EP0938586A1 - Mesure de l'activite de la kinase par polarisation de fluorescence - Google Patents

Mesure de l'activite de la kinase par polarisation de fluorescence

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Publication number
EP0938586A1
EP0938586A1 EP97952171A EP97952171A EP0938586A1 EP 0938586 A1 EP0938586 A1 EP 0938586A1 EP 97952171 A EP97952171 A EP 97952171A EP 97952171 A EP97952171 A EP 97952171A EP 0938586 A1 EP0938586 A1 EP 0938586A1
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EP
European Patent Office
Prior art keywords
measuring
peptide
fluorescence polarization
polarization
enzyme
Prior art date
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EP97952171A
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German (de)
English (en)
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EP0938586A4 (fr
Inventor
Thomas J. Burke
Randall E. Bolger
Gregory Parker
Rebecca P. Schall
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Life Technologies Corp
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Panvera Corp
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Publication of EP0938586A1 publication Critical patent/EP0938586A1/fr
Publication of EP0938586A4 publication Critical patent/EP0938586A4/fr
Withdrawn legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6842—Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the field of the invention is the detection of phosphorylated amino acids using a fluorescence polarization (anisotropy) competition assay.
  • the process of the invention detects and measures protein kinase activities and to monitor phosphatases.
  • the assay can be used to quantitatively measure phosphorylated amino acids in extracts.
  • Kinases are enzymes that catalyze the transfer of a phosphate molecule from a nucleotide triphosphate to a substrate such as an amino acid.
  • a substrate such as an amino acid.
  • the amino acids serine, threonine, histidine, and tyrosine are the phosphate acceptors in proteins and ATP is the phosphate donor.
  • Kinases play a role in virtually all regulated cell pathways, from ion transport to metabolic pathways to DNA replication to developmental differentiation. For example, phosphorylation of a protein may induce activation via a conformational change or it may initiate a cascade of molecular binding events.
  • phosphorylation of a protein may induce activation via a conformational change or it may initiate a cascade of molecular binding events.
  • Several human pathologies such as diabetes, cancer, and the allergic response have been directly linked to faulty kinase regulation in cells.
  • a mutated or missing protein can change a regulated pathway to be constitutively 'on' or OfF, disrupting the steady state balance of biological control.
  • Many of the extracellular receptors transmit information to the nucleus via pathways involving kinases. For example, when interleukins and cytokines bind to the surface of the cell, their effects are typically modulated via kinases.
  • Epidermal growth factor receptor, nerve growth factor receptor, platelet derived growth factor receptor, and fibroblast growth factor receptor all involve signal transduction via kinase pathways. Because kinases play such a fundamental role in cellular control, they are often targets for the development of new therapeutics. New drugs are sought which can restore control to faulty regulatory systems and diminish the pathological effects.
  • kinase activity is measured using radioisotopes such as phosphorus-32 or phosphorus-33.
  • radioisotopes such as phosphorus-32 or phosphorus-33.
  • Using radiation provides for very sensitive assays but creates biological hazards and expensive disposal costs.
  • a radioactive phosphate from radioactive ATP is incorporated into the amino acid via the kinase reaction.
  • the unincorporated radioactive ATP is removed with a filter binding separation.
  • the radioactivity that remains bound to the filter is counted in a scintillation counter and is then used to calculate protein kinase activity.
  • Radioactive method called the SPA (scintillation proximity assay) can also be used for measuring kinase activity.
  • the radioactive substrate is brought is close proximity to a molecule which amplifies the energy emission signal from the radioactive source. While this method is still radioactive and requires several molecules to be covalently labeled, it does not require the separation step of the filter binding assay.
  • Non-radioactive methods have been developed but most require separation or washing steps or some reaction components need to be immobilized.
  • the kinase substrate peptide or protein
  • the kinase reaction is then performed and a tagged antibody is used to detect the phosphorylated amino acid.
  • a detection system is then used to detect the tagged primary antibody and amplify the signal using systems such as alkaline phosphatase or horseradish peroxidase.
  • Time-resolved fluorescent assays have also been developed to measure kinase activity.
  • fluorophores with very long fluorescent lifetimes such as the lanthanide chelates are used because their fluorescence emission allows detection long after most other fluorophores have emitted the light. They also can be coupled with a receptor molecule to capture the energy released during emission.
  • These coupled time-resolved fluorescent assays offer good sensitivity if a laser light source is used.
  • a significant drawback is that two molecules are labeled in this method and the chemicals that are required are not widely available.
  • Fluorescence polarization is a versatile laboratory technique for measuring equilibrium binding, nucleic acid hybridization, and enzymatic activity. Fluorescence polarization assays are homogeneous in that they do not require a separation step such as centrifugation, filtration, chromatography, precipitation or electrophoresis. Assays are done in real time, directly in solution and do not require an immobilized phase. Polarization values can be measured repeatedly and after the addition of reagents since measuring the polarization is rapid and does not destroy the sample. Generally, this technique can be used to measure polarization values of fluorophores from low picomolar to micromolar levels.
  • a fluorescently labeled molecule When a fluorescently labeled molecule is excited with plane polarized light, it emits light that has a degree of polarization that is inversely proportional to its molecular rotation. Large fluorescently labeled molecules remain relatively stationary during the excited state (4 nanoseconds in the case of fluorescein) and the polarization of the light remains relatively constant between excitation and emission. Small fluorescently-labeled molecules rotate rapidly during the excited state and the polarization changes significantly between excitation and emission. Therefore, small molecules have low polarization values and large molecules have high polarization values. For example, a fluorescein-labeled peptide has a relatively low polarization value but when the peptide is bound to a very large protein, it has a high polarization value.
  • Fluorescence polarization is defined as:
  • Int is the intensity of the emission light parallel to the excitation light plane and Intj_ is the intensity of the emission light perpendicular to the excitation light plane.
  • TM being a ratio of light intensities, is a dimensionless number.
  • the rotational relaxation time is small ( ⁇ l nanosecond) for small molecules (e.g. fluorescein) and large ( «100 nanoseconds) for large molecules (e.g. immunoglobulins) (Jolley, 1981). If viscosity and temperature are held constant, rotational relaxation time, and therefore polarization, are directly related to the molecular volume. Changes in molecular volume may be due to interactions with other molecules, dissociation, polymerization, degradation, hybridization, or conformational changes of the fluorescently labeled molecule.
  • fluorescence polarization has been used to measure enzymatic cleavage of large fluorescein labeled polymers by proteases, DNases, and RNases. It also has been used to measure equilibrium binding for protein/protein interactions, antibody/antigen binding, and protein/DNA binding. In this patent, we will show that fluorescence polarization is a simple and economical way to measure protein kinase activity.
  • a method for measuring the presence of a phosphorylated amino acid of a compound by competition comprising: measuring the fluorescence polarization of a reporter complex comprising a first phosphorylated amino acid bound to a binding molecule; adding a substance containing a second phosphorylated amino acid to compete for the binding molecule; incubating the solution; measuring the fluorescence polarization of the solution during step c); and, comparing the fluorescence polarization measurements.
  • a method for measuring enzyme activity for attaching and cleaving a phosphate with a compound by competition comprising: measuring the fluorescence polarization of a reporter molecule comprising a phosphorylated amino acid; adding an enzyme, incubating the solution; measuring the fluorescence polarization of the solution during step c); and, comparing the fluorescence polarization measurements.
  • a kit utilizing the method of claim 1 for measuring the amount of phosphorylated molecules in a mixture comprising: instructions for utilizing fluorescence polarization to identify the amount of phosphorylated amino acids in a mixture, a receptacle containing a reporter molecule; and, a receptacle containing a binding protein.
  • a method for measuring enzyme activity of attaching and cleaving a phosphate with a compound comprising: measuring the fluorescence polarization of a reporter molecule in solution with the phosphate. Then adding an enzyme which is a phosphatase or a kinase in the preferred embodiments. Incubating the solution for a time sufficient to allow enzyme activity and measuring the polarization. Finally, comparing the fluorescence polarization of the solution in the first step with the fluorescence polarization measurements in the last step.
  • a method for measuring enzyme activity of attaching and cleaving a phosphate with a peptide comprising: incubating a first phosphate, the peptide, and an enzyme in solution. Then, adding a reporter molecule to the solution and measuring the fluorescence polarization of the reporter molecule. Incubating the solution for a time sufficient to allow for measurement of the fluorescence polarization and comparing the fluorescence polarization measurements.
  • a kit utilizing the method of claim 1 for measuring enzyme activity comprising: instructions for utilizing fluorescence polarization to identify the enzyme activity; a receptacle containing a reporter molecule; and, a receptacle containing a binding protein.
  • FIG. 1 is a cartoon illustrating the direct detection of phosphorylated amino acids using fluorescence polarization.
  • FIG. 2 is a cartoon illustrating competitive detection of phosphorylated amino acids using fluorescence polarization.
  • FIG. 3 is a cartoon illustrating the detection of amino acid dephosphorylation.
  • FIG. 4 is a line graph indicating that as the antibody binds to a fluorescently labeled phosphopeptide, the polarization value increases.
  • FIG. 5 is a line graph showing that antibody bound to a fluorescently labeled phosphopeptide can be competed off with an unlabeled phosphopeptide.
  • FIG. 6 is a bar graph demonstrating that PKC activity can be measured by using competitive fluorescence polarization.
  • FIG. 7 is a line graph showing the quantitative measurement of kinase activity.
  • FIG. 8 is a line graph demonstrating the binding measurements between different antibodies and fluorescein labeled peptides containing phosphotyrosine.
  • FIG. 9 is a line graph illustrating that a competition assay can be used quantitatively to measure the amount of phosphorylated amino acid in a mixture.
  • FIG. 10 is a scatter graph showing a kinase activity measurement using competitive fluorescence polarization.
  • FIG. 11 is a scatter graph showing the autophosphorylation ofan EGF receptor.
  • FIG. 12 is a scatter graph showing a tyrosine kinase assay performed with and without the inhibitor EDTA.
  • FIG. 13 is a scatter graph illustrating a high polarization mixture containing an antibody bound to a fluorescein labeled phosphopeptide.
  • This invention provides a simple, homogeneous, nonradioactive method for detecting phosphorylated amino acids which allows for quantitative measurement of protein kinase and phosphatase activity and for identification of enzyme inhibitors.
  • the method is based on the discriminate recognition of phosphorylated versus unphosphorylated amino acids by proteins such as antibodies or SH2 domains. It is also based on the ability of fluorescence polarization measurements which distinguish between a small fluorescently labeled molecule and the same molecule when it is bound to a large antibody or other protein.
  • Our method depicts a change in fluorescence polarization signal with a change in concentration of phosphorylated amino acid.
  • the change in concentration can be produced by: 1) a protein kinase adding phosphate groups, 2) a phosphatase removing phosphate groups, or 3) utilizing different amounts of an extract which contains phosphorylated amino acids.
  • Detection of modified amino acids is performed using either a direct assay or a competition assay.
  • a direct assay a small fluorescently labeled substrate is phosphorylated and then bound to an antibody. As more of the substrate in the reaction is phosphorylated, then more of it binds to the antibody, and the higher the polarization rises.
  • the increase in the fluorescence polarization signal is directly proportional to the amount of phosphorylated peptide, which in turn is directly proportional to the amount of kinase activity present.
  • a competition assay is performed.
  • the kinase reaction uses standard reagents with no labels for fluorescence or capture and no limitations on substrate size or concentration.
  • the synthesis of the phosphorylated peptide during the reaction is detected by adding a high polarization complex to the reaction.
  • the complex comprises a fluorescently labeled phosphopeptide bound to an antibody.
  • the antibody will reach equilibrium between the phosphorylated amino acids from both sources.
  • the polarization value goes down in proportion to the amount of phosphorylated amino acids made by the kinase reaction.
  • the fluorescence polarization signal is inversely proportional to the amount of kinase activity present.
  • our method can be used to measure the removal of phosphate groups from amino acids.
  • the phosphatase assay is a direct assay: this means that a fluorescently labeled peptide is treated with the phosphatase; an antibody is then added which binds to the peptide; if the phosphate group is still present, the polarization value rises; if the phosphate group is removed, then the polarization remains low.
  • This assay can also be monitored using a simple complete assay where all of the components, including the high polarization mixture, are added to a test tube except the kinase. The kinase may then be added and the starting high polarization decreases as the phosphatase removes phosphate from the peptide.
  • Binding molecule - A molecule that has an affinity for another specific molecule.
  • a binding molecule can be a protein or, more specifically, an antibody specific for a phosphorylated amino acid.
  • Peptide - a molecule made up of 2 or more amino acids.
  • the amino acids may be naturally occurring or synthetic.
  • Reporter complex a fluorescence-emitting compound attached to a phosphorylated amino acid bound to a binding molecule.
  • Reporter molecules - Chemical (organic or inorganic) molecules or groups capable of being detected and quantitated in the laboratory.
  • Reporter molecules include fluorescence-emitting molecules (which include fluoresceins, rhodamines, pyrenes, lucifer yellow, BODLPY®, malachite green, coumarins, dansyl derivatives, mansyl derivatives, dabsyl drivatives, NBD flouride, stillbenes, anthracenes, acridines, rosamines, TNS chloride, ATTO-TAGTM, LissamineTM derivatives, eosins, naphthalene derivatives, ethidium bromide derivatives, thiazole orange derivatives, ethenoadenosines, CyDyesTM, aconitine, Oregon Green, Cascade Blue, and other fluorescent molecules).
  • the reporter molecule comprises a fluorescence-emitting peptide molecule.
  • the pp60c-src C-terminal phosphoregulatory peptide (BIOMOL; Madison Meeting, PA) was fluorescently labeled according to the instructions included with the fluorescein amine labeling kit (PanVera Corporation; Madison, WI). Briefly, 50 mg of the peptide was labeled at 37°C for one hour in a 50 mL reaction containing 5 mL lOx coupling buffer (1 M KPO4, pH 7.0) and 5 mL 20 mM fluorescein. The reaction was then quenched with 5 mL 1 M Tris-HCl (pH 8.0) and incubated at room temperature for 30 minutes. Fluorescein-labeled products were then separated by thin layer chromatography.
  • a short peptide is fluorescently labeled and used as a substrate in the kinase reaction.
  • the kinase adds a phosphate group to the amino acid and then a protein which binds to the phosphate is added to the reaction.
  • the starting fluorescent peptide has a low polarization value and when the protein binds to it, it has a high polarization value. This is a good assay for determining whether or not a peptide is a substrate for a kinase.
  • the substrate for the kinase can be any peptide or protein and size is not a limitation. Also, there is no limitation on the concentration of the peptide or protein.
  • a high polarization complex is the added to the reaction. This complex contains a fluorescently labeled phosphorylated peptide bound to protein.
  • the reaction and high polarization complex are mixed, the phosphorylated amino acids in the reaction compete for binding to the proteins in a high polarization mix.
  • the polarization value of the peptide goes down. In this case the shift in polarization is from a high polarization complex to a low polarization free phosphopeptide.
  • both the kinase reaction and the high polarization complex are mixed together at the beginning of the reaction. As the reaction proceeds, the polarization value goes down.
  • Example 3 the reaction begins with a high polarization mixture, which is a binding protein attached to a fluorescently labeled phosphopeptide.
  • a phosphatase an enzyme that removes phosphate groups from other molecules, is added to the reaction and the polarization value goes down.
  • Phosphatase enzymes are important in cellular regulation because they perform the opposite role of kinases.
  • the shift in polarization (FIG. 4) of a fluorescein-labeled 11 mer peptide (F- RRRVTpSARRS) and fluorescein-labeled 6mer (F-pSAARRS ) upon addition of antiphosphopeptide antibody (anti-GFAP-P, MBL clone YC10) was measured.
  • the anti- GFAP-P was serially diluted in 100 mM potassium phosphate, 100 ⁇ g ml bovine gamma globulin (BGG) in two sets from 4 ⁇ l to 0.016 ⁇ l per reaction.
  • the fluorescein-labeled 6mer and 1 lmer were added to sets 1 and 2, respectively to a final concentration of approximately 0.5 nM.
  • Polarization values were measured after a 30 minute incubation and plotted versus the Log of fluorescein-peptide concentration.
  • FIG. 4 shows that peptide is a substrate for PKC, and the phosphorylated peptide contains phosphoserine. It also shows that by decreasing the size of the peptide, it is possible to get a higher shift in polarization.
  • the monoclonal antibody was supplied by MBL
  • the phosphoserine and phosphothreonine are small molecules compared to phosphotyrosine. To make antibodies against these phosphopeptides, the phosphotyrosine is large enough that it can constitute an epitope for antibody recognition almost by itself, with little recognition of the amino acids surrounding phosphate containing amino acids. In contrast, an antibody prepared against a phosphoserine containing peptide will also recognize the amino acids adjacent to the phosphorylated amino acids. This means that antibodies used in the kinase reactions are very specific to individual peptides.
  • Example 5 An experiment (FIG. 5) was performed to measure the ability of the unlabeled phosphopeptide (GFAP-P) to compete with the fluorescein-labeled GFAP-P (GFAP-P-F) for binding to Anti-GFAP-P.
  • GFAP-P was serially diluted in 16 tubes from 100 ng to 0.2 ng in 100 ⁇ L final volumes. Then 50 pg GFAP-P-F and 1 ⁇ l Anti-GFAP-P were added to each reaction tube. Fluorescence polarization was measured at 10 minutes, 1, and 2 hours in all tubes, data was plotted as polarization versus Log peptide concentration.
  • the peptide contains a phosphoserine, and when bound to an antibody has a high polarization value (100 mP). As increasing amounts of the unlabeled peptide are added, the polarization value decreases. The decrease in polarization is directly proportional to the amount of competitor peptide added.
  • PKC isozymes Beta2, Delta, and Epsilon
  • Kinase reactions were performed containing PKC or buffer, 63 ⁇ g/ml GFAP peptide, 63 ⁇ M ATP, 30 mM HEPES, 6 mM MgC12, 63 ⁇ M CaCl2, phosphatidyl serine (100 ⁇ g/ml) and diacylglycerol (100 ⁇ g/ml). Reactions were incubated at 30°C for 30 minutes, then placed on ice.
  • the amount of phosphopeptide produced was measured by competition with a anti-GFAP-P and GFAP-P-F.
  • a negative control with no reaction added and positive control with no reaction and no antibody added were also performed.
  • Polarization values were measured.
  • the presence of GFAP- P, produced in the kinase reactions was demonstrated by the reduced polarization values in the tubes containing the kinase reactions.
  • the tubes containing no reaction or a mock reaction showed no drop in polarization, while the tube containing no antibody showed the polarization value of the free GFAP-P-F.
  • the duplicate reactions were set up with one reaction being spiked with radioactive ATP and the other reaction performed by fluorescence polarization.
  • the reaction which had no enzyme added did not show incorporation of the radioactive nucleotide.
  • Three of the different PKC isoforms were tested in these reactions. All three of the enzymes showed activity in the radioactive assay.
  • the reactions were allowed to proceed and then the high polarization complex of antibody and phosphopeptide were added to the reactions. The phosphorylated amino acids which were made by the kinase reactions competitively bound to the antibody, thereby releasing the fluorescein labeled phosphopeptide. This caused a decrease in the fluorescence polarization value.
  • the radioactive data for figure 6 showed that the PKC beta II enzymes phosphorylated 0.82% of the substrate, PKC delta phosphorylated 3.27% of the substrate, and PKC epsilon phosphorylated 1.47% of the substrate.
  • the background level where no PKC was added to the reaction showed a level of 0.05% of the substrate being phosphorylated.
  • the method for the radioactive measurement follows:
  • the PKC activity assay was performed by a traditional radioactive protocol to compare with a competitive fluorescence polarization assay.
  • the radioactive ( 32 P) was performed according to the standard protocol with the following alterations: All final concentrations of reagents were identical (although lOO ⁇ l reactions were utilized, not 60 ⁇ l) with the following exceptions: Final ATP concentration of lO ⁇ M (rather that lOO ⁇ M) No EGTA was added Final concentration of 100 ⁇ M CaCl 2
  • Peptide substrate utilized was the GFAP peptide at lOO ⁇ g/ml final
  • This protocol is to provide the reader with the conditions that we use to determine the activity and phospholipid dependence of Protein Kinase C ⁇ .
  • This protein is purified to near homogeneity and may behave differently from crude preparations.
  • the calculated molecular weight of the protein is 83.5 kDa. Apparent molecular weights of 89-96 kDa have been reported in the literature, and the protein may run as a doublet (1).
  • PKC ⁇ is classified as a novel protein kinase C because it does not show calcium dependence, and is activated in vitro by phorbol esters. With the assay described in this protocol, we typically see a two-fold phosphatidylserine stimulation of activity.
  • One unit is defined as the amount of enzyme necessary to transfer 1 nmol of phosphate to the PKC Epsilon ( ⁇ ) substrate peptide (supplied by PanVera, Inc.) in 1 minute at 30° C. Activity Calculation
  • Phospholipid Dependence Phospholipid dependence is shown by following the procedure above and simultaneously running reactions to which no lipid has been added.
  • the reaction volume is made up by adding lipid resuspension buffer in section 3.3 instead of lipid.
  • the reactions without lipid are processed identically to those with lipid.
  • PKC B2 reactions were performed.
  • the reactions contained PKC Beta2 or buffer, 63 ⁇ g/ml GFAP peptide, 63 ⁇ M ATP, 30 mM HEPES, 6 mM MgC12, 63 ⁇ M CaC12, phosphatidyl serine (100 ⁇ g/ml) and diacylglycerol (100 ⁇ g/ml).
  • Reactions were incubated at 30°C for 30 minutes, then placed on ice.
  • the reactions were serially diluted in BGG/P buffer. 10 ⁇ l of a complex of F-GFAP-P and antibody was added to each tube in both sets (enzyme and no enzyme) and then the polarization was measured.
  • the mock reaction had no effect on the complex, while the polarization drop in the complete reaction was dose-dependent on the amount of reaction added.
  • a single kinase reaction was set up and allowed to proceed in phosphorylated the peptide substrate. Different amounts of the kinase reaction were then ended to tubes which contains the high polarization antibody - peptide mixture. As figure 7 shows, larger amounts of the reaction mixture caused a lower polarization value. As a control, the buffer alone was substituted for the kinase reaction mixture. This did not show any significant change in the polarization value.
  • Figures 1-7 dealt with kinases which add phosphate groups to serine and threonine amino acids and with antibodies that binds these phosphopeptides. As shown in FIG. 7, some of the antibodies bind the phosphopeptide more tightly and cause a higher shift in polarization. The higher shift in polarization produces a better assay, because it is easier to measure the decreasing polarization using a competition assay.
  • Figure 4 shows that the different length of peptide can affect the polarization value. This figure shows that different antibodies can also have a significant effect. Therefore, to optimize the assay is necessary to find the best combination of antibody and peptide.
  • Fluorescence polarization changes (FIG. 8) caused by four commercially available anti- phosphotyrosine antibodies were measured using 7.35 nM of the pp60c-src C-terminal phosphoregulatory peptide (BIOMOL; Madison Meeting, PA) as the fluorescent tracer. Each serial dilution was done in final volume of 100 mL in BeaconTM-grade phosphate- buffered saline (PanVera Corporation; Madison, WI) which contains 1.2 mM monobasic potassium phosphate, 8.1 mM dibasic sodium phosphate, 2.7 mM KC1, 138 mM NaCl, 0.02% sodium azide (pH 7.5). Each sample was read at 25°C after a 15 minute equilibration. The antibodies were from Upstate Biotechnology (Lake Placid, NY), Zymed (South San Francisco, CA), and Transduction Labs (Lexington, KY).
  • Figure 8 demonstrates that different antibodies against phosphotyrosine have different binding affinities and can cause different shifts in polarization values.
  • the optimal antibodies give the highest shift at the lowest concentration, and therefore the most sensitive assays.
  • a peptide competition standard curve was generated in final volume of 100 mL BeaconTM- grade phosphate-buffered saline (PanVera Corporation; Madison, WI) which contains 1.2 mM monobasic potassium phosphate, 8.1 mM dibasic sodium phosphate, 2.7 mM KC1, 138 mM NaCl, 0.02% sodium azide (pH 7.5).
  • Each tube also contained 20 nM 4G10 anti-phosphotyrosine monoclonal antibody (Upstate Biotechnology; Lake Placid, NY), 10 nM fluorescent pp60c-src C-terminal phosphoregulatory peptide (BIOMOL; Madison Meeting, PA), and serially diluted, non-phosphopeptide (same sequence as the pp60c-src C-terminal phosphoregulatory peptide) as the competitor peptide (BIOMOL; Plymouth Meeting, PA). Each tube was analyzed at 25°C after a 15 minute equilibration.
  • EGF epidermal growth factor
  • FIG. 10 three reactions were performed using increasing amounts of the EGF receptor, a tyrosine kinase.
  • the reaction and the detection were performed simultaneously. All the kinase reaction components and the high polarization mixture were added to the tube, the reaction started, and a polarization value was measured every few seconds for 30 minutes. As the kinase reaction proceeded, the polarization value went down. The polarization value was lower when higher levels of the kinase were used.
  • these assays can be performed as end-point assays. The results shown in this figure shows that this is the simplest way to perform the assay as long as the presence of the antibody and the fluorescently labeled peptide do not interfere with the kinase reaction.
  • Autophosphorylation of the EGF receptor can also be detected using fluorescence polarization.
  • the 100 mL reaction was assayed under the following conditions: -12.5 nM EGF receptor, 20 mM Hepes (pH 7.4), 2 mM MgC12, 5 mM MnC12, 50 mM Na3VO4, 50 mM ATP, 20 nM 4G10 anti-phosphotyrosine monoclonal antibody (Upstate Biotechnology; Lake Placid, NY), and 10 nM fluorescent pp60c-src C-terminal phosphoregulatory peptide (BIOMOL; Madison Meeting, PA).
  • the change in polarization was measured every 10 seconds (after the addition of the kinase) in Kinetic Mode on a BeaconTM 2000 instrument (PanVera Corporation; Madison, WI) running at 30°C.
  • the kinase adds the phosphate group to another molecule such as a peptide or protein.
  • a peptide or protein a molecule that is called autophosphorylation.
  • autophosphorylation As shown in FIG. 11, no peptide substrate was added to the reaction and the production of phosphotyrosine was measured using the competitive fluorescence polarization assay. A previous control experiment showed that the antibody was not a significant substrate for this kinase (data not shown).
  • EDTA inhibits the EGF receptor kinase activity.
  • the 100 mL reactions (with or without 50 mM BeaconTM-grade EDTA (PanVera Corporation; Madison, WI) were assayed under the following conditions: -12.5 nM EGF receptor, 20 mM Hepes (pH 7.4), 2 mM MgC12, 5 mM MnC12, 50 mM Na3VO4, 50 mM ATP, 20 nM 4G10 anti- phosphotyrosine monoclonal antibody (Upstate Biotechnology; Lake Placid, NY), and 10 nM fluorescent pp60c-src C-terminal phosphoregulatory peptide (BIOMOL; Madison Meeting, PA).
  • the change in polarization was measured every 10 seconds (after the addition of the kinase) in Kinetic Mode on a BeaconTM 2000 instrument (PanVera Corporation; Madison, WI) running at 30°C.
  • the assay could be used to screen for selective inhibitors of kinase activity, especially in a high throughput screen format. These inhibitors could chelate the metal ions like EDTA, or bind to the active site of the enzyme, or bind to the ATP binding site on the kinase.
  • Fluorescence polarization can also be used to measure phosphatase activity.
  • the conditions of each 100 mL assay were: 0.5 U T-cell protein tyrosine phosphatase (New England Biolabs; Beverly, MA), 25 mM imidazole, 50 mM NaCl, 2.5 mM Na 2 EDTA, 5 mM DTT, 100 mg/mL BSA (pH 7.0), 5 nM fluorescent pp60c-src C-terminal phosphoregulatory peptide (BIOMOL; Beverly Meeting, PA), 20 nM 4G10 anti- phosphotyrosine monoclonal antibody (Upstate Biotechnology; Lake Placid, NY).
  • One assay received 1.0 mM of the phosphatase inhibitor Na 3 VO while a another assay did not receive enzyme.
  • the change in polarization was measured every 10 seconds (after the addition of the phosphatase) in Kinetic Mode on a BeaconTM 2000 instrument (PanVera Corporation; Madison, WI) running at 30°C.
  • Phosphatase enzyme was added to a high polarization mixture and as it removed the phosphate from the peptide, the antibody was released, and the polarization value went down, shown in FIG. 13.
  • Two control experiments were performed. One control showed that when no phosphatase was added, the polarization value remained constant. The second control showed that when a phosphatase inhibitor, vanadate, was added to the reaction, the polarization value also remained constant.
  • This assay was performed with all of the reaction and detection components present in a test tube. However, this reaction could also be done as an endpoint assay with aliquots of the reaction taken at incremental time points, or with different reactions started at the same time but terminated at incremental time points.

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Abstract

L'invention concerne un procédé de quantification de l'activité enzymatique de la phosphorylation et la déphosphorylation d'un substrat peptidique ou protéique, qui consiste à mesurer au moyen du phosphate la polarisation de fluorescence d'une molécule reporter à fluorescence dans une solution; à ajouter une enzyme, une kinase ou une phosphatase et à laisser incuber la solution. Il consiste ensuite à mesurer la fluorescence de la solution après que l'enzyme a réagi avec le substrat peptidique ou protéique.
EP97952171A 1996-10-28 1997-10-28 Mesure de l'activite de la kinase par polarisation de fluorescence Withdrawn EP0938586A4 (fr)

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US2983196P 1996-10-28 1996-10-28
US29831P 1996-10-28
PCT/US1997/019570 WO1998018956A1 (fr) 1996-10-28 1997-10-28 Mesure de l'activite de la kinase par polarisation de fluorescence

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EP1036192B1 (fr) 1997-12-05 2003-02-26 PHARMACIA & UPJOHN COMPANY Essais de criblage a haut rendement a base de fluorescence pour proteines kinases et phosphatases
US6656696B2 (en) 1999-02-26 2003-12-02 Cyclacel Compositions and methods for monitoring the phosphorylation of natural binding partners
US6287774B1 (en) 1999-05-21 2001-09-11 Caliper Technologies Corp. Assay methods and system
IL146375A0 (en) * 1999-05-21 2002-07-25 Caliper Techn Corp Fluorescence polarization assays involving polyions
US6472141B2 (en) 1999-05-21 2002-10-29 Caliper Technologies Corp. Kinase assays using polycations
EP1418239B1 (fr) * 1999-05-21 2006-07-12 Caliper Life Sciences, Inc. Essais fluorescents impliquant des polyions pour la mesure des activités enzymatiques
EP1701163A1 (fr) * 1999-05-21 2006-09-13 Caliper Life Sciences, Inc. Essais de polarisation fluorescente impliquant de polyions
GB9923208D0 (en) * 1999-10-01 1999-12-08 Cambridge Drug Discovery Ltd Assay
AU4067301A (en) * 2000-03-02 2001-09-12 Morphochem Ag Process for obtaining inhibitors/activators of an enzyme
GB0007915D0 (en) * 2000-03-31 2000-05-17 Univ Bristol Protein kinase assay
WO2001094614A2 (fr) * 2000-06-07 2001-12-13 Cyclacel Limited Proceder pour observer l'activite d'une enzyme
US6808874B2 (en) 2000-06-13 2004-10-26 Cyclacel Ltd. Methods of monitoring enzyme activity
US20090263821A1 (en) 2004-12-01 2009-10-22 Proteologics, Inc. Ubiquitin Ligase Assays And Related Reagents

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DANDLIKER, WALTER B. ET AL: "Equilibrium and kinetic inhibition assays based upon fluorescence polarization" METHODS ENZYMOL. (1981), 74(IMMUNOCHEM. TECH., PT. C), 3-28, 1981, XP001029241 *
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Seethala K., "A Fluorescence Polarization Tyrosine Kinase Assay for High Throughput Screening", 3rd Annual Conference of The Socienty for Biomolecular Screening, San Diego, CA, Sept. 22-25, 1997 XP002968015 & US 6 203 994 B1 (EPPS DENNIS E ET AL) 20 March 2001 *
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WO1998018956A1 (fr) 1998-05-07

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