Dioxin-Binding Material and Method of Detecting or Quantifying Dioxin

Abstract

A principal object of the present invention is to provide low-cost techniques for readily detecting or quantifying dioxins using substances which are inexpensive and can be easily produced, and extracting dioxins using such substances. Dioxin binding peptides of the present invention are highly selective to dioxins, and are therefore capable of detecting or quantifying dioxins in test samples containing impurities. Dioxin binding peptides of the present invention are also capable of selectively extracting dioxins from impurity-containing test samples, and are therefore useful in simple pretreatment for quantifying and analyzing dioxins.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows steps of detecting or quantifying dioxin.

FIG. 2 shows steps of screening dioxin binding peptides.

FIG. 3 shows the structures of NBD-labeled dichlorophenol; 2,3,7-TriCDD; and 2,3,7,8-TeCDD.

FIG. 4 shows a fluorescence microscope image of a fluorescently stained dioxin binding bead, wherein the round object is the fluorescently stained dioxin binding bead.

FIG. 5 shows the results of on-bead competitive quenching tests for the DB2 peptide according to SEQ ID No. 3, wherein the round objects are the fluorescently stained dioxin binding beads.

FIG. 6 shows the results of on-bead competitive quenching tests for the DB2 peptide according to SEQ ID No. 3 conducted in a solvent containing 30% 1,4-dioxane, wherein graph (A) shows the fluorescence intensity of the beads, and graph (B) shows the quenching ratio of the beads, with the black circles representing the test results for 2,3,7,8-TeCDD, and the white circles representing the test results for 2,3,7-TriCDD.

FIG. 7 shows a graph of the relationship between dioxin concentration and the time it takes for fluorescent staining and competitive quenching, using 2,3,7-TriCDD having a concentration of 10 nM, wherein the black circles represent 10 nM NBD-labeled dichlorophenol; the black triangles represent 5 nM NBD-labeled dichlorophenol; the black squares represent 1 nM NBD-labeled dichlorophenol; the solid lines represent NBD-labeled dichlorophenol only; and the broken lines represent mixtures of NBD-labeled dichlorophenol at the aforementioned concentrations with 2,3,7-TriCDD.

FIG. 8 shows the structures of substituted amino acid side chains in the one amino acid-substituted peptide library.

FIG. 9 shows a graph illustrating the degree of NBD-labeled dichlorophenol staining of each peptide in the amino acid-substituted peptide library.

FIG. 10 shows the evaluation results of the dioxin binding capabilities of single residue-substituted peptides by the method of competitive dioxin binding on bead, wherein 1 Cha is the peptide according to SEQ ID No. 5; 5 Phg is the peptide according to SEQ ID No. 22; 5 Leu is the peptide according to SEQ ID No. 23; and 5 Nva is the peptide according to SEQ ID No. 24.

FIG. 11 shows the structures of test samples used in evaluating the peptide specificities by the method of competitive dioxin binding on bead.

FIG. 12 shows the results of binding specificity tests for the DB1 peptide according to SEQ ID No. 2 and the substituted peptides confirmed to have a capability of binding to 2,3,7,8-TeCDD equal to or more than that of the DB2 peptide according to SEQ ID No. 3 by the method of competitive dioxin binding on bead using a 30% 1,4-dioxane solvent, wherein 1 Cha is the peptide according to SEQ ID No. 5; 5 Phg is the peptide according to SEQ ID No. 22; 5 Leu is the peptide according to SEQ ID No. 23; and 5 Nva is the peptide according to SEQ ID No. 24; the horizontal axis of each graph representing the decrease in fluorescence intensity.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is further described in detail by the following Examples, which are not intended to limit the disclosure of the invention.

EXAMPLE 1

Obtaining Dioxin Binding Oligopeptides

A peptide library was constructed by split-and-pool synthesis, one of the typical techniques of combinatorial chemistry, using beads for peptide solid-phase synthesis. According to the present method, a peptide of one kind of sequence is synthesized on a single bead. As shown in FIG. 2, screening consisted of two stages. Primary screening involved screening of fluorescently stained peptide beads, using a composite (FIG. 3) obtained by labeling 3,4-dichlorophenol, which has an analogous structure to dioxins, with NBD as a fluorescent material. Secondary screening involved screening of peptide beads fluorescently decreased by competition with 2,3,7-trichlorodibenzo-p-dioxin, i.e., peptide beads having an affinity to dioxin(s), from the peptide beads which were stained with the fluorescently labeled dichlorophenol.

About 2.5 million peptide beads were used for screening, the number being equal to the number of combinations of all the sequences of peptides comprising 5 amino acid residues. The primary screening was conducted in a screening solvent (a 10 mM phosphate buffer containing 20% 1,4-dioxane (pH: 8)) containing 4 nM NBD-labeled dichlorophenol. 20 ml of the buffer solution was first mixed with about 50 mg of the peptide beads, and the mixture was subsequently incubated overnight, with mild shaking, in a petri dish at room temperature. A fraction of fluorescently stained peptide beads observed by a fluorescent microscope was collected with a micropipette. These peptide beads were transferred into a micro test tube containing 50 or 100% 1,4-dioxane, and were then incubated overnight at room temperature, i.e., washing with 50 or 100% dioxane overnight at room temperature. Peptide beads which were unable to be washed, on which the NBD-labeled dichlorophenol was unspecifically adsorbed, were excluded. Peptide beads that could be washed were re-stained with 1 nM NBD-labeled dichlorophenol. In order to measure the fluorescence intensity, fluorescent microscope images of the peptide beads were recorded with a digital camera (FIG. 4).

The aforementioned washable peptide beads were put into 1 ml of a screening solvent containing 1 nM NBD-labeled dichlorophenol and 10 or 100 nM 2,3,7-trichlorodibenzo-p-dioxin (2,3,7-TriCDD). The mixture was incubated overnight with mild shaking at room temperature, and was then transferred onto a glass petri dish, and fluorescent microscope images thereof were recorded. The obtained images were compared with the images recorded in the previous test so as to screen decreased beads. As shown in FIG. 5, two peptide beads were confirmed to be decreased under the competitive condition of NBD-labeled dichlorophenol (1 nM) with 10 fold concentration of 2,3,7-TriCDD (10 nM). The term “Reference” in FIG. 5 denotes the beads which were determined as not being fluorescently stained in the primary screening. The amino acid sequences of the peptides on the screened beads were determined with a protein sequencer. As a result, the amino acid sequences of the dioxin-binding peptide beads for which quenching by competition with 10 nM 2,3,7-TriCDD was confirmed proved to be Phe-Leu-Asp-Gln-Ile and Phe-Leu-Asp-Gln-Val. The Phe-Leu-Asp-Gln-Ile peptide bead was named DB1, and the Phe-Leu-Asp-Gln-Val peptide bead was named DB2.

EXAMPLE 2

Evaluation of Binding Capabilities of Dioxin Binding Peptides

Using dioxin-binding peptide beads, the dioxin binding capabilities of the peptides were evaluated in terms of affinity and specificity.

Method of Competitive Dioxin Binding on Bead

1 ml of a screening solvent containing 4 nM NBD-labeled dichlorophenol and 0-100 nM of a substance to be detected was prepared in a glass vial, and then three dioxin-binding peptide beads were put into the screening solvent. The resulting mixture was incubated, with mild shaking, overnight at room temperature, and then fluorescent microscope images thereof were recorded. The average fluorescence intensity of each bead was calculated from the recorded images. Calculation of the average fluorescence intensity was performed with an image analysis/measurement software, “Image-Pro Plus” (Planetron, Inc.). When competitive binding of the dichlorophenol with a dioxin occurs, the fluorescence intensity of each bead decreases depending on the concentration of the dioxin. Such a method of measuring the dioxin concentration utilizing the quenching phenomenon of the beads was termed “the method of competitive dioxin binding on bead”.

The DB2 peptide was evaluated for affinity to dioxins by the method of competitive dioxin binding on bead. FIGS. 6 (A) and 6 (B) show the results of tests conducted in a solvent containing 30% 1,4-dioxane. The relationship between dioxin concentration and average fluorescence intensity is plotted in FIG. 6 (A). As shown in the figure, declining sigmoid curves, which are characteristic of the competitive binding, were obtained. The curves were fitted using the four-parameter logistic equation, y=(a−d)/(1+(x/c)^b)+d, which is an empirical equation typically employed in competitive ELISA (Eiji Ishikawa, “Enzyme Immunoassay (3rd Edition)”, IGAKU-SHOIN). Concentration-dependent quenching was observed for both 2,3,7-TriCDD and 2,3,7,8-TeCDD, and the results showed that 1 nM (about 0.3 ng/ml) 2,3,7,8-TeCDD can be detected using 30% 1,4-dioxane.

FIG. 6 (B) shows a graph which plots the quenching ratio of the beads determined from the average fluorescence intensity. The quenching ratio was calculated using the equation shown below. The beads were evaluated for affinity with the substances detected based on the values of quenching ratios.

quenching ratio=(fluorescence intensity 1−fluorescence intensity 2)/fluorescence intensity 1×100(%)

The binding constants of the DB2 peptide were calculated using the results of the above equation. Fitting was performed based on the theoretical equation for one-to-one binding of a receptor with a ligand as shown below:

Y=((Ymax/2e−9)*(1/2)*((2e−9+X*1e−9+1/Ka)−((2e−9+X*1e−9+1/Ka){circumflex over (])}2−4*2e−9*X*1e−9)̂0.5))−Ymin

Ka(binding constant)=10⁹(2,3,7,8-TeCDD), 10⁸(2,3,7-TriCDD)

Ymax (maximum quenching ratio)=0.25 (2,3,7,8-TeCDD), 0.25 (2,3,7-TriCDD)

Ymin (minimum quenching ratio)=−0.01 (2,3,7,8-TeCDD), −0.02 (2,3,7-TriCDD)

The results of fitting with these initial values showed that the peptides had affinities as high as 1.7×10⁹ M⁻¹ for 2,3,7,8-TeCDD and 2.0×10⁸ M⁻¹ for 2,3,7-TriCDD.

EXAMPLE 3

Method of Detecting Dioxin by Method of Competitive Dioxin Binding on Bead

A sample of known concentration (1 μl) was reacted with 1 μM NBD-labeled dichlorophenol (1 μl) and three dioxin-binding peptide beads in 1 ml of a 10 mM phosphate buffer solution (pH: 8) containing 20-30% 1,4-dioxane. After the reaction, fluorescence microscope images of the beads were recorded to establish a calibration curve.

Next, a test sample was reacted with the labeled dichlorophenol and dioxin binding beads, and then the obtained results were compared with the calibration curve, so as to give the dioxin concentration in the test sample.

The results of the competitive quenching shown in FIGS. 6 (A) and 6 (B) can be regarded as calibration curves for dioxin detection, the results being useful in detection using the method of competitive dioxin binding on bead.

FIG. 7 shows the time required for staining. The results of the competition of 1-10 nM NBD-labeled dichlorophenol with 10 nM 2,3,7-TriCDD showed that incubation of 15 hr or more is necessary to detect clear quenching.

EXAMPLE 4

Analysis of Dioxin Binding Peptide Sequences

A library of twenty-one kinds of single amino acid-substituted peptides shown in FIG. 8 was constructed in order to evaluate the importance of each amino acid of the obtained oligopeptides, and to optimize the sequences. Those having analogous properties to the original amino acids, including unnatural amino acids, were used as substituting amino acids. DB1 and DB2 were added to these twenty-one kinds of peptides, and using these twenty-three kinds of peptides, evaluation was made based on staining with NBD-labeled dichlorophenol and quenching by dioxin binding. Table 1 shows all the sequences of the twenty-one amino acid-substituted peptides other than DB1 and DB2. The amino acid sequences in Table 1 are shown in the Sequence Listing with their sequence identification numbers corresponding to those shown in Table 1.

	TABLE 1
	SEQ ID No.
	First Residue Substitution
	SEQ ID No. 4	Nal (1)	Leu Asp Gln Val
	SEQ ID No. 5	Cha	Leu Asp Gln Val
	Second Residue Substitution
	SEQ ID No. 6	Phe Ala Asp Gln Val
	SEQ ID No. 7	Phe Phe Asp Gln Val
	SEQ ID No. 8	Phe Ile Asp Gln Val
	SEQ ID No. 9	Phe Met Asp Gln Val
	SEQ ID No. 10	Phe Nle Asp Gln Val
	SEQ ID No. 11	Phe Asn Asp Gln val
	Third Residue Substitution
	SEQ ID No. 12	Phe Leu Ala Gln Val
	SEQ ID No. 13	Phe Leu Leu Gln Val
	SEQ ID No. 14	Phe Leu Nva Gln Val
	SEQ ID No. 15	Phe Leu Asn Gln Val
	SEQ ID No. 16	Phe Leu Glu Gln Val
	Fourth Residue Substitution
	SEQ ID No. 17	Phe Leu Asp Ala Val
	SEQ ID No. 18	Phe Leu Asp Leu Val
	SEQ ID No. 19	Phe Leu Asp Nle Val
	SEQ ID No. 20	Phe Leu Asp Glu Val
	SEQ ID No. 21	Phe Leu Asp Asn Val
	Fifth Residue Substitution
	SEQ ID No. 22	Phe Leu Asp Gln Phg
	SEQ ID No. 23	Phe Leu Asp Gln Leu
	SEQ ID No. 24	Phe Leu Asp Gln Nva

As shown in FIG. 9, because all the amino acid-substituted peptides tested, including those substituted with alanine, showed no fluorescent staining, the amino acids playing important roles in binding were found to be leucine as the second residue, aspartic acid as the third residue, and glutamine as the fourth residue. With 30% 1,4-dioxane, phenylalanine as the first residue could be replaced by 1-naphthylalanine or cyclohexylalanine. Valine or isoleucine as the fifth residue could be replaced by leucine or phenylglycine. With 30% 1,4-dioxane, the fifth residue could also be replaced by norvaline.

EXAMPLE 5

Sequence Suitable for Detecting Tetrachlorodibenzo-p-dioxin

Tests were conducted on the above-mentioned peptides by the method of competitive dioxin binding on bead described in Example 2, using a 30% 1,4-dioxane solvent. The results confirmed that the peptide having cyclohexylalanine substituting for the first residue (N-terminal) amino acid (SEQ ID No. 5); peptide having phenylglycine substituting for the fifth residue (SEQ ID No. 22; 5 Phg); peptide having leucine substituting for the fifth residue (SEQ ID No. 23; 5 Leu); and peptide having norvaline substituting for the fifth residue (SEQ ID No. 24; 5 Nva) when stained each exhibit fluorescence intensity equal to or greater than that of the DB2 peptide (SEQ ID No. 3) (FIG. 10). All the four kinds of single residue-substituted peptides were evaluated for dioxin binding capability by the method of competitive dioxin binding on bead. The results showed that the peptide comprising 5 Phg (SEQ ID No. 22) is capable of detecting 0.15 nM (0.05 ng/ml) 2,3,7,8-TeCDD while providing a detection sensitivity about ten times greater than that of DB2.

EXAMPLE 6

Changes in Binding Specificities of Substituted Peptides

The specificity of the DB2 peptide was evaluated by the method of competitive dioxin binding on bead. FIG. 11 shows the structure of each of the substances to be detected used in evaluation.

DB1 (SEQ ID No. 2) and the four substituted peptides (SEQ ID No. 5, 22, 23 and 24) confirmed to have a capability of detecting 2,3,7,8-TeCDD equal to or more than that of DB2 were evaluated for their binding specificities by the method of competitive dioxin binding on bead using a 30% dioxane solvent. Twenty substances in total, including dioxin isomers and other like substances, were used as the substances to be detected. For each peptide, “the change in fluorescence intensity” is shown (FIG. 12), which was obtained by subtracting the fluorescence intensity of the peptide after the addition of the substance to be detected from the maximum fluorescence intensity thereof when the substance had not been added. The greater the amount of change in the fluorescence intensity, the higher the affinity of the peptide to the substance. Consequently, the binding specificities of the substituted peptides to dioxin with a toxic equivalency factor (TEF) and the other tested chemicals varied compared to DB2.

Claims

1. An oligopeptide represented by Formula (I) shown below: A1-Leu-Asp-Gln-A2-(X)n  (I)where A1 represents a hydrophobic amino acid residue having a side chain with a cyclic group; A2 represents a hydrophobic amino acid residue having an aliphatic hydrocarbon group or an aromatic hydrocarbon group; n is zero or one; and X represents an amino acid residue.

2. A linearly-linked peptide formed by linking two or more oligopeptides represented by Formula (I) as a repeating unit via a spacer, if necessary.

3. An oligopeptide complex formed by using a linker to the C-terminal of the oligopeptide according to claim 1.

4. An oligopeptide according to claim 1, wherein A1 is represented by Formula (II) shown below:

5. An oligopeptide according to claim 1, wherein A1 is phenylalanine, 1-naphthylalanine, or cyclohexylalanine.

6. An oligopeptide according to claim 1, wherein A2 is represented by Formula (III) shown below:

7. An oligopeptide according to claim 1, wherein A2 is valine, norvaline, leucine, or phenylglycine.

8. An oligopeptide according to claim 1, comprising Phe-Leu-Asp-Gln-Ile.

9. An oligopeptide according to claim 1, comprising Phe-Leu-Asp-Gln-Val.

10. An oligopeptide according to claim 1, comprising Phe-Leu-Asp-Gln-Phg, where Phg represents a phenylglycine residue.

11. A process for detecting or quantifying dioxin in a sample wherein the oligopeptide, linearly-linked peptide and oligopeptide complex according to claim 1 is used for detecting or quantifying dioxin in said sample.

12. A peptide immobilizing support formed by linking (a) the oligopeptide according to claim 1, (b) a linearly-linked peptide formed by linking two or more oligopeptides according to claim 1 or (c) an oligopeptide complex formed by using a linker to the C-terminal of the oligopeptide according to claim 1to (d) a support.

13. A peptide immobilizing support according to claim 12, wherein the support is a bead.

14. A method of detecting or quantifying dioxin is selected from the group consisting of (A) the method comprising the steps of:(1) bringing the peptide immobilizing support according to claim 12 into contact with a labeled dummy and a test sample which may contain dioxin; and(2) detecting or quantifying dioxin based on the amount of the labeled dummy bound to the support which is determined in Step (1); and(B) the method comprising the steps of:(1) bringing the peptide immobilizing support according to claim 12 into contact with a test sample containing dioxin to bind the dioxin to the support: and(2) separating the dioxin bound to the support obtained in Step (1) using a solvent.

15. A method according to claim 14, wherein in (A) the labeled dummy is NBD-labeled 3,4-dichlorophenol.

16. A method of extracting dioxin according to claim 14, wherein said method is (A).

17. A method of extracting dioxin according to claim 14, wherein said method is (B).