JP2006153637A - New glutamic acid biosensor utilizing phenomenon of fluorescent energy moving - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To measure the concentration of glutamic acid in a specimen at a high speed with high sensitivity, using a small amount of the specimen. <P>SOLUTION: A heterodimer, which is a fluorescence-labeled LBD protein heterodimer, prepared by respectively bonding two kinds of fluorescent substances to extracellular ligand bonded domain protein (LBD) of a metabolic type glutamic acid acceptor and where two kinds of the fluorescent substances are a donor and an acceptor in fluorescence resonance energy transfer (FRET), is brought into contact with the specimen and irradiated with exciting light to measure the intensity of the fluorescence from the donor fluorescent substance and the acceptor fluorescent substance. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a novel glutamate biosensor, and more particularly to a glutamate biosensor and a glutamate concentration measurement method using a change in the three-dimensional structure of the extracellular region of the receptor that occurs when glutamate binds to a metabotropic glutamate receptor.

Glutamic acid is also a major umami ingredient in foods, and is used as a seasoning in various foods such as being used directly in cooking and added to artificially produced seasonings. In addition to food, it is widely used for soap, shampoo treatments, cosmetics and supplements. Therefore, in producing these processed products, development has been made of a method for detecting and quantifying glutamic acid for taste inspection as a taste sensor for seasonings and quality control / inspection of products.
Glutamic acid is a protein-constituting amino acid in vivo and is a non-essential amino acid. However, due to increased catabolism such as metabolic stress, the amount of biosynthesis in the body may be insufficient, and it is treated as a semi-essential amino acid. In some cases. In the body, it is present in large quantities throughout the brain and is the main transmitter of the central nervous system. Therefore, it has become clear that the physiological activity in the body has a very close relationship with the intake. In particular, in the medical field, detection and quantification of trace amounts of glutamic acid such as diagnostic methods and test methods as glutamic acid sensors are required. The glutamic acid sensor currently being researched and developed is mainly a combination of an enzyme reaction and an electrode using glutamic acid dehydrogenase, glutamic acid oxidase and the like (Non-patent Document 1). However, these sensors detect the resulting current by causing the electrons generated in the initial enzymatic reaction to eventually reach the electrode through several chemical reaction steps, which is a fairly indirect measurement method. There are many factors that cause the sensor performance to become unstable during use. In addition, since an electrode is used, a certain amount of sample is required, and it is relatively difficult to achieve high throughput such as measuring multiple samples simultaneously.
Metabotropic glutamate receptors are 7-transmembrane GPCRs that, unlike channel-type receptors, cause cells to have a longer-term response. Recently, the three-dimensional structure of the extracellular ligand binding domain (about 520 amino acids) of this receptor has been revealed (Non-patent Document 2). This reveals that the ligand-binding domain of this receptor forms a homodimer due to disulfide bonds and hydrophobic interactions, and is activated by greatly changing the relative position between dimers with glutamate binding. It was suggested that
Okayama University Faculty of Agriculture Vol. 91, 15-22 (2002) Nature Vol. 407 971-977 (2000)

  An object of the present invention is to easily and quickly quantify the glutamic acid concentration in a specimen. Another object of the present invention is to make it possible to measure multiple specimens simultaneously.

In the present invention, when an extracellular ligand-binding domain protein (hereinafter abbreviated as LBD) of a metabotropic glutamate receptor forms a homodimer having a specific three-dimensional structure, glutamate as a ligand is added thereto, and glutamate binds to LBD. This is based on the knowledge that the three-dimensional structure of the LBD homodimer changes. When LBD is labeled with two different types of fluorescent substances, LBD heterodimers are formed, and as the three-dimensional structure of LBD heterodimers changes due to the binding of glutamic acid, the distance between the two types of fluorescent substances changes and the efficiency of fluorescence energy transfer also increases. Change. It is the principle of the present invention to measure the concentration of glutamic acid from the fluorescence intensities from two kinds of fluorescent materials by utilizing this. Here, “fluorescence energy transfer” (FRET) means that when two types of fluorescent substances (donor and acceptor) whose absorption spectra overlap each other are present within a distance of about 100 angstroms, the excitation energy of the donor is It refers to a phenomenon in which fluorescence is emitted from an acceptor dye that moves to an acceptor and is not directly excited (see Non-Patent Document 2). A “donor” in FRET refers to a fluorescent material that absorbs excitation light, emits part of its energy as fluorescence, and the other part moves to an adjacent acceptor. An “acceptor” in FRET refers to a fluorescent substance that absorbs energy from a donor and emits fluorescence without receiving direct excitation light.
Stryer, L .: Fluorescence energy transfer as a spectroscopic ruler. Annu. Rev. Biochem. 47 p819-846 (1978)

That is, the present invention
A fluorescently labeled LBD protein heterodimer in which two types of fluorescent substances are bound to the extracellular ligand binding domain protein (LBD) of metabotropic glutamate receptors, and the two kinds of fluorescent substances are fluorescence resonance energy transfer. ) Heterodimers that are donors and acceptors in (FRET),
About.
“Extracellular ligand-binding domain protein (LBD) of metabotropic glutamate receptor” is an extracellular portion of the receptor consisting of about 520 amino acid residues at the N-terminal of metabotropic glutamate receptor. Is the part that joins. In the present invention, the number of amino acid residues of LBD need not be exact.

The present invention also provides
1) contacting the specimen containing the heterodimer and glutamic acid;
2) Irradiate excitation light and measure the fluorescence intensity from the donor fluorescent material and the acceptor fluorescent material;
The present invention relates to a method for measuring the concentration of glutamic acid in a sample.

The present invention measures the glutamic acid concentration in a specimen from the change in the three-dimensional structure of the protein, and is completely different from the conventional method for measuring glutamic acid concentration, and has the following advantageous effects.
1) Since the binding of glutamic acid is directly converted into an optical signal, high sensitivity can be expected. Depending on the photo detector, current photo detection technology can detect single photons.
2) In principle, the glutamic acid concentration of a very small amount of sample of 1 microliter or less can be quantified. In this case, the sensor molecule of the present invention is immobilized on the microbead surface, and the bead is observed by microscopic imaging using a CCD camera.
3) Glutamate binding to metabotropic glutamate receptors is a response on the order of milliseconds, and is very fast, so it can be expected to speed up detection. In addition, since light detection is used without using electrodes, it is easy to achieve high throughput for quickly measuring multiple samples.

There are two ways to fluorescently label LBD. One is to make a fusion protein of LBD and fluorescent protein, and the other is to bind a low molecular weight fluorescent substance to the amino acid side chain of LBD. In either case, two fluorescent materials, FRET donor and FRET acceptor, are required. The fluorescence wavelength of the FRET donor must be shorter than the fluorescence wavelength of the FRET acceptor. In addition, the fluorescence spectrum of the fluorescent substance serving as a donor must overlap with the absorption spectrum of the fluorescent substance serving as an acceptor.
Selection of fluorescent proteins

The combination of FRET donor and acceptor fluorescent proteins includes CFP (Cyan fluorescent protein: yellow fluorescent protein). YFP (yellow fluorescent protein: yellow fluorescent protein) GFP (green fluorescent protein: a fluorescent protein that emits green fluorescence) / RFP (red fluorescent protein: a fluorescent protein that emits red or longer wavelength fluorescence).
As Cyan fluorescent protein, ECFP manufactured by Clontech can be used. As yellow fluorescent protein, Evrogen's Phi-Yellow can be used in addition to Clontech's YFP. As Green fluorescent protein, Evrogen Cop-Green can be used in addition to Clontech EGFP. As a red fluorescent protein, Evrogen HcRed-Tandem can be used in addition to Clontech DsRed2 and HcRed1.
(2) Method for producing heterodimer of fusion protein of LBD and fluorescent protein

A fusion protein of LBD and fluorescent protein can be conveniently produced by genetic recombination as follows.
A fluorescent protein gene serving as a FRET donor and acceptor is fused to the 3 ′ end of the gene of the extracellular ligand binding domain (LBD) (SEQ ID NO: 1) of the metabotropic glutamate receptor. It is inserted into two expression gene cloning sites of Invitrogen's pFastBac Dual vector. (Alternatively, insert one LBD-fluorescent protein fusion gene into the pFastBac Dual vector, and prepare two for the donor and acceptor.) Transform the plasmid into E. coli DH10Bac for bacmid preparation and contain the gene of interest from there. Purify the bacmid. The bacmid is transfected into insect cells Sf9 using Cellfectin reagent of Invitrogen, and cultured at 27 ° C. for several days to recover the culture supernatant. The culture supernatant contains baculovirus with the target gene. Furthermore, in order to prepare more viruses, Sf9 cells are prepared in large quantities and subjected to virus amplification, and then the culture supernatant is stored as a virus solution for protein expression. In order to perform protein expression, the virus solution is infected into HighFive cells, which are insect cells for protein expression, cultured for several days, and then the culture supernatant is collected. (If the donor fusion gene and acceptor fusion gene are separately cloned into the pFastBac dual vector, two viruses prepared from both vectors can be simultaneously infected into the same cell.) Infecting cells, the target protein is produced in the cells and secreted into the culture supernatant. Since LBD has the property of becoming a dimer, donor-fused LBD and acceptor-fused LBD become heterodimers and are secreted into the culture supernatant. However, at this stage, many donor-fused LBDs and acceptor-fused LBDs form a homodimer are included, so the donor acceptor heterodimer is finally prepared using affinity chromatography. For example, if a FLAG tag is added to the donor fusion gene and a His tag is added to the acceptor fusion gene, and protein purification is performed using these two affinity tags, the resulting protein is in principle the donor fusion LBD. And acceptor-fused LBD heterodimer.

  FIG. 1 shows an example of a conceptual diagram of a heterodimer of a fusion protein of LBD and fluorescent protein of the present invention. In this example, the FRET donor protein is CFP and the FRET acceptor protein is YFP. The His tag is attached to the LBD / CFP fusion protein, and the FLAG tag is attached to the LBD / YFP fusion protein.

In another embodiment of the present invention, a fusion protein of an enzyme that catalyzes bioluminescence, such as luciferase, and LBD may be used instead of the FRET donor protein and the LBD fusion protein. In this case, luciferin, which is a substrate for luciferase, is excited by the free energy accompanying oxidation by oxygen molecules and emits light, but part of the energy moves to the FRET acceptor protein and emits fluorescence.
(3) Selection of low-molecular fluorescent substances

  Another method of fluorescently labeling LBD is to attach a small fluorescent substance to the amino acid side chain of LBD. Low molecular fluorescent substances that can serve as FRET donors include naphthalene derivatives such as 1,5-IAEDANS, IAANS, MIANS, pyrene derivatives such as pyrenemaleimide, pyreneiodoacetamide, CPM, DCIA, DACIA, DACM, MDCC, IDCC, etc. Coumarin derivatives, fluorescein maleimide, 5-iodoacetamide fluorescein, 5-bromomethylfluorescein, Oregon green 488 maleimide, Oregon green 488 iodoacetamide and other fluorescein derivatives, Alexa 488 maleimide, Alexa 532 maleimide, Alexa 546 maleimide, Alexa 568 maleimide Alexa derivatives such as Alexa 594 maleimide, Alexa 555 maleimide, BODIPY493 / 503 bromomethyl, BODIPY499 / 508 maleimide, BODIPY507 / 545 There are BODIPY derivatives such as door acetamide, BODIPY530 / 550 iodoacetamide, rhodamine derivatives such as tetramethyl rhodamine maleimide, tetramethyl rhodamine iodoacetamide, rhodamine red maleimide, Texas red derivatives such as Texas red maleimide, Texas red iodoacetamide, etc. .

  Low molecular fluorescent substances that can serve as FRET acceptors include fluorescein derivatives such as fluorescein maleimide, 5-iodoacetamide fluorescein, 5-bromomethylfluorescein, Oregon Green 488 maleimide, Oregon Green 488 iodoacetamide, Alexa 488 maleimide, Alexa 532 maleimide, Alexa derivatives such as Alexa 546 maleimide, Alexa 568 maleimide, Alexa 594 maleimide, Alexa 555 maleimide, Alexa 633 maleimide, Alexa 647 maleimide, Alexa 660 maleimide, Alexa 680 maleimide, BODIPY493 / 503 bromomethyl, BODIPY499 / 508 maleimide, BODIPY507 / 545 , BODIPY 530/550 iodoacetamide, BODIPY577 / 618 maleimide, BODIPY630 / 650 maleimide, etc. , Tetramethylrhodamine maleimide, tetramethylrhodamine iodoacetamide, Rhodamine Red Rhodamine derivatives such as maleimide, Texas Red-maleimide, and the like Texas Red derivatives such as Texas Red iodoacetamide.

Examples of preferred FRET donor / acceptor combinations include pyrenemaleimide / furorecein maleimide, CPM / fluorescein maleimide, fluorescein maleimide / Texas red maleimide, Alexa 488 maleimide / Texas red maleimide, tetramethylrhodamine maleimide / Alexa 633 maleimide and the like.
(4) Binding method of LBD and low molecular fluorescent substance

  The LBD and the low-molecular fluorescent substance are bound by targeting a cysteine residue introduced into the LBD in a site-specific manner. First, in the LBD ligand binding domain, the position where the fluorescent dye is to be bound is determined from the three-dimensional structure, and the mutation that replaces the amino acid residue at that site with cysteine, and the mutation that replaces the preceding and subsequent amino acids with arginine, It introduce | transduces into LBD gene using PCR. The reactivity of the target cysteine is increased by making arginine residues on both sides. A baculovirus is prepared from the LBD gene using the Bac-to-Bac system, and the target protein is expressed using the virus and insect cells HighFive. The protein is purified using a FLAG tag, concentrated to about 10 μM, and buffer exchanged (20 mM HEPES pH 7.4, 50 mM NaCl). Next, TCEP is added to activate the target cysteine, a molar fluorescent donor such as protein is added thereto, and the mixture is reacted at room temperature for 30 minutes with stirring. After the reaction, the label protein and the remaining fluorescent substance are separated by gel filtration, and the protein is concentrated again. Next, an acceptor fluorescent substance of about 10 equivalents of protein is similarly reacted at room temperature, and the labeled protein and the remaining fluorescent dye are separated and purified again by gel filtration. The label protein obtained here is a protein (LBD) in which the donor and the acceptor are double-labeled (however, the label rate of the donor is smaller than the label rate of the acceptor).

(5) Glutamic acid concentration measurement liquid phase method:
An aqueous solution of this sensor molecule (fluorescently labeled heterodimer) is mixed with a sample containing glutamic acid (sensor molecule 0.5 μM in 20 mM HEPES pH 7.4, 50 mM NaCl. The sample is about 1 μL for about 500 μL of the sensor solution. Irradiate the excitation light of the donor with a fluorometer (the wavelength of the excitation light is shorter than the fluorescence wavelength of the donor fluorescent substance), and the fluorescence intensity of each fluorescent substance at a wavelength at which it peaks alone Measure the fluorescence intensity and take the ratio. On the other hand, a glutamic acid solution with a known concentration is prepared, and a calibration curve is prepared by taking the ratio of fluorescence intensities in the same manner. By using this calibration curve, the glutamic acid concentration of the specimen can be determined.

Solid phase method:
This fluorescent double-labeled LBD is coated on the surface of a suitable solid support, for example, a FLAG antibody agarose bead having a diameter of about 100 micrometers, a specimen containing glutamic acid is brought into contact with the beads, and the beads are observed with a fluorescence microscope. On the detector side, a W-view system capable of observing both donor and acceptor images at the same time is introduced, and the image is captured in a computer using a digital CCD camera. The fluorescence intensity ratio of both images of each channel of the two fluorescent substances is calculated, and the difference between the values is imaged as a difference in color. The color of the beads changes depending on the glutamic acid concentration.
Since the volume of beads to be used is 1 μl or less, it is expected that the glutamic acid concentration can be quantified with a minimal amount of the sample using this method.

Example 1
Preparation of LBD-CFP fusion protein / LBD-YFP fusion protein heterodimer and measurement of its fluorescence spectrum Fluorescent protein CFP (SEQ ID NO: 3) at the C-terminus of ligand binding domain (LBD) (SEQ ID NO: 1) of metabotropic glutamate receptor In order to fuse YFP (SEQ ID NO: 2), each fluorescent protein gene portion was amplified by PCR using Clontech pECFP and pEYFP as templates. An XbaI site was inserted into both the Forward and Reverse primers for PCR and ligated to the C-terminus of the LBD gene using this restriction enzyme site. A stop codon was designed at the C-terminus of the Reverse primer. A FLAG-tagged hemagglutinin secretion signal sequence was inserted at the N-terminus of the LBD fused with YFP, and a secretory signal originally possessed by this receptor was inserted at the N-terminus of the LBD fused with CFP. A histidine tag was added to the Reverse primer for amplifying the CFP gene. The LBD used is a gene encoding up to amino acid number 522 of metabotropic glutamate subtype 1. LBD gene (NotI-XbaI region) and each fluorescent protein (XbaI-XbaI region) are linked using XbaI site to create LBD-fluorescent protein fusion gene (LBD-CFP and LBD-YFP, NotI-XbaI region) did. This was cloned into pFastBac dual vector of Invitrogen using NotI-XbaI site. This plasmid was transformed into DH10BAC, which is a bacmid-producing Escherichia coli, and an E. coli colony producing a bacmid containing the target gene was cloned by blue-white selection on a Luria-agar plate. A bacmid was prepared from this E. coli by the plasmid miniprep alkaline method. Next, in order to produce a virus from this bacmid, the bacmid was transfected into insect cell Sf9 using Cellfectin reagent. After culturing at 27 ° C. for 3 days, the culture supernatant was recovered. In addition, Sf9 cells at this time were recovered, and it was confirmed that the target protein was expressed in the cells by Western blotting using SDS-PAGE, anti-FLAG antibody, and anti-Histag antibody. Since the virus was still present in the culture supernatant at this stage, a larger amount of Sf9 cells were prepared, and this virus was infected there to propagate the virus encoding the target gene. The LBD-CFP virus and LBD-YFP virus prepared as described above were infected with appropriate amounts of the protein expression insect cells HighFive (100 microliters of virus solution was added to one 225 cm ² plate), 4 days The culture supernatant was collected and used for protein purification (the culture supernatant collected at 6000 rpm for 15 minutes was centrifuged to remove the remaining cells). First, a cocktail of protease inhibitors was added to the culture supernatant (PMSF, leupeptin, pepstatin, aprotinin), and it was directly passed through an agarose resin column (about 1 ml) to which an Anti-FLAG antibody was crosslinked. The flow rate was about 1 mL / min. After passing all the culture supernatants through the column, the column was washed with 10 mL of buffer (10 mM Tris-HCl pH 7.5, 20 mM NaCl). Thereafter, the target protein was eluted from the column with 150 μg / mL FLAG peptide (sequence: DYKDDDDK, 20 mM HEPES pH 7.4 300 mM NaCl). Next, the sample eluted from the anti-FLAG antibody column was passed through about 1 mL of Ni-agarose at a flow rate of 1 mL / min. After passing through all samples, the column was washed with a buffer of 25 mM imidazole and 0.5 M NaCl, and then the target protein was eluted with a buffer of 200 mM imidazole and 0.5 M NaCl. Western blotting was performed using the eluted sample, and it was confirmed that the obtained protein was a heterodimer of LBD-CFP and LBD-YFP. By this method, about 0.5 to 1 mg of the target protein can be prepared from 1 liter of the culture supernatant.

Example 2
Measurement of fluorescence spectrum of LBD-CFP fusion protein / LBD-YFP fusion protein heterodimer The fluorescence spectrum of the obtained sample was measured using a fluorescence spectrophotometer (F-4500) manufactured by Hitachi, Ltd. The excitation light was 430 nm light, and the fluorescence spectrum from 450 nm to 600 nm was recorded on a computer. The measurement buffer was 20 mM HEPES pH 7.4, 50 mM NaCl. The spectrum when this heterodimer solution was excited with light of 430 nm was as shown by the solid line in FIG. 2A. CFP fluorescence is observed at a peak around 480 nm, and YFP fluorescence is observed at a peak around 530 nm. It was confirmed that when 1 mM glutamic acid was added thereto, the fluorescence spectrum changed as indicated by the dotted line, the CFP fluorescence increased, and the YFP fluorescence decreased (FIG. 2A). This means that due to the binding of glutamic acid, a structural change occurs in the molecule, and the fluorescence energy transfer efficiency between CFP and YFP changes.
On the other hand, even when S-MCPG (s-alphamethylcarboxyphenylglycine), which is an antagonist of metabotropic glutamate receptors, was added, such a spectral change was not observed (FIG. 2B). Can be said to be agonist specific.

Example 3
Preparation of calibration curve When the ratio of 480 nm and 530 nm of the fluorescence spectrum is taken at various glutamic acid concentrations in Example 2 and plotted, the ratio of 480/530 clearly changes depending on the glutamic acid concentration from 1 nM to 2 mM. (Figure 3). If this is a calibration curve, the glutamic acid concentration in the sample can be quantified by such a method using a fluorometer. In this measurement, 0.5 μl of the ligand solution was added to 500 μl of the sample solution, and the fluorescence spectrum was measured about 1 minute later. Final data was calculated taking into account the change in sample solution volume with ligand addition.

Example 4
Quantification of glutamic acid concentration by solid-phase method As a method of quantifying glutamic acid concentration in a small amount of sample, this molecule is coated on the surface of a FLAG antibody agarose bead having a diameter of about 100 micrometers, contacted with the sample, and the beads are observed with a fluorescence microscope. did. A sensor molecule solution having a concentration of about 300 μg / ml and 50 microliters of anti-FLAG antibody-coated agarose beads (manufactured by Sigma) were mixed and stirred by inverting at 4 ° C. for 12 hours to coat the sensor molecules on the bead surface. Beads were precipitated using a microcentrifuge (15000 rpm, 30 seconds), the supernatant was discarded, and beads were prepared by repeatedly adding Wash buffer (20 mM HEPES pH 7.4, 50 mM NaCl) three times. About 10 μl of the solution was observed with a fluorescence microscope, and the objective lens was 10 × (Olympus) The change in bead fluorescence when a ligand was added was obtained by adding about 0.5 μl of the ligand solution to the bead solution drop. On the detector side, a W-view system (manufactured by Hamamatsu Photonics) that can observe both CFP and YFP images at the same time was introduced, and an image was captured into a computer using a digital CC camera. Aqua Cosmos Imaging System (manufactured by Hamamatsu Photonics) CFP and YFP channels The fluorescence intensity ratio (CFP image intensity / YFP image intensity) of these two images was calculated, and the difference in the values was imaged as a difference in color, confirming that the color of the beads changed according to the glutamic acid concentration ( 4A, B).

  It can be used to detect, quantify, and measure the concentration of glutamic acid in taste inspections of foods and the like, quality control / inspection of products containing glutamic acid, diagnosis in the medical field, blood tests, and the like.

. An example of the conceptual diagram of the heterodimer of the fusion protein of LBD of this invention and fluorescent protein is shown. In this example, the FRET donor protein is CFP and the FRET acceptor protein is YFP. The LBD / CFP fusion protein has a His tag and the LBD / YFP fusion protein has a FLAG tag. A: A fluorescence spectrum of a glutamic acid sensor molecule (heterodimer of fusion protein of LBD and fluorescent protein) is shown. A solid line indicates a case where glutamic acid is not added, and a broken line indicates a case where 1 mM glutamic acid is added. B: The solid line indicates the case where glutamic acid is not added, and the broken line indicates the case where 1 mM S-MCPG1 is added. The fluorescence spectrum change depending on the glutamic acid concentration is shown. A: A Ratio image of agarose beads coated with a glutamic acid sensor molecule is shown. The bead color changes depending on the glutamate concentration. B: A graph of a Ratio image at each glutamic acid concentration.

Claims (15)

  A fluorescently labeled LBD protein heterodimer in which two types of fluorescent substances are bound to an extracellular ligand binding domain protein (LBD) of a metabotropic glutamate receptor, and the two kinds of fluorescent substances are fluorescence resonance energy transfer. ) Heterodimers that are donors and acceptors in (FRET).   The heterodimer according to claim 1, wherein the fluorescently labeled protein is a fusion protein in which the fluorescent protein is fused to LBD.   The heterodimer according to claim 2, wherein the donor fluorescent protein is cyan fluorescent protein (CFP), and the acceptor fluorescent protein is yellow fluorescent protein (YFP).   The heterodimer according to claim 2, wherein the donor fluorescent protein is green fluorescent protein (GFP) and the acceptor fluorescent protein is red fluorescent protein (RFP).   The heterodimer according to claim 1, wherein the fluorescently labeled protein has a low-molecular fluorescent substance bound to the side chain of the amino acid residue of LBD.   The heterodimer according to claim 5, wherein the donor low molecular fluorescent substance is pyrenemaleimide and the acceptor low molecular fluorescent substance is fluorescein maleimide.   The heterodimer according to claim 5, wherein the donor low-molecular fluorescent substance is CPM and the acceptor low-molecular fluorescent substance is fluorescein maleimide.   The heterodimer according to claim 5, wherein the donor low-molecular fluorescent substance is fluorescein maleimide and the acceptor low-molecular fluorescent substance is Texas red maleimide.   6. The method of claim 5, wherein the donor small molecule fluorescent material is Alexa 488 maleimide and the acceptor small molecule fluorescent material is Texas Red maleimide.   6. The method according to claim 5, wherein the donor low molecular fluorescent substance is tetramethylrhodamine maleimide and the acceptor low molecular fluorescent substance is Alexa 633 maleimide. 1) contacting the heterodimer according to any one of claims 1 to 10 with a specimen containing glutamic acid;
2) Irradiate excitation light and measure the fluorescence intensity from the donor fluorescent material and the acceptor fluorescent material;
A method for measuring the concentration of glutamic acid in a specimen.   The method according to claim 11, wherein the contact between the heterodimer and the specimen is performed in a liquid phase.   The method according to claim 12, wherein the fluorescence intensity is measured using a fluorescence spectrophotometer.   The method according to claim 11, wherein the heterodimer is immobilized on a solid phase and contacted with a specimen.   The method according to claim 14, wherein the fluorescence intensity is measured using a microscope imaging technique in which a fluorescence microscope and a CCD camera are combined.

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