JP2005232132A - Labeling reagent specific to dansyl group-bearing thiol group, method for producing the same and labeling method using the labeling reagent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a labeling reagent that can specifically bond to thiol group in protein and can extract labeled protein by the reactivity with the antibody having the labeling reagent itself and the fluorescence property. <P>SOLUTION: The labeling reagent for a dansyl group-bearing thiol group is represented by formula (I) (wherein X is a halogen, m is an integer of 1 through 3, n is an integer of 2 through 8). <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a thiol group-specific labeling reagent using a halogenated acetic acid derivative having a dansyl group, a production method thereof, and a labeling method using the labeling reagent.

  The labeling of biomolecules such as proteins and peptides (hereinafter collectively referred to as “proteins”) is a technique frequently used in the fields of medicine, agricultural chemistry, biochemistry, molecular biology, and the like. It is useful for identification of properties and addition of functions.

  The thiol group possessed by cysteine and the disulfide formed by cysteine are important for maintaining the three-dimensional structure of the protein and exerting its enzyme activity. For example, it is known that many proteins such as ribonuclease cannot form a correct three-dimensional structure when the intramolecular or intermolecular disulfide bond is cleaved, resulting in loss of enzyme activity. Enzymes such as glucose oxidase and papain contain a thiol group in the active site, and enzymes such as protein disulfide isomerase exhibit activity by exchanging thiol and disulfide with cysteine residues in the active site. That is, these enzymes are known to involve thiols and disulfides when exhibiting their activities. From the above, identifying the positions of thiol groups and disulfides in a protein is a very important factor in examining the structure and activity of the protein.

  The protein thiol group or disulfide position is determined by (1) cleaving the test protein with a protease, (2) extracting only peptide fragments having a thiol group from various peptide fragments decomposed by the protease, (3) It is identified by determining the primary structure of the extracted peptide fragment by means such as an amino acid sequence. For this “purification of a peptide fragment having a thiol group”, a method using a thiol group-specific labeling reagent can be considered.

Patent Document 1 discloses a fluorescent thiol group labeling reagent (acridine derivative), a protein labeling method using the labeling reagent, and a protein (labeled protein) labeled with the labeling reagent by post-column high-performance liquid chromatography. The method of extracting by is disclosed. Patent Document 2 and Non-Patent Documents 1 to 5 describe fluorescent thiol group labeling reagents (maleimide derivatives, N- (2-bromoacetylamino) ethyl-2- (methylamino) benzamide, difluorescein disulfide, monobromide. Mobiman, N- (iodoacetylaminoethyl) -naphthylamine sulfate, N- (p- (2-benzoxazoyl) phenyl) maleimide) and a protein labeling method using the labeling reagent are disclosed in Patent Document 3 and Non-Patent Document 6. And 7 include a radionuclide-containing thiol group labeling reagent (maleimide-based compound, deaminolysine (N ⁶ -N-maleoyl-β-alanine) -8-arginine vasopressin, 3- (2-pyridyldithio) propionylarginyl- [ ¹²⁵ I] - monoiodotyrosine) and labeled side of proteins using the labeled reagent But labeling method of proteins using thiol group labeling reagent (N-(4-azido-phenyl-thio) phthalimide) and said labeling reagent having an ultraviolet absorbing effect is disclosed in Non-Patent Document 8.

  Accordingly, the test protein is labeled in advance with these labeling reagents, the protein is cleaved with a protease, and a plurality of peptide fragments decomposed by the protease are utilized to make use of the difference in physical or chemical properties of each peptide fragment. It is also conceivable to extract peptide fragments having a thiol group by separation by means such as liquid chromatography.

  However, when several to several tens or more kinds of proteins are contained in the sample, the separation by the liquid chromatography or the like is as follows. (1) Depending on the properties of each protein contained in the sample, a plurality of proteins may be divided into the same fraction. That only the protein to be labeled cannot be reliably extracted, and (2) a sufficient yield cannot be obtained when there is only a very small amount of the target protein in the sample. Therefore, in practice, it was impossible to use an extraction fraction such as liquid chromatography as a sample of an amino acid sequence. This is most problematic in the identification of the position of the thiol group, particularly including the operation of cleaving the protein into a plurality of peptide fragments by protease.

  On the other hand, Non-Patent Document 9 discloses that anti-dansyl possessed by a labeling reagent by binding a test protein and a specific labeling reagent (monodansyl cadaverine) for transglutaminase-reactive glutamine residues in the presence of transglutaminase. A method for specifically extracting a labeled protein by antibody affinity chromatography utilizing the reactivity with a base antibody is disclosed. Further, this document describes that the labeled protein extracted by the method can be used as a sample for an amino acid sequence, and that the primary structure of the labeled protein can be determined by the amino acid sequence.

As described above, if the labeling reagent itself has properties such as reactivity with an antibody, it is possible to extract the protein to be labeled specifically and in large quantities using the properties. However, a labeling reagent for a thiol group having such properties is not known.
JP 9-249663 A JP-A-6-122679 JP-A-5-194384 Pouchnik, DJ et al. Anal. Biochem. 235: 26-35 (1996) Wingender, E. et al., Anal. Biochem. 127: 351-360 (1982) Kosower, NS et al., Proc. Natl. Acad. Sci. USA 76: 3382-3386 (1979) Hudson, EN et al., Biochemistry 12: 4154-4161 (1973) Kanaoka et al., Chem. Pharm. Bull. (Tokyo) 15: 1738-1743 (1967) Pavo, I. et al., FEBS Lett. 316: 59-62 (1993) Krueger, RJ et al., Anal. Biochem. 198: 165-173 (1991) Moreland, RB et al. Anal. Biochem. 121: 321-326 (1982) Takagi, J. et al., Eur. J. Biochem. 232: 773-777 (1995)

  Accordingly, it has been desired to develop a labeling reagent that can specifically bind to a thiol group of a protein and extract a protein to be labeled due to the properties of the labeling reagent itself.

（式中、Ｘはハロゲンを、ｍは１〜３の整数を、ｎは２〜８の整数を意味する）
で表される化合物を含有するチオール基標識用試薬を提供することである。
さらに本発明の他の目的は、上記式（Ｉ）で表される化合物の製造方法を提供することである。
さらにまた、本発明の他の目的は上記式（Ｉ）で表される化合物を用いるタンパク質の標識方法を提供することである。 In order to solve the above problems, the present inventor has proposed a monohalogenated carboxylic acid having reactivity with a thiol group and an amino group, and a dansyl group having reactivity and fluorescence with a commercially available anti-dansyl group antibody. And developed a reagent for labeling thiol groups that combines these properties.
That is, the object of the present invention is to formula (I)

(Wherein X represents halogen, m represents an integer of 1 to 3, and n represents an integer of 2 to 8)
It is providing the reagent for label | marking the thiol group containing the compound represented by these.
Still another object of the present invention is to provide a method for producing the compound represented by the above formula (I).
Furthermore, another object of the present invention is to provide a protein labeling method using the compound represented by the above formula (I).

式中、Ｘ、ｍ、ｎは上述したとおりであり、Ｒ₁、Ｒ₂はそれぞれ独立して、メチル基、エチル基、プロピル基、ブチル基、ヘキシル基またはシクロヘキシル基であり、さらにＲ₂は、

または

であり、ここで、ｐは１〜４の整数であり、Ｒ₃ およびＲ₄ はそれぞれ独立してメチル基、エチル基、プロピル基、ブチル基、ヘキシル基またはシクロヘキシル基を意味する。 [Method for Producing Compound Represented by Formula (I) of the Present Invention]
The compound represented by the formula (I) of the present invention is reacted with the halogenated carboxylic acid (III) in the presence of carbodiimide (IV) as shown in the following scheme. Can be easily produced in a one-step reaction. The produced compound (I) can be purified by a known separation method based on the difference in physical and chemical properties between the product and the starting material. Such purification methods include chromatography, fractional extraction, fractional crystallization or fractional distillation.

In the formula, X, m and n are as described above, R ₁ and R ₂ are each independently a methyl group, an ethyl group, a propyl group, a butyl group, a hexyl group or a cyclohexyl group, and R ₂ is ,

Or

Where p is an integer from 1 to 4 and R ₃ And R ₄ Each independently represents a methyl group, an ethyl group, a propyl group, a butyl group, a hexyl group or a cyclohexyl group.

As a specific example of the general formula (II),
1- (aminoethylsulfamyl) -5- (dimethylamino) naphthalene,
1- (aminopropylsulfamyl) -5- (dimethylamino) naphthalene,
1- (aminobutylsulfamyl) -5- (dimethylamino) naphthalene,
1- (aminopentylsulfamyl) -5- (dimethylamino) naphthalene,
1- (aminoheptylsulfamyl) -5- (dimethylamino) naphthalene,
1- (aminooctylsulfamyl) -5- (dimethylamino) naphthalene,
Etc.

  Examples of the halogenated carboxylic acid (III) include water-soluble halogenated compounds such as haloacetic acids such as iodoacetic acid, bromoacetic acid, chloroacetic acid and fluoroacetic acid, 3-bromopropanoic acid, 4-bromobutanoic acid and 3-chloropropanoic acid. Carboxylic acid is mentioned. The halogenated carboxylic acid (III) is used in a ratio of 1 to 3 equivalents per 1 equivalent of the compound of the formula (II).

または

であり、ここで、ｐは１〜４の整数であり、Ｒ₃ およびＲ₄ はそれぞれ独立してメチル基、エチル基、プロピル基、ブチル基、ヘキシル基またはシクロヘキシル基を意味する）が挙げられる。カルボジイミド（IV）は式（II）の化合物１当量に対し１〜３等量の割合で用いられる。 Examples of the carbodiimide used in the present invention include, for example, the general formula (IV)
R ₁ —N═C═N—R ₂ (IV)
(Wherein, R _1, R ₂ are each independently a methyl group, an ethyl group, a propyl group, a butyl group, a hexyl group or a cyclohexyl group, further R ₂ is

Or

Where p is an integer from 1 to 4 and R ₃ And R ₄ Each independently represents a methyl group, an ethyl group, a propyl group, a butyl group, a hexyl group or a cyclohexyl group). Carbodiimide (IV) is used in a proportion of 1 to 3 equivalents per equivalent of the compound of formula (II).

  The solvent used in the reaction is not particularly limited as long as it does not affect the reaction, but halogenated hydrocarbons (for example, chloroform, dichloromethane, carbon tetrachloride, etc.), ether solvents (for example, diethyl ether, diisopropyl ether, etc.) , Ketones (for example, acetone, methyl ethyl ketone, etc.) or organic solvents such as acetonitrile can be used alone or in combination.

Although the reaction time depends on the kind and amount of the starting material used, the completion of the reaction can be finally confirmed by thin phase chromatography. The reaction temperature is preferably about room temperature.
The separation operation following the above-mentioned amidation step can be performed by chromatography, fractional extraction, fractional crystallization, fractional distillation or a combination thereof.

  In the case of fractional extraction, the solvent used in the organic layer is not particularly limited unless it is uniformly mixed with the aqueous solvent used in the aqueous layer. Hydrocarbons (for example, benzene, toluene, xylene, hexane, heptane, etc.), Halogenated carbons (chloroform, dichloromethane, carbon tetrachloride, etc.) can be used alone or in combination. Moreover, as a solvent used for the aqueous layer, an acidic aqueous solvent adjusted to pH 2 to 3 (for example, hydrochloric acid aqueous solution, acetic acid aqueous solution, citric acid aqueous solution, etc.) and a basic aqueous solvent adjusted to pH 10 to 11 (for example, hydroxylated) Sodium aqueous solution, sodium carbonate aqueous solution, etc.) can be used. Furthermore, a glycine buffer or the like whose pH is adjusted to 2-3 or 10-11, respectively, can also be used.

  In the case of performing chromatography, the column carrier used includes, for example, silica gel, and the mobile phase used is not particularly limited as long as it does not affect the separation, and hydrocarbons (for example, benzene, toluene, xylene, hexane, heptane). Etc.), halogenated carbons (eg, chloroform, dichloromethane, carbon tetrachloride, etc.), ethers (eg, diethyl ether, diisopropyl ether, etc.), ethyl acetate, etc. can be used alone or in combination.

  The compound (I) thus separated and purified is dried at 20 ° C. to 100 ° C. (preferably 80 ° C.) using an evaporator or the like, and is stored in the form of a powder at −20 ° C. and protected from light. Is desirable.

  Next, a method for reacting the compound (I) of the present invention with a thiol group in a protein and labeling them will be described.

[Method for labeling protein with compound (I) of the present invention]
The labeling of the protein using the compound (I) of the present invention can be carried out under mild conditions. For example, SDS, Triton can be added to a protein having a thiol and 1 equivalent of the thiol group of the protein as necessary. What is necessary is just to make it react with 2-4 equivalents of compound (I) solubilized with surfactants, such as X, a chaps, and Tween 20, in a buffer solution of room temperature and pH 6-8 under shading. The reaction time may be 30 minutes or longer, and even if the reaction is performed overnight or longer, it does not react with groups other than thiol groups in the protein (for example, amino groups), but the standard reaction time is 45 minutes to 2 minutes. It's time. As long as the reaction solution does not inhibit the reaction between the compound (I) and the protein, a preservative, a surfactant, and a stabilizer may coexist.

  The protein labeled with the compound (I) is not particularly limited as long as the protein has a thiol group. When the protein is an insoluble protein, the protein can be labeled with a surfactant such as SDS, Triton X, Chaps, or Tween20. It can be solubilized and labeled.

  Compound (I) has specific reactivity with a thiol group in a protein and does not react with disulfide, but when it is desired to label a cysteine residue forming a disulfide bond, dithiothreitol, 2- Such cysteine residues can be labeled by reducing the disulfide bond to a thiol group in advance with a reducing agent such as mercaptoethanol.

[Method for detecting and extracting protein labeled with compound (I) of the present invention (labeled protein)]
The labeled protein thus obtained can be specifically identified by means such as antibody affinity chromatography utilizing the reactivity of the dansyl group contained in compound (I) with the anti-dansyl group antibody. Can be extracted. Furthermore, detection by a highly sensitive immunological technique such as Western blotting or immunoassay is possible. Further, the protein to be labeled can also be detected by measuring fluorescence under the known dansyl group fluorescence measurement conditions, for example, under ultraviolet irradiation, using the fluorescence of the dansyl group.

  As an application of the present invention, for example, the thiol-specific reactivity of compound (I) can be used to confirm whether the thiol group present in the test protein exists in an oxidized form or a reduced form. For example, after reacting a protein having an oxidized form and a reduced form, such as glutathione, with a compound (I) under specific conditions, the molecular weight is measured by a mass spectrometer or the like. It can be confirmed whether it exists in a state.

  Another application of the present invention is, for example, extraction of a protein to be labeled by antibody affinity chromatography. That is, by passing the labeled protein labeled as described above through an affinity column to which an anti-dansyl antibody is added, only the labeled protein labeled with the compound (I) can be extracted.

  Furthermore, another application of the present invention is, for example, detection of a protein to be labeled by Western blotting or immunoassay utilizing reactivity with anti-dansyl group antibodies. Since these methods have a higher detection sensitivity than the chromatographic method, it becomes possible to detect the labeled protein with a small number of samples.

  Further, as another application example of the present invention, for example, the labeled protein labeled as described above is separated by thin layer chromatography, and the fluorescence of the dansyl group is detected to detect the thiol group in the test protein. The existence can be confirmed.

  As another application of the present invention, for example, use of so-called post column high performance liquid chromatography can be considered. That is, the test protein can be labeled with the compound (I) of the present invention, separated by the separation method such as the chromatograph, and the labeled protein can be extracted by fluorescence measurement using a fluorescence detector.

  Furthermore, as another application of the present invention, for example, extraction of a labeled protein combining anti-dansyl antibody affinity chromatography and high performance liquid chromatography can be mentioned. That is, when a plurality of types of labeled proteins are contained in the antibody affinity chromatography extract, it is possible to further separate them by high performance liquid chromatography and to isolate the labeled proteins.

  Unlike the halogenated carboxylic acid, the compound (I) of the present invention specifically recognizes a thiol group contained in a protein and does not bind to an amino group or a disulfide group. It can be specifically labeled. That is, by utilizing such a property of compound (I), a protein having a thiol group can be specifically detected, and further, a cysteine residue in the protein exists in an oxidized form or a reduced form. You can check if you want to.

  Moreover, since the dansyl antibody contained in the compound (I) has reactivity with the anti-dansyl group antibody, the labeled protein can be extracted by means such as antibody affinity chromatography which could not be performed conventionally. That is, only the protein to be labeled can be prepared specifically and in large quantities, and the preparation of the sample for amino acid sequence is facilitated, which contributes to the identification of the position of the cysteine residue in the protein. Furthermore, by combining antibody affinity chromatography and high performance liquid chromatography, even when there are a plurality of types of labeled proteins, it is possible to isolate each type of labeled protein.

  In addition to the detection means using the fluorescence possessed by dansyl groups, it has reactivity with anti-dansyl group antibodies, so it can be used for Western blotting or immunoassay as a highly sensitive immunological detection means. It is possible to use such means. That is, even when only a very small amount of the protein to be labeled exists, it can be easily detected by using this method.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated concretely, this invention is not restrict | limited at all by these Examples.

Example 1 Synthesis of iodoacetyl dansyl cadaverine iodoacetamide (11.1 mg, final concentration 20 mM), monodansyl cadaverine (10.1 mg, final concentration 10 mM) and N, N′-dicyclohexylcarbodiamide (6.2 mg, final concentration) 10 mM) was stirred in dichloromethane (3 mL) at room temperature for 15 minutes.
After confirming the completion of the reaction by thin-layer chromatography using hexane / ethyl acetate (1: 1), the reaction solution was alternately mixed with glycine buffer (3 mL) at pH 3.0 and pH 10.0. Partitioned twice in two layers. After washing, the organic layer containing iodoacetyl dansyl cadaverine was collected, dried by a rotary evaporator, and redissolved in chloroform (3 mL). This solution was washed twice with a solution of chloroform / methanol / glycine buffer (3 mL, pH 3.0) (40: 20: 7), and the organic layer containing iodoacetyl dansyl cadaverine was collected again and dried to dryness using a rotary evaporator. did. It was dissolved in a solution of hexane / ethyl acetate (1: 1) and purified by a silica gel column (diameter 12 mm, length 250 mm) to obtain 7.2 mg of iodoacetyl dansyl cadaverine (yield 48%). The analytical values of this substance were as follows.
¹ H-NMR (CDCl ₃ ) ppm: δ1.07-1.42 (6H, m), 2.88 (2H, dt, J = 5.6 Hz), 2.89 (6H, s), 3.10 (2H, dt, J = 6.3 Hz) ), 3.66 (2H, s), 4.6 (1H, s), 6.74 (1H, s), 7.19 (1H, d, J = 7.6 Hz), 7.50-7.59 (2H, m), 8.22 (1H, d, J = 8.6 Hz), 8.31 (1H, d, J = 8.6 Hz), 8.54 (1H, d, J = 8.6 Hz)

[Example 2] Labeling of reduced glutathione Reduced glutathione (30 µg, final concentration 10 µM), which is a tripeptide having a thiol group, was dissolved in 50 mM Tris buffer (pH 7.5, 1 mL), and then iodoacetyldansylcadaverine (5 µg). The final concentration was 10 μM) and the reaction was allowed to proceed at room temperature for 45 minutes while protected from light. After completion of the reaction, the solution was diluted with a methanol / water (1: 1) solution containing 1% acetic acid, and the molecular weight of the reaction product was measured using an electrospray ionization mass spectrometer.
As a result, a small amount of a compound having a molecular weight of 683.3 in which hydrogen ions are added to the reaction product of reduced glutathione and iodoacetyldansylcadaverine and an unreacted reduced glutathione (molecular weight of 308.2) to which hydrogen ions have been added are detected. (A in FIG. 1). On the other hand, as a result of conducting the same experiment using oxidized glutathione having no thiol group, iodoacetyl dansyl cadaverine and oxidized glutathione did not react, and as a result of mass spectrometry, hydrogen ions were added to this compound. A compound in which hydrogen ion (molecular weight 613.3) and sodium ion (molecular weight 634.9) were added to the compound (molecular weight 504.2) and oxidized glutathione was detected (B in FIG. 1).
This result reveals that iodoacetyl dansyl cadaverine does not react with disulfide, but reacts specifically with thiol groups, and further confirms whether thiol groups in proteins exist in oxidized or reduced forms. It was considered possible.

[Example 3] Detection of labeled reduced ribonuclease by thin layer chromatography 20 μg of ribonuclease having all cysteine residues in the molecule as thiol groups was prepared with dithiothreitol, and in 50 mM Tris buffer (1 mL, pH 7.5). The reaction was carried out with iodoacetyl dansyl cadaverine (50.3 μg, 0.1 mM) at room temperature for 45 minutes in the dark. Thereafter, by thin-layer chromatography using methanol / acetic acid (19: 1) as a developing solvent, only iodoacetyl dansyl cadaverine was developed in lane 2, and a reaction mixture of iodoacetyl dansyl cadaverine and ribonuclease was developed in lane 1. Analysis with an ultraviolet lamp. As a result, only spots showing iodoacetyl dansyl cadaverine in lane 2 were observed, whereas spots showing unreacted iodoacetyl dansyl cadaverine in lane 1 and spots showing reaction products of ribonuclease not moved by the developing solvent. Was observed (FIG. 2).
From this result, it was revealed that the protein bound by iodoacetyl dansyl cadaverine is fluorescently labeled, and the protein to be labeled can be detected by measuring the fluorescence.

[Example 4] Detection of labeled protein by Western blotting 20 μg each of ribonuclease, lysozyme, and bovine serum albumin having all cysteine residues in the molecule as thiol groups was prepared with dithiothreitol and contained 1% Triton X-100 Iodoacetyl dansyl cadaverine (0.5 mg, final concentration 1 mM), iodoacetyl dansyl cadaverine (0.5 mg, final concentration 1 mM) and 5,5′-dithiobis (2-nitro) in 50 mM Tris buffer (1 mL, pH 7.5) Benzoic acid) (0.4 mg, final concentration 10 mM), iodoacetyl dansyl cadaverine (0.5 mg, final concentration 1 mM) and iodoacetamide (0.2 mg, final concentration 10 mM) were reacted in the dark for 45 minutes at room temperature. . Thereafter, each reaction solution was subjected to Western blotting using an anti-dansyl antibody. As a result, ribonuclease containing all cysteine residues in the molecule, lysozyme, bovine serum albumin and iodoacetyl dansyl cadaverine reacted with each other, and each protein band was detected by Western blotting using an anti-dansyl antibody ( FIG. 3, lane 2). In contrast, when iodoacetyl dansyl cadaverine (0.5 mg, final concentration 1 mM) and 5,5′-dithiobis (2-nitrobenzoic acid) or iodoacetamide were added simultaneously, each reduced protein and iodoacetyl dansyl were added. Reaction with cadaverine was inhibited (FIG. 3, lanes 3 and 4). In addition, even when ribonuclease, lysozyme, and bovine serum albumin not treated with dithiothreitol were reacted with iodoacetyldansylcadaverine, almost no band of each protein was detected by Western blotting (FIG. 3, lane 1).
From this result, iodoacetyl dansyl cadaverine does not bind to the amino group in the protein, it reacts specifically only with the thiol group, the labeled protein has reactivity with the anti-dansyl group antibody, and this property It has become clear that labeled proteins can be detected by immunological techniques.

[Example 5] Identification of a peptide fragment containing a cysteine residue derived from glyceraldehyde 3-phosphate dehydrogenase According to the following method, a peptide fragment containing a cysteine residue derived from glyceraldehyde 3-phosphate dehydrogenase was identified. Identification was performed.
Glyceraldehyde 3-phosphate dehydrogenase (1 mg, final concentration 30 μM) is dissolved in 50 mM Tris buffer (1 mL, pH 7.5) containing 1 mM dithiothreitol, 1% Triton X-100, 4 M urea, and room temperature. And reacted for 2 hours in the dark. Thereafter, iodoacetyl dansyl cadaverine (2.5 mg, final concentration 5 mM) was added, and the mixture was further reacted for 1 hour. The reaction solution was dialyzed sequentially with 50 mM Tris buffer (pH 7.5) and 50 mM Tris buffer (pH 8.8), and then lysyl endopeptidase (16.5 μg, final concentration 0.6 μM) and urea (0.24 mg, final concentration 4 M). ), Sodium dodecyl sulfate (1 mg, final concentration 0.1%) was added and allowed to react at 37 ° C. for 20 hours in the dark, and glyceraldehyde 3-phosphate dehydrogenase labeled with iodoacetyldansylcadaverine was converted to protease. Digested. Peptide fragments in this solution were separated by high performance liquid chromatography loaded with a C-18 hydrophobic column, and absorbance at 205 nm (A in FIG. 4) and fluorescence (excitation wavelength 360 nm, fluorescence wavelength 510 nm) (B in FIG. 4). Detected by. As a result, many peaks were detected in the absorption chromatograph, while several peaks were detected in the fluorescence chromatograph.

Further, this reaction solution was passed through an antibody affinity column to which an anti-dansyl antibody was added, and only the peptide fragment labeled with iodoacetyldansylcadaverine was adsorbed and eluted. The eluate was similarly separated by high performance liquid chromatography loaded with a C-18 hydrophobic column, and detected by absorbance at 205 nm (A in FIG. 5) and fluorescence (excitation wavelength 360 nm, fluorescence wavelength 510 nm) (B in FIG. 5). did. As a result, the absorption and fluorescence chromatographs almost coincided, and four peaks were obtained. The peaks 1 to 4 in FIGS. 5A and 5B were collected, and the amino acid sequence of the peptide was analyzed by a protein sequencer. As a result, the sequences corresponded to the underlined sequences indicated by 1 to 4 in FIG.
From this result, it was revealed that the labeled peptide can be extracted specifically by this method, and the position of the cysteine residue in the protein molecule can be identified using the peptide extract.

The result of the mass spectrum which measured the reaction solution of iodoacetyl dansyl cadaverine and glutathione with the electrospray ionization mass spectrometer is shown. In the figure, A shows the result when reduced glutathione is used, and B shows the result when oxidized glutathione is the test peptide. The results of separation of labeled ribonuclease by thin layer chromatography and detection with an ultraviolet lamp are shown. The result of detecting various proteins to be labeled (ribonuclease (upper), lysozyme (middle), bovine serum albumin (lower)) by Western blotting is shown. The analysis result by the high performance liquid chromatography of the peptide fragment of the labeled glyceraldehyde 3-phosphate dehydrogenase fragmented with lysyl endopeptidase is shown. In the figure, A is an elution curve when the absorbance at 205 nm is measured, and B in the figure is an elution curve when fluorescence is measured. The peptide fragment of labeled glyceraldehyde 3-phosphate dehydrogenase fragmented with lysyl endopeptidase was passed through an anti-dansyl antibody column, and the eluate received from the anti-dansyl antibody column was analyzed by high performance liquid chromatography. Show. In the figure, A is an elution curve when the absorbance at 205 nm is measured, and B in the figure is an elution curve when fluorescence is measured. 1 shows the primary structure of chicken muscle-derived glyceraldehyde 3-phosphate dehydrogenase.

Claims (4)

Formula (I)

(Wherein X represents halogen, m represents an integer of 1 to 3, and n represents an integer of 2 to 8).   2. The thiol group labeling reagent according to claim 1, wherein m is 1 and n is 5. Formula (II)

(Wherein n represents an integer of 2 to 8), the compound represented by formula (III)

A compound represented by the formula (IV), wherein X represents a halogen and m represents an integer of 1 to 3;
R ₁ —N═C═N—R ₂ (IV)
(Wherein, R _1, R ₂ are each independently a methyl group, an ethyl group, a propyl group, a butyl group, a hexyl group or a cyclohexyl group, further R ₂ is

Or

Where p is an integer from 1 to 4 and R ₃ And R ₄ Each independently reacts with a compound represented by a methyl group, an ethyl group, a propyl group, a butyl group, a hexyl group or a cyclohexyl group), and the reagent for labeling a thiol group according to claim 1 Manufacturing method. Formula (I)

(Wherein X represents a halogen, m represents an integer of 1 to 3, and n represents an integer of 2 to 8), and a protein represented by reacting with a thiol group in the protein Sign method.

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