JP3345646B2 - Reagent for detecting chlorinated aromatic compound having amino group and tetrapeptide used therefor - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a reagent for detecting a chlorinated aromatic compound having an amino group and an oligopeptide used therefor.

[0002]

2. Description of the Related Art In recent years, organochlorine pesticides remaining in the environment such as soil, water, and air, which have become a social problem, for example, 3- (3,4-dichlorophenyl)-
The rapid and accurate detection and measurement of chlorinated aromatic pesticides having an amino group such as 1,1-dimethylurea (DCMU), atrazine, linuron, simazine, etc. is for ensuring the safety of the environment and food. Needless to say, it is necessary. Conventionally, precision analysis devices such as gas chromatography and high performance liquid chromatography have been generally used as a method for detecting and measuring such organic chlorine compounds. However, although these methods are excellent in accuracy and sensitivity, there is a problem that a large and complicated apparatus is required, sample preparation becomes complicated, and much labor and time are required. In order to improve such a method, a method utilizing an antigen-antibody reaction exhibited by the agricultural chemical component and a method utilizing the fluorescence emission characteristics of the agricultural chemical component have been proposed, but at present it is still insufficient.

[0003]

DISCLOSURE OF THE INVENTION The present invention has been made under the above circumstances, and is directed to a novel detection reagent capable of rapidly and accurately detecting a chlorinated aromatic compound having an amino group, and a tetrade used therefor. It is intended to provide a peptide.

[0004]

Means for Solving the Problems The present inventors diligently studied the above-mentioned problems also using a combinatorial chemistry technique, and as a result, it was found that a tetrapeptide having a specific amino acid sequence obtained from a random tetrapeptide library was effective. Having found that there is, the present invention has been achieved. That is, according to the present invention, first, there is provided a reagent for detecting a chlorinated aromatic compound having an amino group, the reagent comprising at least one tetrapeptide selected from the following formulas A1 to A5. A1 Asp-Thr-Tyr-Tyr (SEQ ID NO: 1) A2 Asp-Phe-Tyr-Ala (SEQ ID NO: 2) A3 Asp-Asn-Ile-Tyr (SEQ ID NO: 3) A4 Asp-Val- Ile-Val (SEQ ID NO: 4) A5 Asp-Gln-Phe-Leu (SEQ ID NO: 5) Second, at least one selected from the above formulas A1 to A5 used in the detection reagent according to the first invention Are provided. Third, at least one tetrapeptide selected from the following formulas A1 to A5 is provided. A1 Asp-Thr-Tyr-Tyr (SEQ ID NO: 1) A2 Asp-Phe-Tyr-Ala (SEQ ID NO: 2) A3 Asp-Asn-Ile-Tyr (SEQ ID NO: 3) A4 Asp-Val- Ile-Val (SEQ ID NO: 4) A5 Asp-Gln-Phe-Leu (SEQ ID NO: 5)

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION The reagent for detecting a chlorinated aromatic compound having an amino group according to the present invention comprises at least one tetrapeptide having a specific amino acid sequence represented by the following formulas A1 to A5. A1 NH2-Asp-Thr-Tyr-Tyr A2 NH2-Asp-Phe-Tyr-Ala A3 NH2-Asp-Asn-Ile-Tyr A4 NH2-Asp-Val-Ile-Val A5 NH2-Asp-Gln-Phe-Leu For such peptides, first, a combinatorial chemistry technique was used to construct a random tetrapeptide library, and then these peptides exhibited the presence or absence of affinity with a chlorinated aromatic compound having an amino group.
It is obtained by identification and screening by a fluorescence index method or the like.

Specifically, a peptide library consisting of four amino acids is constructed by split solid phase synthesis, which is a kind of combinatorial chemistry, using 19 natural amino acids except cysteine, and synthesized on beads. The prepared peptide is prepared. Next, as shown in FIG. 1, a chlorinated aromatic compound having an amino group, for example, 3,4-dichloroaniline is used as a fluorescent labeling agent, for example, 4-fluoro-7-nitrobenzofuralazane (N
BD-F) to prepare a dispersion / solution. Next, the peptide beads are suspended in the dissolved / dispersed liquid of the labeled product, and the fluorescent-stained peptide beads are washed and screened, and the washing and screening steps are repeated as appropriate to obtain a desired peptide. You.

The chlorinated aromatic having an amino group to be detected in the present invention includes, for example, dichloroaniline, 3
-(3,4-dichlorophenyl) -1,1-dimethylurea (DCMU), atrazine, linuron, simazine and the like are included, but DCMU, atrazine and the like are preferably used.

[0008]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail with reference to embodiments.

Example 1 Dichloroaniline was labeled with a fluorescent labeling agent, 4-fluoro-7-nitrobenzofuralazane (NBD-F) (FIG. 1). Note that dichloroaniline and NBD
4-Amino-n-butyric acid was used as a spacer between -F. Subsequently, a peptide library consisting of four residues using 19 natural amino acids excluding cysteine was constructed by solid phase synthesis using the split synthesis method. This solid-phase library was screened for dichloroaniline binding using NBD fluorescence as an index (FIG. 2). Specifically, 18
A 10 mM phosphate buffer (pH 7.0) containing 5% ethanol and 100 mM NaCl was prepared by adding 0 nM concentration of NBD-dichloroaniline.
As a result of screening about 420,000 peptide beads used for the screening, about 400 beads to be fluorescently stained were obtained. In order to further exclude those that recognize domains other than the target site (dichloroaniline) in the molecule, beads that were competitively quenched by addition of an excessive amount of 3,4-dichloroaniline were searched. Five peptides were obtained. A1 NH2-Asp-Thr-Tyr-Tyr A2 NH2-Asp-Phe-Tyr-Ala A3 NH2-Asp-Asn-Ile-Tyr A4 NH2-Asp-Val-Ile-Val A5 NH2-Asp-Gln-Phe-Leu

Example 2 [Test for binding ability of chlorinated organic compound] Cysteine was introduced into the C-terminal of the peptide represented by the formula A1 to synthesize a peptide A1C.
The concentration was adjusted to 3 mM with M acetate buffer (pH 5.0). The ability of this A1C peptide to bind chlorinated organic compounds was measured using a surface plasmon resonance (SPR) measuring device, BIACORE X
(Biacore). CM5 with carboxymethyl dextran on the surface of the sensor chip
Using a sensor chip (Biacore), 3,4-dichloroaniline derivative bound to the peptide was measured. 0.
05 M N-hydroxysuccinimide (NHS) /0.2 M
By injecting an aqueous solution of N-ethyl-N,-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) into a flow path of an SPR device equipped with a CM5 sensor chip at a flow rate of 5 μl / min for 8 minutes, CM5 Dextran was activated. Then, 0.1 M borate buffer (pH 8.30) containing 0.8 mM 2- (2-pyridinyldithio) ethanamide (PDEA).
Disulfide was introduced by loading 5) for 16 minutes. The dichloroaniline-recognizing tetrapeptide A1C solution prepared by the method described above was applied to the chip at a flow rate of 5
Immobilization was carried out by loading four times for 20 minutes at μl / min. The amount of the peptide thus immobilized was about 1 ng. As the chlorinated organic compound, 3- (3,4) represented by the following formula, which is a 3,4-dichloroaniline derivative:
-Dichlorophenyl) -1,1-dimethylurea (DC
MU) (herbicide), its peptide A1C and DCM
The amount bound to U was measured by SPR.

Embedded image 3- (3,4-Dichlorophenyl) -1,1-dimethylurea As a result, the SPR signal indicating the amount of binding between peptide A1C and DCMU showed a concentration-dependent increase (FIG. 3).
FIG. 3 shows that this peptide A1C has a property of specifically recognizing and binding to DCMU which is a chlorinated aromatic compound having an amino group.

Example 3 In Example 2, instead of DCMU (herbicide), 2-chloro-4-ethylamino-6- represented by the following formula was used.
The amount of binding to peptide A1C was measured by SPR in the same manner as in Example 2 except that isopropylamino-1,3,5-triazine (atrazine: a triazine herbicide) was used.

Embedded image As a result, the SPR signal indicating the binding amount between peptide A1C and atrazine showed a concentration-dependent increase (FIG. 3). From FIG. 3, it can be seen that this peptide A1C has a property of specifically recognizing and binding to atrazine, which is a chlorinated aromatic compound having an amino group.

Comparative Example 1 In Example 2, instead of DCMU (herbicide), a chlorinated organic compound represented by the following formula and having no amino group, (RS) -2 (4-chloro-o- The amount of binding to peptide A1C was determined in the same manner as in Example 2 except that (triloxy) propionic acid (MCPP: phenoxy herbicide) was used.
Measured by SPR.

Embedded image As a result, the SPR signal indicating the amount of binding between peptide A1C and MCPP did not show a concentration-dependent increase phenomenon (FIG. 3). From FIG. 3, this peptide A1C recognizes MCPP which is a chlorinated aromatic compound having no amino group,
It turns out that it does not show the binding property.

[0013]

The peptide of the present invention selected from the above formulas A1 to A5 has the property of specifically recognizing and binding to a chlorinated organic compound having an amino group. Since chlorinated aromatic pesticides having an amino group remaining in the environment such as the atmosphere can be detected quickly and accurately, safety in the environment and food can be easily ensured.

[0014]

[Sequence List] SEQUENCE LISTING <110> Director-General of National Institute of Advanced Industrial Science and Technology, Ministry of Economy, Trade and Industry <120> Chemical detection reagent, peptides binding chlorinated aromatic compounds having amino group. <130> 117F011 <140> JP P2000-200565 <141> 2000-7-3 <150> <151> <160> 5 <170> <210> 1 <211> 4 <212> PRT <400> 1 Asp Thr Tyr Tyr <210 > 2 <211> 4 <212> PRT <400> 2 Asp Phe Tyr Ala <210> 3 <211> 4 <212> PRT <400> 3 Asp Asn Ile Tyr <210> 4 <211> 4 <212> PRT <400> 4 Asp Val Ile Val <210> 5 <211> 4 <212> PRT <400> 5 Asp Gln Phe Leu

[Brief description of the drawings]

FIG. 1 is an explanatory diagram of NBD-labeled dichloroaniline.

FIG. 2 is an explanatory diagram of a screening flowchart.

FIG. 3. DCMU detected by A1C peptide,
Atrazine and MCPP concentration correlation diagram.

Continuation of the front page (56) References JP-A-9-187296 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) G01N 31/00-31/22 C07K 5/113 REGISTRY ( STN)

Claims (3)

(57) [Claims] 1. A reagent for detecting a chlorinated aromatic compound having an amino group, comprising at least one tetrapeptide selected from the following formulas A1 to A5. A1 Asp-Thr-Tyr-Tyr (SEQ ID NO: 1) A2 Asp-Phe-Tyr-Ala (SEQ ID NO: 2) A3 Asp-Asn-Ile-Tyr (SEQ ID NO: 3) A4 Asp-Val- Ile-Val (SEQ ID NO: 4) A5 Asp-Gln-Phe-Leu (SEQ ID NO: 5) 2. At least one tetrapeptide selected from the formulas A1 to A5 used in the detection reagent according to claim 1. 3. At least one tetrapeptide selected from the following formulas A1 to A5. A1 Asp-Thr-Tyr-Tyr (SEQ ID NO: 1) A2 Asp-Phe-Tyr-Ala (SEQ ID NO: 2) A3 Asp-Asn-Ile-Tyr (SEQ ID NO: 3) A4 Asp-Val- Ile-Val (SEQ ID NO: 4) A5 Asp-Gln-Phe-Leu (SEQ ID NO: 5)