PHOSPHATASE OR KINASE ACTIVITY DETECTION COMPOSITION AND DETECTION METHOD

Abstract

The present invention relates to a composition for detecting phosphatase or kinase activity and a method of detecting phosphatase or kinase activity. The kinase or phosphatase activity may be quantitatively measured in real time by using the composition of the present invention.

Claims

1. A composition for measuring kinase or phosphatase activity, the composition comprising (a) zinc ions (Zn.sup.2+), (b) a zinc ion receptor comprising a chelating ligand, and (c) a kinase or phosphatase peptide substrate, and exhibiting a phosphorylation or dephosphorylation detection signal according to a change in a phosphorylation state of the peptide substrate.

2. The composition of claim 1, wherein the peptide substrate comprises any one selected from (a) a fluorescence signal-generating donor fluorophore and (b) a fluorophore acceptor that quenches a fluorescence signal by causing fluorescence resonance energy transfer (FRET) with the donor fluorophore, and the zinc ion receptor comprises any other one not selected from a donor fluorophore and a fluorophore acceptor.

3. The composition of claim 2, wherein the peptide substrate is immobilized on a support.

4. The composition of claim 3, wherein the peptide substrate further comprises biotin, and the support is NeutrAvidin agarose beads.

5. The composition of claim 1, wherein the peptide substrate comprises a polyhistidine, and the zinc ion receptor comprising a chelating ligand is metal nanoparticles surface-modified with a chelating ligand.

6. The composition of claim 5, wherein the metal nanoparticles are any one selected from the group consisting of gold, silver, copper, platinum, palladium, nickel, and iron, or a mixture of two or more thereof.

7. The composition of claim 5, wherein the metal nanoparticles have an average diameter of 2 nm to 50 nm.

8. The composition of claim 5, wherein the chelating ligand is any one or more selected from the group consisting of nitrilotriacetic acid (NTA), ethylene diamine tetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), diethylene triamine pentaacetic acid (DTPA), phenanthroline (PHEN), N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), N-(2-hydroxyethyl)ethylenediamine-N,N′,N′-triacetic acid (HEDTA), and 1,3-bis[bis(pyridin-2-ylmethyl)amino]propan-2-olato (Phos-tag).

9. The composition of claim 5, wherein the polyhistidine consists of 4 to 10 histidines.

10. A method of measuring phosphatase activity, the method comprising the following processes: (i) preparing a composition for measuring phosphatase activity, wherein the composition comprises (a) zinc ions, (b) a fluorophore acceptor as a zinc ion receptor comprising a chelating ligand, and (c) a phosphorylated phosphatase peptide substrate to which a donor fluorophore is bound, and a fluorescence signal of the donor fluorophore is quenched by an interaction between the zinc ions and the fluorophore acceptor; and (ii) detecting recovery of the fluorescence signal having been quenched by FRET by contacting the composition with a phosphatase.

11. A method of measuring kinase activity, the method comprising the following processes: (i) preparing a composition for measuring kinase activity, wherein the composition comprises (a) zinc ions, (b) a fluorophore acceptor as a zinc ion receptor comprising a chelating ligand, and (c) a kinase peptide substrate to which a donor fluorophore is bound, and the kinase peptide substrate comprises one or more dephosphorylated phosphorylation site peptides; and (ii) detecting quenching of a fluorescence signal by FRET by contacting the composition with a kinase.

12. A method of measuring phosphatase activity, the method comprising the following processes: (i) preparing a composition for measuring phosphatase activity, wherein the composition comprises: (a) zinc ions; (b) metal nanoparticles surface-modified so as to have a chelating ligand; and (c) a phosphorylated phosphatase peptide substrate comprising a polyhistidine, and the zinc ions, the metal nanoparticles, and the peptide substrate are self-assembled by interactions therebetween; and (ii) detecting the disassembly of the self-assembled structure of the composition by contacting the composition with a phosphatase.

13. The method of claim 12, wherein the metal nanoparticles comprise any one selected from the group consisting of gold, silver, copper, platinum, palladium, nickel, and iron, or a mixture of two or more thereof.

14. A method of measuring kinase activity, the method comprising the following processes: (i) preparing a composition for measuring kinase activity, wherein the composition comprises: (a) zinc ions; (b) metal nanoparticles surface-modified so as to have a chelating ligand; and (c) a kinase peptide substrate comprising a polyhistidine, and the kinase peptide substrate comprises one or more dephosphorylated phosphorylation site peptides; and (ii) detecting an increase in a self-assembled structure of the composition by contacting the composition with a kinase.

15. The method of claim 14, wherein the metal nanoparticles comprise any one selected from the group consisting of gold, silver, copper, platinum, palladium, nickel, and iron, or a mixture of two or more thereof.

16. A method of screening a phosphatase activity inhibitor, the method comprising the following processes: (i) preparing a composition for measuring phosphatase activity, wherein the composition comprises: (a) zinc ions; (b) metal nanoparticles surface-modified so as to have a chelating ligand; and (c) a phosphorylated phosphatase peptide substrate comprising a polyhistidine, and the zinc ions, the metal nanoparticles, and the peptide substrate are self-assembled by interactions therebetween; and (ii) simultaneously contacting the composition, a phosphatase inhibiting candidate, and a phosphatase, and analyzing whether or not phosphatase activity is decreased; wherein, when the phosphatase activity is decreased by comparing with a control not treated with a candidate, the candidate is determined as a phosphatase inhibiting material.

Description

DESCRIPTION OF DRAWINGS

[0064] FIG. 1 is a view illustrating a method of measuring kinase or phosphatase activity using the self-assembly of metal nanoparticles.

[0065] FIG. 2 illustrates a method of measuring kinase or phosphatase activity using an FRET signal.

[0066] FIG. 3 illustrates measurement results of the size of gold nanoparticles surface-modified with NTA through light scattering.

[0067] FIG. 4 illustrates test results of the self-assembly of metal nanoparticles for a peptide including a polyhistidine and/or a phosphate group or excluding any one thereof.

[0068] FIG. 5 illustrates test results of the self-assembly of metal nanoparticles for various types of metal ions.

[0069] FIG. 6 illustrates test results of the self-assembly of metal nanoparticles according to peptide substrate concentration.

[0070] FIG. 7 illustrates test results of the self-assembly of metal nanoparticles according to zinc ion concentration.

[0071] FIG. 8 illustrates test results of an absorbance ratio (520 nm/700 nm) according to treatment concentration of a phosphatase.

[0072] FIG. 9A illustrates colorimetric assay results of a self-assembly test of metal nanoparticles according to the type of metal ion solution.

[0073] FIG. 9B illustrates colorimetric assay results of a self-assembly test of metal nanoparticles for a peptide including a polyhistidine and/or a phosphate group or excluding any one thereof.

[0074] FIG. 10 illustrates analysis results of an absorbance ratio (520 nm/700 nm) of pep1 according to phosphatase concentration.

[0075] FIG. 11A illustrates an experimental method of agglomeration of gold nanoparticles using plants exhibiting different expression behaviors of PP2A-A1.

[0076] FIG. 11B illustrates experimental results of agglomeration of gold nanoparticles according to the PP2A-A1 expression behavior of plants.

[0077] FIG. 12A illustrates a process of FRET test between QDs and TAMRA-PEPPKA or TAMRA-PEPPKA(p).

[0078] FIG. 12B illustrates measurement results of FRET spectra and FRET ratios (F580/F525) between QDs and TRAMRA-PEPPKA(p) or TAMRA-PEPPKA according to the type of metal ions. Fluorescent spectra of QDs with either (i) TAMRA-LRRApSLG (TAMRA-PEPPKA(p)); or (ii) TAMRA-LRRASLG (TAMRA-PEPPKA) in the presence of different metal ions; (iii) Effect of metal ions on the QD-FRET ratios of TAMRA-PEPPKA(p) (white bar) and TAMRA-PEPPKA (black bar). The FRET ratio was determined by the acceptor (FA) emission area (integrated from 550 to 650 nm) relative to the donor (F.sub.D) emission area (integrated from 450 to 550 nm).

[0079] FIG. 12C illustrates a change of FRET ratios between QDs and TRAMRA-PEPPKA(p) according to a concentration of Zn2+ ions (i); a change of fluorescence intensities between QD and fluorescence peptide according to a concentration of metal ions. Changes in FRET (i) between QD and TAMRA-PEPPKA(p) as a function of Zn2+ concentration. The relative FRET percentage was calculated by dividing the experimental FRET ratio by the maximal FRET ratio (0.74). Fluorescence intensities of donor (QD); (ii) and acceptor (TAMRA-PEPPKA(p)); (iii) as a function of metal ion (Zn2+, Ni2+, Co2+, and Fe3+) concentration.

[0080] FIG. 12D illustrates a change of FRET ratio between QD and TAMRA-PEPPKA(p) according to time (i), and a change of FRET of QD according to a ratio of TAMRA-PEPPKA and FRET TAMRA-PEPPKA(p). Time-dependent change in the QD-FRET ratio in the presence (black diamond) and absence (black square) of Zn(II) (i); and peptide phosphorylation-dependent change in the QD-FRET ratio (ii).

[0081] FIG. 13A illustrates a process of phosphorylating TAMRA-PEP.sub.HSF-1.

[0082] FIG. 13B illustrates measurement results of an FRET ratio (F.sub.580/F.sub.525) according to the phosphorylated state of PEP.sub.HSF-1.

[0083] FIG. 14A illustrates a test method of detecting an FRET signal using NeutrAvidin agarose beads in Example 11.

[0084] FIGS. 14B and 14C illustrate test results of FRET signal detection using NeutrAvidin agarose beads.

[0085] FIG. 15 is a conceptual view illustrating phosphopeptide detection using nanoparticles and Zn.sup.2+.

EXAMPLES

[0086] Hereinafter, the present invention will be described in further detail with reference to the following examples. It will be obvious to those of ordinary skill in the art that these examples are provided only for illustrative purposes, and are not intended to limit the scope of the present invention according to the essence of the present invention.

Example 1

Characterization of Gold Nanoparticles

[0087] Gold nanoparticles reduced using citrate were modified with polyethylene glycol having a thiol group and a carboxyl group linked to opposite ends thereof, and an amine group-linked nitrilotriactic acid (NTA) was bound thereto through a 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) reaction. The size of the gold nanoparticles surface-modified with NTA was measured by light scattering. As a result of measurement, it was confirmed that the gold nanoparticles had an average size of about 21.3 nm (see FIG. 3).

Example 2

Zn.SUP.2+.- and Phosphopeptide-Dependent Self-Assembly of Gold Nanoparticles

[0088] 5 nM gold nanoparticles, 1 mM zinc ions, and 2.5 μM peptide with a histidine tag and a phosphate group were allowed to react in Tris-HC1 (pH 7.4) buffer for 1 hour, and then absorbance was measured at 520 nm and 700 nm. After measurement, an absorbance ratio of the two wavelengths was calculated, and, as a result of calculation, it was confirmed that gold nanoparticles were agglomerated specifically with respect to only a peptide having both a histidine tag and a phosphate group (see FIG. 4). In FIG. 4, pep1 denotes H.sub.6GLRRAS.sub.(p)LG, pep2 denotes H.sub.6GLRRASLG, and pep3 denotes GLRRAS.sub.(p)LG.

Example 3

Zn.SUP.2+.-Specific Self-Assembly

[0089] 2.5 μM phosphopeptide, 5 nM gold nanoparticles, and various types of metal ions were allowed to react in Tris-HCl buffer (pH 7.4) for 1 hour, and then a ratio of absorbance at 520 nm to absorbance at 700 nm was obtained. As a result, it was confirmed that gold nanoparticles were agglomerated specifically only when reacted with zinc ions (see FIG. 5).

Example 4

Effect of Peptide Concentration

[0090] After a reaction between 5 μM gold nanoparticles, 1 mM zinc ions, and various concentrations of a phosphopeptide, absorbance at 520 nm/700 nm was measured. After measurement, an absorbance ratio of the two wavelengths was obtained, from which it was confirmed that saturation occurred at a phosphopeptide concentration of about 2.5 μM (see FIG. 6).

Example 6

Effect of Zinc Concentration

[0091] 5 nM gold nanoparticles, 2.5 μM phosphopeptide, and zinc ions having a concentration of 1 μM to 100 mM were allowed to react in Tris-HCl buffer (pH 7.4) for 1 hour, and absorbance at 520 nm/700 nm was measured. After measurement, an absorbance ratio of the two wavelengths was obtained, from which it was confirmed that saturation occurred at a zinc ion concentration of 1 mM (see FIG. 7).

Example 6

Phosphatase Analysis

[0092] Various concentrations of a phosphatase, 2.5 μM phosphopeptide, and 1 mM manganese ions were allowed to react for 1 hour, and then 5 nM gold nanoparticles and 1 mM zinc ions were added thereto, and then allowed to react for 1 hour. As a result, it was confirmed that an absorbance ratio of 520 nm/700 nm was gradually recovered in proportion to the concentration of phosphatase (see FIG. 8).

Example 7

Colorimetric Assay Using Gold Nanoparticles and Zn.SUP.2+ Ions (1)

[0093] 2.5 μl of gold nanoparticles surface-modified with NTA, 85 μl of 20 mM Tris buffer (pH 7.4), 10 μl of each of various types of metal ion solutions (10 mM), and 2.5 μl of 100 μM phosphopeptide were mixed in this order in a 96-well plate, and then the mixture was allowed to react at room temperature for about 1 hour, and then absorbance thereof was measured (see FIG. 9A).

[0094] 2.5 μl of gold nanoparticles surface-modified with NTA, 85 μl of 20 mM Tris buffer (pH 7.4), 10 μl of a zinc ion solution (10 mM), and 2.5 μl of each of various types of peptides (100 μM) were mixed in this order in a 96-well plate, and then the mixture was allowed to react at room temperature for about 1 hour, and then absorbance thereof was measured (see FIG. 9B).

[0095] The experimental results were shown as a graph representing a value obtained by dividing an absorbance at 520 nm by an absorbance at 700 nm.

[0096] As a result, changes in the absorbance ratio due to self-assembly were observed only when Zn.sup.2+ was used and when pep1 was used.

Example 8

Colorimetric Assay Using Gold Nanoparticles and Zn.SUP.2+ Ions (2)

[0097] 37.5 μl of 20 mM Tris buffer, 5 μl of a manganese ion solution (10 mM), 2.5 μl of peptide (100 μM), and 5 μl of PP1 according to each concentration were mixed and then allowed to react at 30□ for 1 hour. The reaction product was added to a 96-well plate in which 37.5 μl of 20 mM Tris buffer, 2.5 μl of gold nanoparticles, and 10 μl of a Zn.sup.2+ solution (10 mM) had been previously mixed, and then allowed to react at room temperature for 1 hour, and, thereafter, absorbance was measured.

[0098] As a result, it was confirmed that, when pep1 was used, the self-assembled structure of gold nanoparticles was disassembled as the concentration of PP1 increased (see FIG. 10).

Example 9

Colorimetric Assay Using Gold Nanoparticles and Zn.SUP.2+ Ions (3)

[0099] Protein A agarose beads (100 μl of resin slurry) were divided into 50 μl aliquots, each aliquot was washed with 1 ml of 50 mM Tris buffer, and this process was repeated twice. 4.64 mg/ml of a homemade rabbit polyclonal antibody using 9.28 μg of GFP antigen [EGFP-6His] was added to the beads and bound thereto at 4□ overnight. Wild-type Columbia-0 (Col-0) not expressing PP2A-A1 and a RCN1-type plant, Arabidopsis thaliana, overexpressing PP2A-A1 (expressed as PP2A-A1-YFP) were completely frozen using liquid nitrogen, and then suspended in an extraction buffer (50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 0.3 M sucrose, 1% Triton X-100, 1× protease inhibitor cocktail, and 0.2 mM PMSF). The centrifugation was performed thereon at 4000 rpm for 6 minutes, and then the supernatant was filtered by Miracloth, and then spun down again for 10 minutes. The used antibody is capable of strongly binding to YFP as well as EGFP, and thus the supernatant was mixed with the GFP antibody-bound protein-A agarose beads and bound thereto for 1 hour and 15 minutes. After the reaction, the beads were washed four times with a wash buffer ((50 mM Tris-HCl (pH 7.5), 50 mM NaCl, 0.3 M sucrose, 0.2% Triton X-100, 1× protease inhibitor cocktail, and 0.2 mM PMSF). The washed beads were centrifuged again at 1000 rpm for 5 minutes to remove the supernatant, followed by mixing with 42.5 μl of 20 mM Tris buffer, 5 μl of a manganese ion solution (10 mM), and 2.5 μl of peptide (100 μM) and then allowed to react at room temperature for 1 hour. The supernatant obtained after centrifugation of the reaction product at 1000 rpm for 5 minutes was added to a 96-well plate in which 37.5 μl of 20 mM Tris buffer, 2.5 μl of gold nanoparticles, and 10 μl of a zinc ion solution (10 mM) had been previously mixed, and then allowed to react at room temperature for 1 hour, followed by absorbance measurement (see FIG. 11A).

[0100] As a result, it was confirmed that the self-assembly of the gold nanoparticles strongly occurred in the case of Col-0 without PP2A-A1, while the self-assembly of the gold nanoparticles was suppressed in an extract of the RCN1-type plant overexpressing PP2A-A1, resulting in a high absorbance ratio (see FIG. 11B).

Example 10

Test for FRET Signal Detection Between QDs and TAMRA-PEP.SUB.PKA .or TAMRA-PEP.SUB.PKA(p)

[0101] A synthetic peptide substrate labeled with 5(6)-carboxytetramethylrhodamine at the N-terminus (TAMRA-LRRASLG; TAMRA-PEP.sub.PKA) was compared with its phosphorylated form (TAMRA-LRRApSLG; TAMRA-PEP.sub.PKA(p)) (FIG. 12B). The FRET ratio was determined by the acceptor (F.sub.A) emission area (integrated from 550 to 650 nm) relative to the donor (F.sub.D) emission area (integrated from 450 to 550 nm). The concentrations of QD, TAMRA-PEP.sub.PKA(p) (or TAMRA-PEP.sub.PKA), and ZnCl.sub.2 were 2 nM, 80 nM and 100 μM, respectively. The QD-FRET spectra were obtained at an excitation wavelength of 380 nm.

[0102] While divalent metal ions (Ni(II), Co(II), Cu(II), and Zn(II)) and a trivalent metal ion (Fe(III)) were tested, only Zn(II) ion triggered a strong association between the energy donor and acceptor of the QD-FRET in the presence of TAMRA-PEP.sub.PKA(p) (FIG. 12B (i) and (ii)). Since Cu.sup.2+ completely quenched the fluorescence intensity of QDs, there were no signals in QD-FRET at both acceptors. This Zn(II)-coordination led to a high FRET ratio (F.sub.A/F.sub.D, 0.65), which is defined by the acceptor (F.sub.A) integrated emission relative to donor (F.sub.D) integrated emission (FIG. 1C), whereas other metal ions did not produce a significant FRET ratio. In addition, non-phosphopeptides resulted in a marginal FRET ratio even in the presence of Zn(II). This result strongly indicates that Zn(II) is specifically associated with phosphopeptides on the surfaces of QDs. In the present study, the FRET ratio was saturated at a 1:40 molar ratio of QD to T-pPEP1 in the presence of Zn(II) ion when the concentration of QD was fixed to 2 nM.

Example 11

Test for FRET Signal Detection According to a Concentration of Metal Ions

[0103] The change in FRET between QD and TAMRA-PEPPKA(p) according to a function of Zn.sup.2+ concentration was calculated. Furthermore, the relative FRET percentage was calculated by dividing the experimental FRET ratio by the maximal FRET ratio (0.74). The concentrations of QD and TAMRA-PEP.sub.PKA(p) were 2 nM and 80 nM, respectively. Excitation/emission wavelengths of QD-FRET (FIG. 12C (i)), QD (FIG. 12C (ii)), and TAMRA-PEP.sub.PKA(p) (FIG. 12C (iii)) were obtained at 380/580, 380/525, 530/580 nm, respectively.

[0104] The FRET ratio was also dependent on Zn(II) concentration, where maximum FRET ratio was acquired over the range of >100 μM Zn(II) at a 1:40 molar ratio of QD to TAMRA-PEP.sub.PKA(p) (FIG. 12C (i)). Considering the hydrodynamic diameter (10-20 nm) and multivalent capacity of carboxyl QD525, 40 times more fluorescent peptides were not fully saturated relative to QDs, but this number was optimized between QD/TAMRA-PEP.sub.PKA(p) and QD/TAMRA-PEP.sub.PKA in terms of signal-to-background ratio. Compared to Zn(II)-caged complexes capable of capturing the phenyl phosphate dianion, free Zn(II) ions in this study revealed a relatively low binding affinity of K.sub.d=8.8 μM for TAMRA-PEP.sub.PKA(p), based on the FRET efficiency. When we examined the effect of metal ions (except but Cu.sup.2+) on the florescence intensity of donor QD or acceptor dye, Zn.sup.2+ and Ni.sup.2+ ions did not affect the emission intensities of QD and TAMRA-PEP.sub.PKA(p) (FIG. 12C (ii) and (iii)), whereas Co.sup.2+ and Fe.sup.3+ reduced the emission intensity of QD as their concentrations increased (FIG. 12C (ii)). As a consequence, since free Zn(II) ions even at high concentration did not influence fluorescent intensity of the donor QD or the acceptor TAMRA-PEP.sub.PKA(p), they are favorable for use in the FRET-based method in order to avoid synthesis of complex chemicals, which may adversely affect the fluorophores. Therefore, this result suggests that our Zn(II)-mediated QD-FRET would be useful for detecting phosphopeptides without complex metal-chelating ligands.

Example 12

Test for FRET Signal Detection

[0105] Time-dependent change in the QD-FRET ratio in the presence (black diamond) and absence (black square) of Zn(II) (i); and peptide phosphorylation-dependent change in the QD-FRET ratio (ii) were confirmed. Total concentration of peptides (TAMRA-PEP.sub.PKA(p) and TAMRA-PEP.sub.PKA) was kept constant at 80 nM, while TAMRA-PEP.sub.PKA(p) concentration was varied (0, 25, 75, and 100%). The concentrations of QD and metal ions were 2 nM and 100 μM, respectively. The QD-FRET spectra were obtained at an excitation wavelength of 380 nm.

[0106] To examine kinetics and phosphorylation-dependency of this FRET phenomenon, we examined time-dependent FRET ratio in the presence and absence of Zn(II) ion (FIG. 12D (i)), and compared the FRET ratios at varied proportions of phosphorylated peptide (FIG. 12D (ii)). The maximum FRET ratio was reached within 5 min after addition of Zn(II) ion, whereas no addition of Zn(II) did not change the FRET ratio. Under the same conditions, where the molar ratio of QD to peptide was 1:40 in the presence of Zn(II), the FRET ratio was proportional to the TAMRA-PEP.sub.PKA(p) concentration

Example 13

Test for FRET Signal Detection according to Phosphorylated State of Peptide Substrate

[0107] 71 μl of 20 mM Tris-HCl buffer (pH 7.4), 20 μl of 20 nM Qdot 525 (Invitrogen), and either (8 μl at 1 μM) of TAMRA-PEP.sub.HSF-1 (TAMRA-KEEPPSPPQSPR), TAMRA-PEP.sub.HSF-1(P) (TAMRA-KEEPPSPPQpSPR), or TAMRA-PEP.sub.HSF-1(PP) (TAMRA-KEEPPpSPPQpSPR) were mixed and transferred to a 96-well plate. 1 μl of a 10 mM zinc ion solution were further added to the 96-well plate, and then the aforementioned materials were satisfactorily mixed together in this order and allowed to react at room temperature for about 5 minutes, followed by measurement of a fluorescence signal using a plate reader.

[0108] The initially synthesized TAMRA-PEP.sub.HSF-1 is a substrate in which a phosphor is bound to a peptide derived from Heat shock factor-1 (HSF-1), which is a substrate protein, and the substrate is consecutively phosphorylated by mitogen-activated protein kinase (MAPK) and glycogen synthase kinase-3 (GSK-3). Serine at the C-terminal of the peptide sequence is first phosphorylated by protein kinase A (PKA), and the phosphorylated serine at the C-terminal is recognized by GSK-3 to phosphorylate serine at the N-terminal (see FIG. 13A).

[0109] As the 96-well plate, a FluoroNunc 96-well plate available from Nunc was used, an excitation wavelength was 380 nm, and a measurement wavelength was in the range of 450 nm to 650 nm. A measurement time for each wavelength was 0.8 ms.

[0110] As a result, as illustrated in FIG. 13B, a higher FRET ratio (F.sub.580/F.sub.525) was detected as consecutive phosphorylation proceeded.

Example 14

Test for FRET Signal Detection Using NeutrAvidin Agarose Beads

[0111] Unlike Example 13, to implement a method of effectively removing a substance that affects an FRET signal (salt, metal ions, ATP, DTT, or the like), biotin was bound to one end of a peptide substrate to induce a kinase reaction, and then the substrate having undergone the kinase reaction through NeutrAvidin-biotin binding was effectively isolated, followed by FRET signal detection (see FIG. 14A). TAMRA-PEP.sub.PKA(TAMRA-LRRASLG) was used as an initial peptide substrate, the substrate is a substrate in which a phosphor was bound to a peptide derived from protein Porcine liver pyruvate kinase, and the substrate was phosphorylated by PKA. TAMRA-PEP.sub.PKA-Biotin(biotin-LRRASLG-TAMRA), which is a substrate to which biotin is further bound to the peptide substrate, was synthesized and finally applied to the experiment. In particular, 78 μl of 20 mM Tris-HCl buffer (pH 7.4), 5 μl of 100 μM TAMRA-PEP.sub.PKA-biotin, 10 μl of a PKA reaction buffer (10×), 2 μl of a 10 mM ATP solution, and 5 μl of a 25 U PKA enzyme were sequentially mixed in a 1.5 ml tube, and then allowed to react at 30□ for 1 hour and 30 minutes. At this time, a control not including a PKA enzyme in the 1.5 ml tube was used to conduct a comparative experiment. Thereafter, 50 μl of 50% NeutrAvidin agarose bead slurry were added thereto, and mixed by vortexing for 30 minutes, followed by centrifugation at 1500 rpm for 5 minutes to remove the supernatant therefrom. Afterwards, 50 μl of TAMRA-PEP.sub.PKA-Biotin-tagged NeutrAvidin bead slurry was added to a 96-well plate, 80 μl of 20 mM Tris-HCl buffer (pH 7.4), 20 μl of 20 nM Qdot 525, and 1 μl of a 10 mM zinc ion solution were sequentially added thereto and mixed well, and then the mixture was allowed to react at room temperature for about 5 minutes, followed by measurement of a fluorescence signal using a plate reader.

[0112] As the 96-well plate, a FluoroNunc 96-well plate available from Nunc was used, an excitation wavelength was 380 nm, and a measurement wavelength was in the range of 450 nm to 650 nm. A measurement time for each wavelength was 0.8 ms. The bar graph in FIG. 14C shows calculation results of a ratio of two highest peaks of the measured fluorescence spectrum, which indicates a calculated value obtained by dividing a fluorescence value at 580 nm by a fluorescence value at 525 nm.

[0113] As a result, it was confirmed that the FRET ratio was about 0.16 when the PKA enzyme was absent, while a signal value of 0.59 was observed as the FRET ratio in the tube including the PKA enzyme, which means that PKA enzymatic activity can be effectively detected even in the presence of an inhibitor in a reaction buffer.

[0114] While present invention has been described in detail with reference to exemplary embodiments thereof, it is obvious to those of ordinary skill in the art that these embodiments are provided only for illustrative purposes, and are not intended to limit the scope of the present invention. Thus, the substantial scope of the present invention should be defined by the appended claims and equivalents thereto.

[0115] Characteristics and advantages of the present invention are summarized as follows:

[0116] (a) The present invention provides a composition for measuring kinase or phosphatase activity.

[0117] (b) The present invention provides a method of measuring phosphatase activity and a method of measuring kinase activity.

[0118] (c) The present invention provides a method of screening a phosphatase activity inhibitor.

[0119] (d) When the composition and methods of the present invention are used, the kinase or phosphatase activity may be quantitatively detected in real time.

[0120] (e) When the composition and methods of the present invention are used, a material capable of inhibiting phosphatase activity may be discovered.