (Fig. 3). The nonfluorescent bisamide R-110 substrate is cleaved to give fluorescent monoamide, and further cleavage yields more fluorescent R-110 [6]. The

Bisämide Monoamide Rhodamine- 110

(Non fluorescent) (Fluorescent) (Fluorescent)

Figure 3 Fluorogenic peptidase assay. In this peptidase assay the substrate, a nonfluo-rescent bisamide of rhodamine 110, was cleaved to the fluorescent monoamide and then to the highly fluorescent free rhodamine 110.

Bisämide Monoamide Rhodamine- 110

(Non fluorescent) (Fluorescent) (Fluorescent)

Figure 3 Fluorogenic peptidase assay. In this peptidase assay the substrate, a nonfluo-rescent bisamide of rhodamine 110, was cleaved to the fluorescent monoamide and then to the highly fluorescent free rhodamine 110.

fluorescence intensity of the monoamide derivative and R-110 is constant between pH 3 and pH 9.

3. Comments

Fluorescence intensity assays are simple assays that can be readily miniaturized. Total fluorescence is measured, and these assays give little information for design of assay and quality control. These assays have strong interference from test compound inner-filter and autofluorescence effects, which are difficult to detect and to correct for.

B. Fluorescence Polarization Screens

Fluorescence polarization (FP) is a homogeneous technique in which FP signal is proportional to the molecular size of the fluorescent molecule in defined conditions.

1. Theory of FP

When a fluorescent sample is excited with a polarized light, the emission from the sample is polarized. Polarization of a fluorophore is the result of its orientation relative to the direction of the polarized excitation. When fluorescent molecules in solution are excited with polarized light, the degree to which the emitted light retains polarization reflects the rotation that the fluorophore underwent during the interval between absorbance and subsequent emission. In 1926 Perin first described the utility of FP to study the molecular interactions in solution [8]. FP instrumentation was developed by Weber [9] and adapted to homogeneous assays by Dandliker [10]. FP is a powerful technology for the determination of molecular interactions in solution [9-15]. The polarization value of a molecule is proportional to the molecule's rotational relaxation (correlation) time and is described by the Stokes equation, p=^ (1) RT

where p is rotational relaxation time (the time required to rotate through 68.5°), n is the viscosity of the medium, V is the molecular volume of the molecule, R is the gas constant, and T is the temperature. Therefore if viscosity and temperature are held constant, polarization is directly proportional to molecular volume. Polarization and anisotropy are commonly used to describe the molecular interactions in solution.

The fluorescence polarization (P) is defined as

Anisotropy (r) is defined as

where Ij is the intensity of emission parallel to the plane of excitation light and I± is the intensity of emission perpendicular to the plane of excitation. Polarization and anisotropy can be interconverted using the following equations [2]:

Polarization and anisotropy use the same measurements [Eqs. (2) and (3)]. Although anisotropy values are preferred, FP is the most common technology used in HTS, hence only FP will be discussed here. All the FP readers measure FP in milli P (mP) (1 polarization unit = 1000 mP). The total fluorescence intensity (I) can be determined from the same data from the equation I = Ij + 2I±. Polarization (P), being a ratio of emission light intensities, is a dimensionless entity and does not depend on the intensity of the emitted light or on the concentration of the fluorophore. If the fluorophore is small, it rotates or tumbles faster, and the resulting emitted light is random with respect to the plane of polarization (depolarized) and will have lower FP value (Fig. 4). If the fluorophore is large, it remains relatively stationary, and the emitted light will remain polarized and have higher FP signal [13,14]. Theoretically, the minimum FP possible is 0 and the maximum is 500 mP for fluorescein. However, the real experimental minimum observed is 40-80 mP for small molecules and the experimental maximum for large molecules is 100-300 mP [14,15]. This window of FP signal between the minimum and the maximum is sufficient because the ratiometric FP signal

Figure 4 Principle of fluorescence polarization. Top panel: A small fluorescent molecule in solution rotates rapidly and orients randomly, the emitted light is depolarized, and the polarization value is low. Bottom panel: A small fluorescent molecule binds to a macromole-cule, the resulting fluorescent complex rotates slowly in solution, it orients in the plane of polarization, and the emitted light is polarized giving a high polarization value.

Figure 4 Principle of fluorescence polarization. Top panel: A small fluorescent molecule in solution rotates rapidly and orients randomly, the emitted light is depolarized, and the polarization value is low. Bottom panel: A small fluorescent molecule binds to a macromole-cule, the resulting fluorescent complex rotates slowly in solution, it orients in the plane of polarization, and the emitted light is polarized giving a high polarization value.

is highly reproducible. Unlike other assays where the robustness of the assay depends on the magnitude of the signal-to-noise ratio, in FP assay AP (the highest signal-lowest signal) is important. A AP of 100 mP in a FP assay is considered a good assay and > 150 mP is considered a robust assay.

FP is a homogeneous technology consisting of simple mix reagents and read format. FP can be easily automated. The reactions are very rapid, reaching equilibrium very quickly. Fluorescein derivatives are the most common fluorescent derivatization reagents for covalent labeling. Fluorescein is a well-studied molecule that has high absorptivity, excellent fluorescence quantum yield, and good water solubility, and its excitation maximum closely matches the 488 nm spectral line of the argon-ion laser. A number of other fluorophore (BODIPY®, Texas Red™, Oregon Green®, Rhodamine Red™, Rhodamine Green™, etc.) derivatives are commercially available with various chemistries for making fluorescent bioconjugates [6]. Many biotechnology labs have been synthesizing fluorescent derivatives of custom peptides and nucleic acids, and a few ready made fluorescent compounds are available with some vendors. The reagents are stable, and large batches required for a screen can be prepared at one time. In FP assays only one tracer is needed. FP is free from interferences, independent of intensity, and insensitive to colored compounds because it is a ratiometric measurement.

2. Instrumentation

The first single-tube bench-top instrument, the FPM-1, was developed by Jolley Consulting and Research Inc. (Grayslake, IN) for measuring FP signal for homogeneous assays for drug screening. Beacan 2000 (PanVera Corp., Madison, WI) is another single-tube bench-top instrument. Now FP plate readers from several manufacturers are available. FPM-2 from Jolley Consulting and Research Inc. reads 96-well plates; Polarion from Tecan (Research Triangle Park, NC), Polarstar from BMG LabTechnologies (Durham, NC), and Analyst from LJL Biosystems (Sunnyvale, CA) read both 96- and 384-well plates, and Acqueyst from LJL BioSystems reads 1536-well plates. These FP instruments are very sensitive in measuring fluorescence intensity and FP signals. Polarstar and Analyst are multimode signal detection systems that can read fluorescence intensity, fluorescence polarization, time-resolved fluorescence, and luminescence. All the FP plate readers are robot-friendly for transport and communication.

A simplified schematic diagram for Analyst is given in Fig. 5. In microplate readers, the excitation and emission energy can be focused through the meniscus of the samples in the microplate wells. Analyst achieves almost identical performance in both 96- and 384-well plates by using SmartOptics, which selects and configures light sources, filters, detectors, and optical paths and analyzes light from the same sensed volumes. SmartOptics can place the sensed volume in the middle of the well or at the bottom of the well for cell based assays.

3. Types of FP Assays

FP assays can be classified into three groups. The first group represents assays that acquire FP signal by binding a small molecule containing a fluorophore to a macromolecule and forming a larger fluorophore adduct, e.g., protein-DNA, antigen-antibody, DNA-DNA, DNA-RNA, and protein-protein interactions [12,14-17] and receptor-ligand binding [18-20]. The second group represents

Figure 5 Schematic of optical pathway of Analyst™. The optical path for Analyst™ in fluorescence polarization mode is given. Monochromatic light passes through filter and polarizer and the polarized light excites the sample. The emitted light is passed through horizontal and vertical polarizers and an emission filter and measured in a PMT detector. (Courtesy of LJL Biosystems.)

Figure 5 Schematic of optical pathway of Analyst™. The optical path for Analyst™ in fluorescence polarization mode is given. Monochromatic light passes through filter and polarizer and the polarized light excites the sample. The emitted light is passed through horizontal and vertical polarizers and an emission filter and measured in a PMT detector. (Courtesy of LJL Biosystems.)

the assays that incur loss of polarization signal due to cleavage of relatively larger fluorescent molecules (fluorophore containing macromolecules) to smaller fluorescent molecules, e.g., nuclease, helicase, and protease assays [13,21-23]. The third group represents facilitated (indirect) assays in which the fluorescent re-actant molecule is coupled to antibody with acquisition or loss of FP signal, e.g., protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). FP has been extensively used in the last decade in clinical laboratories in competitive immunoassays for the detection of drugs and hormones [13-15]. FP assays representative of each of these groups are discussed below in detail.

FP Protein Tyrosine Kinase Assay. Seethala and Menzel have developed the first robust FP-PTK assay [14-16]. In the direct FP-PTK assay, the phosphor-ylated fluorescein-peptide (fl-phos-peptide) product formed in the reaction is measured by immunocomplexing with the antiphosphotyrosine antibody (PY antibody) which results in an increase in the FP signal (Fig. 6). The direct FP-PTK assay can only be used with a peptide substrate and requires large amounts of antiphosphotyrosine antibody. To overcome these problems, the FP-PTK competition immunoassay was developed [15,16]. In this assay, phosphorylated pep-tide or protein produced by kinase reaction competes with the fluorescent phos-

Figure 6 FP protein tyrosine kinase assay methods. In the direct FP-PTK assay, a fluo-rescein-peptide substrate is incubated with PTK, Mg2+-ATP, and antiphosphotyrosine (PY) antibody [14]. The phosphorylated fluorescein-peptide (fl-phos-peptide) product is immu-nocomplexed with the PY antibody, resulting in an increase in the FP signal. The signal is proportional to the phosphorylated product formed. In the FP-PTK competition immunoassay [15,16], phosphorylated peptide or protein produced by a kinase reaction will compete with a fluorescent phosphopeptide used as a tracer for immunocomplex formation with PY antibody. In this format kinase activity results in a loss of the FP signal, and the FP signal is inversely proportional to the phosphorylated product formed in the reaction.

Figure 6 FP protein tyrosine kinase assay methods. In the direct FP-PTK assay, a fluo-rescein-peptide substrate is incubated with PTK, Mg2+-ATP, and antiphosphotyrosine (PY) antibody [14]. The phosphorylated fluorescein-peptide (fl-phos-peptide) product is immu-nocomplexed with the PY antibody, resulting in an increase in the FP signal. The signal is proportional to the phosphorylated product formed. In the FP-PTK competition immunoassay [15,16], phosphorylated peptide or protein produced by a kinase reaction will compete with a fluorescent phosphopeptide used as a tracer for immunocomplex formation with PY antibody. In this format kinase activity results in a loss of the FP signal, and the FP signal is inversely proportional to the phosphorylated product formed in the reaction.

phopeptide tracer for PY antibody. In this format, kinase activity results in a loss of the FP signal.

In the FP-PTK competition assays, a peptide substrate (at about Km concentration) is incubated with PTK (Lck), 0.1 mM ATP, 5 nM fl-phosphopeptide as tracer, and PY-54 phosphotyrosine antibody (0.25-1.00 |g) in the assay buffer in a final volume of 25 or 100 |l in a 384- or 96-well plate, respectively. After incubation at room temperature for 30 min, the plate is read in a FP reader. The affinity of fl-phosphopeptide was highest with PY antibody ascites fluid and monoclonal antibody PY54 [14-16] among different monoclonal and polyclonal anti-PY antibodies tested (Fig. 7A). The fl-phos-peptide binding to PY 54 is rapid and reached equilibrium in less than 5 min, and it remained unchanged for at least 60 min (Fig. 7B). The dissociation of PY antibody-fl-phosphopeptide com-

Figure 7 Optimization of FP protein tyrosine kinase assay. (A) FP signal with different PY antibodies. (B) Association of fluorescein labeled tyrosine-phosphorylated Lck-peptide (fl-phos-peptide) with 10 |g of PY antibody at different concentrations of peptide substrate. (C) Dissociation of PY antibody-fl-phos-peptide complex by phos-peptide as a function of time. Dependence of Lck activity on (D) Lck, (E) Lck peptide, and (F) ATP concentrations. (From Ref. 16.)

Figure 7 Optimization of FP protein tyrosine kinase assay. (A) FP signal with different PY antibodies. (B) Association of fluorescein labeled tyrosine-phosphorylated Lck-peptide (fl-phos-peptide) with 10 |g of PY antibody at different concentrations of peptide substrate. (C) Dissociation of PY antibody-fl-phos-peptide complex by phos-peptide as a function of time. Dependence of Lck activity on (D) Lck, (E) Lck peptide, and (F) ATP concentrations. (From Ref. 16.)

plex was rapid, and complete dissociation was achieved with phospho-Lck peptide within 5 min (Fig. 7C). PTK activity showed a good concentration dependence on Lck, ATP, Lck-peptide, and enolase (Figs. 7D-F).

Inhibition by staurosporine, a potent nonspecific PTK inhibitor, and by PP, a specific Lck/Fyn inhibitor competitive with ATP [24], were evaluated both at 5 and 20 |M ATP (Fig. 8). The IC50 for staurosporine was 30 and 110 nM and for PP was 70 and 300 nM at 5 and 20 |M ATP, respectively, and these values are comparable to the values obtained by parallel 32PO4 transfer assay and FP direct assay. In addition, results with a panel of proprietary PTK inhibitors suggested that the FP competition immunoassay can successfully detect inhibitors of Lck with the same rank order of potency.

This FP-PTK assay is very simple, nonradioactive, and highly sensitive and does not involve separation of substrate and product. A variation of this method would also be suitable for the assay of phosphatases and has been successfully so used. The simplicity and speed of this method makes FP-PTK and FP-phosphatase assay ideal for HTS. The advantages of this assay over other more commonly used kinase assays such as 32PO4 transfer assay, ELISA, or DELFIA

Figure 8 FP-PTK assay validation. To validate the FP-PTK competition immunoassay inhibition by staurosporine, a potent nonspecific protein kinase inhibitor, and PP, a specific Lck/Fyn competitive inhibitor, inhibition was evaluated at 5 and 20 ||M ATP and compared with a concurrently run 32PO4 transfer assay. The IC50s obtained by both the FP competition assay and the 32PO4 transfer assay were similar. (From Ref. 16.)

Figure 8 FP-PTK assay validation. To validate the FP-PTK competition immunoassay inhibition by staurosporine, a potent nonspecific protein kinase inhibitor, and PP, a specific Lck/Fyn competitive inhibitor, inhibition was evaluated at 5 and 20 ||M ATP and compared with a concurrently run 32PO4 transfer assay. The IC50s obtained by both the FP competition assay and the 32PO4 transfer assay were similar. (From Ref. 16.)

include the use of nonisotopic substrates and its being a simple one-step assay, without separation, precipitation, washing, or processing steps after incubation. This method can easily be automated for high throughput drug discovery screening. PTK assay kits based on this FP assay described here are now commercially developed by LJL BioSystems and PanVera Corp.

FP Serine/Threonine Kinase Assay. Several nonradioactive PTK assays based on FP, HTRF, DELFIA, ELISA, and ECL formats have been described that utilize the high affinity anti-PY specific antibody [14-16]. These approaches cannot be applied for serine/threonine kinase assays due to the unavailability of a specific, high affinity antibody for phosphoserine and phosphothreonine. Recently, a FP assay for general kinase assay has been described [17] in which the peptide substrate is thiophosphorylated with ATPyS by protein kinase, followed by biotinylation of the thiophosphate group with iodoacetyl LC-biotin, and finally incubation with streptavidin and determination of the FP signal (Fig. 9). The FP signal increased and was directly proportional to the product formed. The Ki obtained for H-89, an ATP-competitive inhibitor of protein kinase A, was calculated to be about 60 nM, which is similar to that reported. The drawback of this FP method is that biotinylation of the thiophosphate group of the peptide takes a long time (~ 8 hr). Nevertheless, the assay can be adapted to the microtiter plate and can be used as a homogeneous assay after some optimization. It is a better alternative to the 32PO4 transfer assay.

Recently, a FP assay for protein kinase C using a specific monoclonal anti-phosphoserine antibody has been described [18]. A selective substrate peptide containing ser or thr is phosphorylated with ATP using PKC along with diacyl-glycerol and phosphatidylserine. After incubation, the reaction is stopped with the addition of EDTA, fluoresceinylated phosphopeptide tracer and antipeptide antibody and read in a plate reader. Phosphorylated product competes with the tracer, and the enzyme activity is inversely proportional to the FP signal. FP-PKC kits are available from LJL BioSystems and PanVera Corp.

FP-RNase H Screen. RNase hydrolizes RNA strands of RNA-DNA hybrid. Fl-RNA-DNA-biotin hybrid substrate is prepared by first synthesizing a 52 mer fluorescein labeled RNA transcript from Hind III linearized pSP65 cDNA using fluorescein 12-UTP. A complimentary 5'-biotin-52-mer oligo DNA is synthesized (bio-DNA) and annealed to the prepared pSP65 fl-RNA. The FP signal of fl-RNA-bio-DNA hybrid, upon binding to avidin, increased from 105 to 350 mP. The AP of 245 mP is a robust signal for a FP assay. RNase H reaction was carried out by incubation of fl-RNA-bio-DNA hybrid with HIV RNase H (HIVreverse transcriptase/RNase H, Worthington) at pH 8.0 and 37°C in a total volume of 50 |L. The reaction was terminated with the addition of stop buffer (50 |L) containing 20 mM MOPS pH 7, 20 mM EDTA and 10 |g of avidin, incubated for a further 15 min, and read in a FP reader. With the enzyme action, the

Fluorescein-Leu-Arg-Arg-Ala-Ser-Leu-Gly -stepl

ATPyS Protein Kinase A

Fluorescein-Leu-Arg-Arg-Ala-Ser-Leu-Gly -step2

Was this article helpful?

0 0

Post a comment