JP2003107004A - Method for modifying binding characteristic of odorant binding protein, chemical substance sensor and method for detecting chemical substance - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for modifying binding characteristic of an odorant binding protein to enable detection chemical substances having different binding characteristic. SOLUTION: An amino acid constituting a central pocket CP of a bovine odorant binding protein (OBPb) is substituted by another amino acid, whereby the degree of hydrophobicity of the central pocket CP is changed to modify the affinity (binding strength) of the OBPb to odorant substances. The odorant substance is bound to a plurality of kinds of OBPb having different affinities. The odorant substance is specified and determined by measuring fluorescence intensity of the plurality of kinds of OBPb.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for modifying the binding characteristics of an odorant binding protein, a chemical substance sensor for detecting a chemical substance contained in a sample, and a method for detecting a chemical substance.

[0002]

2. Description of the Related Art Odor substances are detected as one of the quality control tests for products such as foods. In addition,
Here, the chemical substances that cause odors are collectively called odorants. The detection of this odor substance is, for example, when detecting the odor that occurs when the food or the like that is being manufactured or stored is deteriorated, or when the odor of a container such as a PET bottle is transferred to the internal food. It is used for etc. Also, it has been attempted to detect the location of land mines by detecting the odor of explosives for land mines from the ground surface.

Conventionally, the detection of such an odorant is
For example, a polymer substrate or a film is used, and the change in the conductivity of the substrate or the change in the film potential when the odorant is attached to the substrate or the film is measured.

However, such a method has a problem that it is difficult to distinguish the bond of the odorant and the bond of the substance other than the odorant. Therefore, in order to avoid these problems, a method using an antibody has been developed (“JERoederer and G.J.Bastiaans, Anal. Che.
m., 55 (1983) 2333. "` `KA Davis and TR, Leary, An
al. Chem., 61 (1989) 1227. "` `H. Muramatsu et al., An
al. Chem., 59 (1987) 2760. "` `M. Thompson et al., IEE
ETrans.Ultrasonics, Ferroelectrics, Frequency Contro
L, UFFC-34 (1987) 127. '' `` M. Thompson et al., Anal.
Chem., 58 (1986) 1206. "F. Caruso et al., Colloid I
nterface Sci., 178 (1996) 104. ”). That is, the antibody is immobilized on a polymer substrate or a membrane and only the odorant specific to this antibody is bound. Then, when a specific odorant binds to this antibody, the dielectric constant of the polymer substrate changes or the membrane potential changes, so a specific odorant is detected based on this change, and non-specific It is possible to eliminate the binding of the physical substance.

[0005]

Many of the conventional odor sensors using the above-mentioned polymer substrate or film have low sensitivity (up to ppm). Even if the sensitivity is high (~ p
pb), there are few variations (up to a dozen types at most) in the substrate surface modification necessary for odor adsorption, and the odors that can be handled are limited.

On the other hand, in the detection method using an antibody against an odorant, the antibody is expensive, and therefore the detection is costly. Further, in the detection method using such an antibody, since the specificity in binding between the antibody and the odorant is very high, it is usually necessary to detect each odor composed of a very large number of substances. It is necessary to prepare an antibody corresponding to the substance. Therefore, a method of detecting a plurality of types of odorants using an antibody is not practical in view of the cost required for antibody preparation and the thermal instability of the antibody.

It is an object of the present invention to provide a method of modifying the binding properties of odorant binding proteins to allow detection of chemicals with different binding properties.

Another object of the present invention is to provide a chemical substance sensor capable of performing detection with high sensitivity and low cost and applicable to a plurality of types of chemical substances having different binding characteristics, and detection of the chemical substance. Is to provide a method.

[0009]

Means for Solving the Problems and Effects of the Invention Various animals have olfactory organs, and odors are detected and identified in the olfactory organs. In recent years, molecular biological analysis of the mechanism of this olfactory organ has been advanced. In this analysis, the odorant binds to a specific receptor present in epithelial cells of the olfactory mucosa, etc., and a signal is transmitted to the olfactory cell via this receptor to detect and identify the odorant. It has been revealed that

Further, in the series of detailed elucidation of the mechanism of detection and recognition of odorants, a protein that binds to odorants (hereinafter referred to as odorant binding protein) has been identified and isolated ("J .Pevsner et al., Science, 241
(1988) 336. "). The odorant binding protein is not limited to the odorant, and can bind to a specific chemical substance having no odorant.

The biological action of this odorant binding protein is that it has a role of binding to an odorant substance, carrying this odorant substance to a receptor on a mucosal cell, and transferring the odorant substance to the receptor. Has been speculated. The odorant binding protein has also been analyzed biochemically, and it has been revealed that it has a broad binding ability to substances similar to this odorant, with a peak of the binding force to a certain odorant. (`` J. Pevsner et al., J. Biolog.
Chem., 265 (1990) 6118. '' MA Bianchet et al., Natur
e Struc. Biolog., 3 (1996) 934. "` `M. Tegoni et al., N
ature Struc. Biolog., 3 (1996) 863 "). In particular, some of these odorant binding proteins have a gene cloned (J. Pevsner et al., Supra) and can be mass-produced.

[0012] The present inventor paid attention to such an odorant binding protein and diligently studied a method for detecting a chemical substance using this odorant binding protein. As a result, they have found that it is possible to detect odorants with a certain width with high sensitivity by using odorant binding proteins. Then, the following invention of the present application was devised.

The method for modifying the binding property of an odorant binding protein according to the first aspect of the present invention substitutes at least one amino acid in the binding site of the odorant binding protein for a chemical substance with another amino acid.

Thus, the degree of hydrophobicity at the binding site of the odorant binding protein for the chemical substance can be changed to modify the affinity (binding power) of the odorant binding protein for the chemical substance. Therefore, it is possible to detect chemical substances having different binding properties.

The odorant binding protein has a dimer structure in which two monomeric proteins are bound by a non-covalent bond, and a central pocket is constituted by the portions of the two monomeric proteins. At least one amino acid in the portion of the monomeric protein may be replaced by another amino acid.

The binding of the chemical substance to the odorant binding protein is initiated by the binding of the chemical substance to the central pocket. Therefore, if the chemical substance does not bind to the central pocket, the binding reaction between the odorant binding protein and the chemical substance proceeds. do not do.

Therefore, by changing the kinds of amino acids constituting the central pocket, the degree of hydrophobicity of the central pocket can be changed to modify the affinity (binding strength) of the odorant binding protein for chemical substances. As a result, the overall properties of the odorant binding protein can be modified.

The chemical substance sensor according to the second aspect of the invention comprises a plurality of types of odorant binding proteins and a measuring means for measuring the amount of binding of the chemical substances respectively bound to the plurality of types of odorant binding proteins. Each of the binding proteins has a dimer structure in which two monomeric proteins are bound by a non-covalent bond, and a central pocket is constituted by the two monomeric protein parts, and two monomeric proteins constituting the central pocket are formed. At least one amino acid in the portion of the monomeric protein is different in multiple types of odorant binding proteins.

In the chemical substance sensor, the amount of the chemical substance bound to each of the plurality of types of odorant binding proteins is measured. In this case, the plurality of types of odorant binding proteins have different amino acids that form the central pocket, and thus the odorant binding proteins have different affinities (binding powers) for chemical substances. As a result, the amounts of chemical substances bound to different types of odorant binding proteins differ. Therefore, the chemical substance can be specified and quantified based on the binding amount of the chemical substance to the plurality of types of odorant binding proteins.

The chemical substance sensor may further include a specifying means for specifying the chemical substance based on the binding amount of the chemical substance to the plurality of types of odorant binding proteins measured by the measuring means. Thereby, the chemical substance can be specified.

The chemical substance sensor may further include a quantifying means for quantifying the chemical substance based on the binding amount of the chemical substance to the plurality of types of odorant binding proteins measured by the measuring means. Thereby, the chemical substance can be quantified.

The measuring means includes a light source for irradiating a plurality of types of odorant binding proteins with excitation light, and a fluorescence detection unit for detecting fluorescence obtained from the plurality of types of odorant binding proteins.
A data processing unit that processes data regarding the intensity of the fluorescence detected by the fluorescence detection unit may be included.

The greater the affinity between the chemical substance and the odorant binding protein, the greater the recovery amount (attenuation suppression amount) of the fluorescence intensity. In this way, since there is a correlation between the affinity between the chemical substance and the odorant binding protein and the recovery amount of fluorescence intensity (attenuation suppression amount), it is based on the recovery amount of fluorescence intensity (attenuation suppression amount). The affinity between the chemical substance and the odorant binding protein can be determined, and the chemical substance can be identified and specified based on this difference in affinity.

Further, the greater the amount (concentration) of the chemical substance bound to the odorant binding protein, the greater the recovery amount of the fluorescence intensity. Thus, since there is a correlation between the amount (concentration) of the chemical substance bound to the odorant binding protein and the recovery amount (attenuation suppression amount) of the fluorescence intensity, according to the above method, the fluorescence intensity It is possible to measure the amount (concentration) of the chemical substance based on the recovery amount (attenuation suppression amount), that is, to quantify the chemical substance present in the sample.

The chemical substance sensor according to the third invention has a plurality of storage portions for storing a plurality of types of odorant binding proteins having a dimer structure and a plurality of types of odorant binding proteins stored in the plurality of storage portions. Sample introduction part to introduce sample, light source to irradiate excitation light to multiple types of odorant binding proteins in storage part, fluorescence detection part to detect fluorescence obtained from multiple types of odorant binding proteins, and detection by fluorescence detection part And a data processing unit for processing data regarding the intensity of the generated fluorescence, each of the plurality of types of odorant binding proteins has a dimer structure in which two monomeric proteins are bound by non-covalent bonds and two The central pocket is composed of the monomeric protein,
At least one amino acid in the portions of the two monomeric proteins constituting the central pocket is different in plural types of odorant binding proteins.

When determining the presence or absence of a chemical substance in a sample using the chemical substance sensor according to the present invention, first, a plurality of types of odorant binding proteins having a dimer structure in a plurality of storage parts of the chemical substance sensor. To store. Then, the excitation light is emitted from the light source to excite the plurality of types of odorant binding proteins, and the fluorescence obtained from the plurality of types of odorant binding proteins is detected by the fluorescence detection unit, and the data regarding the fluorescence intensity is obtained by the data processing unit. To process.

In the above-mentioned chemical substance sensor, when irradiation of the excitation light to the odorant binding protein is continued in this way,
The fluorescence intensity obtained from the odorant binding protein is attenuated. Here, in this case, when the fluorescence intensity is attenuated as described above, the sample is introduced into the odorant binding protein accommodated in the accommodating section through the sample introducing section to bring the odorant binding protein into contact with the sample.

When the sample is contacted with the odorant binding protein as described above, when the sample contains a chemical substance, the chemical substance in the sample is bound to the odorant binding protein. As a result, the above-described attenuation of the fluorescence intensity is suppressed and the fluorescence intensity is recovered. Such recovery of fluorescence intensity (suppression of attenuation) is detected by the fluorescence detection unit, and data relating to the detected fluorescence intensity is processed by the data processing unit. On the other hand, when the sample does not contain a chemical substance, the fluorescence intensity of the odorant binding protein does not recover and remains attenuated. Such decay of the fluorescence intensity is detected by the fluorescence detection unit, and the data relating to the detected fluorescence intensity is processed by the data processing unit.

In this case, the plurality of types of odorant binding proteins differ in the amino acids constituting the central pocket, and therefore the odorant binding proteins have different affinities (binding powers) for chemical substances. As a result, the amounts of chemical substances bound to different types of odorant binding proteins differ. Therefore, it is possible to specify and quantify the chemical substance by measuring the amount of the chemical substance bound to the plurality of types of odorant binding proteins based on the fluorescence.

As described above, when the above-mentioned chemical substance sensor is used, the amount of chemical substance in the sample is detected based on the recovery amount (attenuation suppression amount) of the fluorescence intensity when the sample is brought into contact with a plurality of types of odorant binding proteins. The presence or absence can be determined, and the chemical substance in the sample can be detected with high sensitivity. The method using such a chemical substance sensor can be carried out at a lower cost than the conventional method using an antibody.

A first wavelength selection section that selectively transmits light of a specific wavelength among the excitation light emitted from the light source, and light of a specific wavelength of fluorescence obtained from a plurality of types of odorant binding proteins are selectively emitted. It may further include a second wavelength selection unit that transmits the light.

In this case, of the excitation light emitted from the light source, only the light of a specific wavelength is selectively transmitted through the first wavelength selection section and is applied to the odorant binding protein. Further, of the fluorescence obtained from the odorant binding protein, only the light having the specific wavelength is selectively transmitted through the second wavelength selection unit and detected by the fluorescence detection unit.

The odorant binding protein may be a bovine odorant binding protein. Such bovine odorant binding proteins include terpenoids, esters,
Has a wide range of affinity with aldehydes and aromatics. Therefore, by using such a bovine-derived odorant binding protein for the above-mentioned chemical substance sensor, it becomes possible to detect a wide range of chemical substances as listed above with high sensitivity.

The fluorescence obtained from the odorant binding protein is preferably fluorescence derived from tryptophan which constitutes the odorant binding protein. Since the fluorescence obtained from tryptophan has a high intensity, it becomes possible to detect easily and accurately. Therefore, by utilizing such fluorescence from tryptophan, it becomes possible to easily perform measurement with high sensitivity and high accuracy.

The data processing unit stores in advance the relationship between the chemical substances and the fluorescence intensities obtained from the plurality of types of odorant binding proteins, and the fluorescence intensities of the plurality of types of odorant binding proteins detected by the fluorescence detection unit and the stored values. The chemical substance contained in the sample may be detected based on the relationship. In this case, the chemical substance can be detected easily and accurately.

The method for detecting a chemical substance according to the fourth invention is
The method comprises a step of bringing a chemical substance into contact with a plurality of types of odorant binding proteins, and a step of measuring the amount of binding of the chemical substance that binds to each of a plurality of types of odorant binding proteins. The monomeric protein has a dimer structure in which it is bound by a non-covalent bond, and a central pocket is constituted by two monomeric protein portions, and at least one of the two monomeric protein portions constituting the central pocket is formed. One amino acid is different in multiple odorant binding proteins.

In the method of detecting the chemical substance, the amount of the chemical substance bound to each of the plurality of types of odorant binding proteins is measured. In this case, the plurality of types of odorant binding proteins have different amino acids that form the central pocket, and thus the odorant binding proteins have different affinities (binding powers) for chemical substances. Thereby,
The amounts of chemical substances bound to different types of odorant binding proteins are different. Therefore, the chemical substance can be specified and quantified based on the binding amount of the chemical substance to the plurality of types of odorant binding proteins.

The method for detecting a chemical substance may further include the step of specifying the chemical substance based on the measured binding amounts of the chemical substance to the plurality of types of odorant binding proteins. Thereby, the chemical substance can be specified.

The method for detecting a chemical substance may further comprise a step of quantifying the chemical substance based on the measured binding amount of the chemical substance to the plurality of kinds of odorant binding proteins. Thereby, the chemical substance can be quantified.

In the measuring step, a step of irradiating a plurality of types of odorant binding proteins with excitation light, a step of detecting fluorescence obtained from a plurality of types of odorant binding proteins, and processing of data relating to the intensity of the detected fluorescence are carried out. And a process may be included.

The greater the affinity between the chemical substance and the odorant binding protein, the greater the recovery amount (attenuation suppression amount) of the fluorescence intensity. In this way, since there is a correlation between the affinity between the chemical substance and the odorant binding protein and the recovery amount of fluorescence intensity (attenuation suppression amount), it is based on the recovery amount of fluorescence intensity (attenuation suppression amount). The affinity between the chemical substance and the odorant binding protein can be determined, and the chemical substance can be identified and specified based on this difference in affinity.

Further, the greater the amount (concentration) of the chemical substance bound to the odorant binding protein, the greater the recovery amount of the fluorescence intensity. Thus, since there is a correlation between the amount (concentration) of the chemical substance bound to the odorant binding protein and the recovery amount (attenuation suppression amount) of the fluorescence intensity, according to the above method, the fluorescence intensity It is possible to measure the amount (concentration) of the chemical substance based on the recovery amount (attenuation suppression amount), that is, to quantify the chemical substance present in the sample.

A method of detecting a chemical substance according to the fifth invention is
A step of contacting the sample with a plurality of kinds of odorant binding proteins having a dimer structure, a step of irradiating the plurality of kinds of odorant binding proteins with which the sample has contacted with excitation light to obtain fluorescence from the plurality of kinds of odorant binding proteins, A step of detecting fluorescence obtained from a plurality of types of odorant binding proteins, and a step of analyzing a chemical substance contained in the sample based on the intensity of the detected fluorescence, each of the plurality of types of odorant binding protein, The two monomeric proteins have a dimer structure in which they are bound by a non-covalent bond, and a central pocket is composed of the two monomeric protein parts. At least one amino acid is different from each other in plural kinds of odorant binding proteins.

In the method for detecting a chemical substance according to the present invention, first, a sample is brought into contact with a plurality of types of odorant binding proteins having a dimer structure. Here, in the case where the sample contains a chemical substance, the chemical substance in the sample binds to a plurality of types of odorant binding proteins. When excitation light is applied to a plurality of types of odorant binding proteins to which a chemical substance is bound in this manner, the fluorescence intensity obtained from the plurality of types of odorant binding proteins is suppressed from being attenuated. On the other hand, when the sample does not contain a chemical substance, the chemical substance does not bind to a plurality of types of odorant binding proteins, so the fluorescence intensity of the fluorescence obtained from the odorant binding protein is attenuated.

In this case, the plurality of types of odorant binding proteins differ in the amino acids constituting the central pocket, and therefore the odorant binding proteins have different affinities (binding powers) for chemical substances. As a result, the amounts of chemical substances bound to different types of odorant binding proteins differ. Therefore, it is possible to specify and quantify the chemical substance by measuring the amount of the chemical substance bound to the plurality of types of odorant binding proteins based on the fluorescence.

From the above, in the above-described method for detecting a chemical substance, the presence or absence of the chemical substance in the sample can be determined based on the amount of suppression of fluorescence intensity decay when the sample is brought into contact with the odorant binding protein. This makes it possible to detect chemical substances in a sample with high sensitivity. Such a method for detecting a chemical substance can be performed at a lower cost than a conventional method using an antibody.

The step of analyzing includes a step of storing a relationship between a chemical substance and fluorescence intensities obtained from a plurality of types of odorant binding proteins in advance, and a step of storing a relationship between the detected fluorescence intensities of a plurality of types of odorant binding proteins and the stored relationships. Based on the detection of chemical substances contained in the sample. In this case, the chemical substance can be detected easily and accurately.

The odorant binding protein may be a bovine odorant binding protein. Bovine odorant binding proteins have a wide range of affinity with terpenoids, esters, aldehydes, aromatics and the like. Therefore, by using the bovine odorant binding protein in the above method, it becomes possible to detect a wide range of chemical substances as mentioned above with high sensitivity.

The fluorescence obtained from the odorant binding protein is preferably fluorescence derived from tryptophan which constitutes the odorant binding protein. Since the fluorescence obtained from tryptophan has high intensity, it can be easily and accurately detected. Therefore, by using such fluorescence from tryptophan, it becomes possible to easily perform measurement with high sensitivity and high accuracy.

[0050]

BEST MODE FOR CARRYING OUT THE INVENTION A method for modifying the binding property of an odorant binding protein in one embodiment of the present invention will be described below.

FIG. 1 is a schematic diagram showing the structure of the odorant binding protein. The odorant binding protein in FIG. 1 is a bovine-derived odorant binding protein (OBP; odorant binding protein).
protein, J. Pevsner et. al., J. Biolog. Chem. 265 (1990) 6
118). Hereinafter, this bovine-derived odorant binding protein is referred to as OBPb.

Such OBPb has an affinity for various terpenoids, cyclopentane and jasmine derivatives, esters, musks, aldehydes and aromatics, and can bind to these substances. Therefore, OBPb can be suitably used when specifically detecting these various odorants.

In FIG. 1, OBPb30 has a dimer structure in which monomeric proteins 31 are linked by non-covalent bonds. As shown in FIG. 1, a central pocket CP is formed between the proteins 31 constituting the OBPb30. An internal cavity IC is formed in each monomer protein 31.

Amino acids constituting the central pocket CP include those shown in Table 1, for example.

[0055]

[Table 1]

The numbers in Table 1 indicate the positions of amino acids, and A and B indicate one and the other monomers, respectively. Further, (+) and (-) indicate polarities, which means that they are hydrophilic amino acids. (NP) is non-polar (no
n-polar), which means that it is a hydrophobic amino acid. (UP) is slightly polar (uncharged po
lar).

In FIG. 1, the C-terminal amino acid of protein 31 is Glu159 (glutamate at position 159).

Amino acids constituting the internal cavity IC are, for example, those shown in Table 2.

[0059]

[Table 2]

As will be described later, the odorant substance (odorant molecule) is bound to OBPb by binding to the central pocket CP regardless of the type of the odorant substance. If they do not bind, the binding reaction between OBPb and the odorant does not proceed.

Therefore, by changing the kind of amino acids constituting the central pocket CP, the degree of hydrophobicity of the central pocket CP can be changed to modify the affinity (binding power) of OBPb for the odorant. Thereby, the overall properties of OBPb can be modified.

At least one protein 3 shown in FIG.
1 Leu122 (the 122nd leucine) was replaced with 7 in Table 3.
7 kinds of OBPb by substituting with different kinds of amino acids
To make.

[0063]

[Table 3]

Further, by replacing Pro156 (proline at position 156) of at least one protein 31 shown in FIG. 1 with 9 kinds of amino acids shown in Table 4, 9 kinds of OBPb are prepared.

[0065]

[Table 4]

Plural kinds of OBs produced in this way
Pb has different binding characteristics (affinity).
Therefore, the odorant selectivity is different for each OBPb. A library composed of these plural kinds of OBPb is prepared, and an odorant is detected using this library.

FIG. 2 is a schematic diagram showing an example of a method for detecting an odorant using a library composed of plural kinds of OBPb.

In the example of FIG. 2, Leu1 of protein 31
A library consisting of 7 types of OBPb in which 22 (leucine at the 122nd position) is substituted with the amino acid shown in Table 3 is used. The amount of binding between OBPb substituted with each amino acid and the odorant is plotted on each axis of FIG. The measurement result of the binding amount of one odorant is shown by a solid line, and the measurement result of the binding amount of another odorant is shown by a dotted line.

The patterns of the amount of binding shown in FIG. 2 are measured in advance using a library for various odorants and registered in the database. Thereby, the type of the odorant can be specified by measuring the pattern of the binding amount of the unknown odorant using the library and comparing it with the pattern registered in the database.

In addition, each OBP in the pattern of the binding amount
It is also possible to quantify the odorant based on the amount of binding for b.

FIG. 3 is a schematic configuration diagram showing an example of a method of detecting an odorant according to one embodiment of the present invention. The chemical substance sensor 100 shown in FIG. 3 includes a microplate 1 having a plurality of holes 2 for accommodating a reaction solution 3, an outside air introduction unit 4, a light source 5, a first wavelength selection unit 6, and a second wavelength selection unit. 7, a fluorescence detector 8 and a data processor 9.

In the chemical substance sensor 100, the reaction solutions 3 of different types are stored in the respective holes 2 of the microplate 1. The reaction solution 3 is prepared by dissolving OBPb in a phosphate buffered saline (PBS).

In this case, the reaction solutions 3 each containing OBPb having different binding characteristics produced as described above are housed in a plurality of holes 2. That is, in this case, a plurality of types of OBP
b is used and different types of O
A plurality of types of reaction solutions 3 containing BPb are prepared, and the holes 2
It is stored in.

The OBPb as described above does not need to be a naturally isolated protein. For example, when the DNA constituting OBPb has been isolated or cloned, this gene was translated in vitro. It is also possible to use proteins.

Furthermore, it is also possible to use a gene in which the base sequence of the gene encoding OBPb is modified to change the affinity for odorants. As a method for modifying this gene, a known point mutation introduction method, random mutagenesis PCR, kunkel method, or the like can be preferably used.

When detecting an odor substance using the chemical sensor 100, first, the light source 5 emits light. Of the emitted light, only the light having the specific wavelength passes through the first wavelength selection unit 6. The first wavelength selection unit 6 is composed of, for example, a monochrome meter or a wavelength selection filter. In this case, light with a wavelength of 295 nm selectively passes through the first wavelength selection unit 6.

The wavelength 295 nm selected in this way
Of the O light stored in each hole 2 of the microplate 1.
Each reaction solution 3 containing BPb is irradiated. This irradiation excites tryptophan, which is one of the amino acids forming OBPb, in each reaction solution 3, and fluorescence is generated from the excited tryptophan.

Of the fluorescence generated from the tryptophan of OBPb of the reaction solution 3, only the fluorescence of a specific wavelength passes through the second wavelength selection section 7. The second wavelength selection unit 7 is composed of, for example, a monochrome meter or a wavelength selection filter. In this case, the fluorescence having the wavelength of 340 nm selectively passes through the second wavelength selection unit 7. The fluorescence having the wavelength of 340 nm selected in this way is detected by the fluorescence detection unit 8, and further, the data processing unit 9 processes the data of the intensity of the detected fluorescence.

In addition to tryptophan among the amino acids constituting OBPb, phenylalanine and tyrosine also show fluorescence characteristics. However, tryptophan has a higher fluorescence intensity than phenylalanine and tyrosine and can be easily detected. Therefore,
Here, the measurement is performed based on the fluorescence from tryptophan.

As described above, the fluorescence intensity of tryptophan of each OBPb contained in each reaction solution 3 is measured.
FIG. 4A schematically shows an example of the fluorescence intensity of tryptophan of OBPb of the reaction solution 3 thus measured.

When the above reaction solution 3 is continuously irradiated with the light of 295 nm, the intensity of the fluorescence generated from tryptophan of OBPb in the reaction solution 3 is reduced (attenuated) as shown in FIG. 4 (b).

Here, in this case, the outside air containing a plurality of odorants is introduced into each reaction solution 3 whose fluorescence intensity is lowered as described above through the outside air introducing section 4.

The outside air introducing section 4 may be composed of, for example, a fan and a tube, and the outside air blown by the fan may be sent to each hole 2 through the tube. Note that the outside air may be introduced into the reaction solution 3 by directly exposing the microplate 1 to the outside air without providing the outside air introduction part 4. In this case, the hole portion 2 acts as the outside air introduction portion 4.

By introducing the outside air into each reaction solution 3 as described above, the outside air containing a plurality of odorants and the OB
Each reaction solution 3 containing Pb comes into contact with each other, and each of the odorants contained in the outside air binds to OBPb of the corresponding reaction solution 3. The binding of OBPb and the odorant in this way increases the intensity of fluorescence generated from tryptophan of OBPb in the reaction solution 3. As a result, as shown in (c) of FIG. 4, the fluorescence intensity lowered in (b) of FIG. 4 is recovered. The principle of such recovery of fluorescence intensity will be described later with reference to FIG.

In the method of detecting an odorant according to the present embodiment, the odorant is detected by detecting such a change in fluorescence intensity due to the binding between the odorant and OBPb.

That is, in this case, OBPb
The presence or absence of various odorants is determined from the recovery amount (attenuation suppression amount) of the fluorescence intensity in the reaction solution 3 containing For example, when the fluorescence intensity of the reaction solution 3 is recovered, it is determined that the odorant corresponding to OBPb contained in the reaction solution 3 is detected. On the other hand, when the fluorescence intensity of the reaction solution 3 does not recover, OBPb contained in the reaction solution 3
The odorant corresponding to is determined to be undetected.

In the method for detecting an odorous substance by fluorescence measurement using OBPb as described above, it is possible to realize a measurement sensitivity as high as or higher than the conventional method using an antibody. Since such a detection method does not need to use an expensive antibody, it can be carried out at low cost.

OBPb has a wide binding force for odorants similar to this odorant with the peak of the binding force for a specific odorant. For this reason,
According to the above detection method using OBPb, it is possible to detect a wide range of odorants.

In particular, according to the detection method using OBPb, even if a substance newly needs to be detected, the O
It can be easily dealt with by amino acid substitution of BPb and expression in E. coli, and the substance can be detected. For this reason, it becomes possible to perform detection more easily than the conventional method using an antibody.

Further, it is possible to identify the odorant by changing the binding property of OBPb to the odorant and detecting the odorant using a plurality of types of OBPb which have respectively changed the binding properties. Become. It is also possible to specify the odorant.

In the above method using OBPb, the greater the affinity between the odorant and OBPb, that is, the easier the bond between the odorant and OBPb, the greater the recovery amount of the fluorescence intensity in the reaction solution 3. Become. Thus, there is a correlation between the affinity between the odorant and OBPb and the recovery amount of the fluorescence intensity. From this, according to the above method, the affinity between the odorant and OBPb can be obtained based on the recovery amount of the fluorescence intensity, and the odorant can be identified based on the difference in the affinity. Become. It is also possible to specify the odorant.

Further, in the above method using OBPb, the greater the amount (concentration) of the odorant bound to OBPb, the greater the recovery amount of fluorescence intensity. Thus, there is a correlation between the amount (concentration) of the odorant bound to OBPb and the recovery amount of the fluorescence intensity. From this,
According to the above method, it is possible to measure the amount (concentration) of the odorous substance in the outside air based on the recovery amount of the fluorescence intensity, that is, to quantify the odorant substance present in the outside air.

Next, the principle that the fluorescence intensity of OBPb is restored by the binding of the odorant will be described.

As shown in FIG. 5 (a), the OBPb30 used in the above-mentioned detection method is a dimer in which monomeric proteins 31 are linked by non-covalent bonds.
It has a structure. As shown in FIGS. 5A and 5B, a central pocket CP interface (dimer-inte) formed between the proteins 31 constituting the OBPb30 is formed.
rface), tryptophan 32 having a benzene ring
Exists. Note that FIG. 5B is a schematic enlarged view showing the structure of the tryptophan 32.

As shown in FIGS. 5A and 5B, when the OBPb30 is irradiated with light having a wavelength of 295 nm,
The light excites the tryptophan 32 of the OBPb 30, and the tryptophan 32 emits fluorescence with a wavelength of 340 nm as the center. The state in which fluorescence is generated from the tryptophan 32 in this way corresponds to the state shown in FIG.

In the state where the odorant is not bound to OBPb30, the periphery of tryptophan 32 is in a hydrophilic environment. However, since the odorant molecule has a strong hydrophobicity, when the odorant molecule binds to the central pocket CP, the periphery of the tryptophan 32 becomes hydrophobic. Since the fluorescence intensity of tryptophan increases as the hydrophobicity increases, the fluorescence from tryptophan 32 also increases due to the binding of odor molecules.

The change in the structure of OBPb as described above can be confirmed by measuring the CD (circular dichroism) spectrum.

As described above, O due to the bond of the odorant
By detecting the change in the structure of BPb as the change in fluorescence intensity, it becomes possible to detect whether or not the odorant is bound to OBPb. That is, the odorant can be detected by the fluorescence measurement method using OBPb.

In the above embodiments, the case of using bovine odorant binding protein (OBPb) has been described, but it has a dimer structure and the structure is changed by the odorant binding as described above. It is also possible to use an odorant binding protein other than that derived from bovine as long as it is an odorant binding protein whose fluorescence intensity changes.

In the above description, the case of detecting an odorant using OBPb has been described.
According to the above method using b, it is possible to detect chemical substances other than the odorant, such as environmental hormones.

As an example of the chemical substance detected by the above detection method, dimethyloctanol (DM
O), citronellal, citronellol, geraniol, bisphenol A (BPA; bis-phenol A), dibutyl phthalate, diethylhexyl phthalate, octylphenol, hexylphenol, nonylphenol and the like.

[0102]

[Example 1] In this example, four types of odorants, dimethyloctanol (DMO), citronellal, citronellol and geraniol, were identified by a fluorescence method using a bovine odorant binding protein (OBPb). An experiment was conducted.

In this embodiment, first, the O used for detection is used.
The BPb solution was prepared and the odor solution was prepared. Next, a sample solution was prepared using the prepared OBPb solution and odor solution, and the fluorescence intensity of this sample solution was measured. The details of this embodiment will be described below.

1. Preparation of OBPb Solution and Odor Solution When preparing the OBPb solution used in this example, first, bovine odorant binding protein (OBPb) was dissolved in a phosphate buffer solution (PBS; phosphate buffered saline), and the OBPb solution was further added to the OBPb solution. PBS was added to dilute the solution to a concentration of 250 nM. The composition of PBS used here is as shown in Table 5.

[0105]

[Table 5]

On the other hand, when the odorant solution used in this example was prepared, four kinds of odorants, dimethyloctanol (DMO), citronellal, citronellol and geraniol, were dissolved in ethanol solution (EtOH), and the concentration of each odorant was determined. A 10 ⁻² M odor solution was prepared.

2. Measurement of fluorescence intensity 300 μl of OBPb solution having a concentration of 250 nM and 0.3 μl of each odor solution having a concentration of 10 ⁻² M prepared as described above
Were mixed with each other to prepare sample solutions used for fluorescence intensity measurement for each of the four types of odorants. In this case, the final concentration of the odorant in each sample solution is 10 μM. Furthermore, here, as a sample for comparison, a sample solution containing only OBPb containing no odorous substance, that is, a mixture of an OBPb solution and an ethanol solution was also prepared.

Next, each sample solution prepared as described above was put into a quartz cell for fluorescence and the opening was sealed to prevent evaporation. Then, the above cell was placed in a cell holder of a fluorescence measuring device (F-4000 manufactured by Hitachi, Ltd.) whose temperature was previously set to 25 ° C., and left for 10 minutes for temperature equilibration.

Subsequently, the fluorescence intensity was measured as follows for each of the four kinds of sample solutions containing the odorant and the sample solution containing only OBPb containing no odorant using the above-mentioned fluorescence measuring apparatus. .

When measuring the fluorescence intensity of each sample solution, first, the shutter of the device was opened to irradiate the sample solution with a light having a wavelength of 280 nm from a slit having a width of 10 nm.
The OBPb tryptophan in the sample solution was excited. Then, the fluorescence having a wavelength of 340 nm obtained from the thus excited tryptophan was taken out through a slit having a width of 10 nm, and the intensity of the obtained fluorescence was measured.

In this case, the shutter opening / closing time of the apparatus was set to 2 seconds, the shutter was opened / closed at 5 minute intervals, and the fluorescence intensity of the sample solution was measured for 30 minutes.

FIG. 6 is a diagram showing the measurement results of the above fluorescence intensity. FIG. 6A is a diagram showing the relationship between the elapsed time from the start of measurement and the fluorescence intensity of the sample solution. Also,
FIG. 6B is a schematic diagram showing a bar graph of the fluorescence intensity of each sample solution at the time point 10 minutes after the start of the measurement shown in FIG. 6A.

In FIG. 6 (b), the relative value of the easiness of binding (affinity) between the odorant and OBPb in each sample solution is indicated by a circle.

Here, in FIG. 6, the time point at which the measurement is started is 0 minute, and the fluorescence intensity of the sample solution at this time point is 100%. As described above, in FIG. 6, the fluorescence intensity at each elapsed time of 0 minutes, that is, the measurement start time is set to 100% for each sample solution, and the fluorescence intensity at each time point every 5 minutes is set on the basis of this reference value. The relative value of was calculated.

As a result of the measurement of the fluorescence intensity, as shown in FIG.
As shown in (b), it was revealed that the change in the fluorescence intensity was different depending on the type of odorant. From this, it was clarified that different types of odorants can be identified by utilizing the change in fluorescence intensity.

Further, as shown in FIG. 6 (b), among the four kinds of odorants, DMO which has the highest affinity with OBPb, that is, DMO which most easily binds with OBPb, has the smallest decrease in fluorescence intensity. , Geraniol, which has the lowest affinity for OBPb, that is, which has the least affinity for OBPb, showed the greatest decrease in fluorescence intensity. in this way,
It was revealed that the higher the affinity between OBPb and the odorant, the smaller the decrease in fluorescence intensity.

The results of citronellal and citronellol in this case are within the error range, which does not contradict the above conclusion.

From the above, it can be seen that there is a difference in the change in fluorescence intensity depending on the type of odorant as described above.
It was revealed that the affinity between the odorant and OBPb differs depending on the type of the odorant. In addition, it was revealed that it is possible to identify each odorant based on such difference in affinity.

Example 2 In this example, dimethyloctanol (DMO) and bisphenol A (BPA; bis-phenol A) can be quantified by a fluorescence method using bovine odorant binding protein (OBPb). I examined if there was any.

In this embodiment, first, O used for detection is used.
Preparation of BPb solution, DMO solution and BP
The solution A was prepared. In this case, D
Plural kinds of solutions having different concentrations were prepared for each of MO and BPA. Subsequently, a sample solution was prepared using the OBPb solution, the DMO solution, and the BPA solution thus prepared, and the fluorescence intensity of this sample solution was measured. The details of this embodiment will be described below.

1. OBPb solution, DMO solution and BP
Preparation of solution A When preparing the OBPb solution used in this example,
An OBPb solution having a concentration of 250 nM was prepared by the same method.

On the other hand, when preparing the DMO solution and the BPA solution used in this example, first, 2 parts of DMO and BPA were prepared.
Each type of odorant was dissolved in ethanol (EtOH) to prepare a solution having a concentration of 10 ^-2 M for each odorant. Furthermore, the concentration of 1 thus prepared
0 ^-2 M of DMO solutions and BPA solution, respectively EtO
Diluted with H to obtain concentrations of 10 ^-3 , 10 ^-4 , 10 ^-5
And 10 ⁻⁶ M solutions were prepared. As described above,
DMO solutions and BPA solutions having concentrations of 10 ⁻³ , 10 ⁻⁴ , 10 ⁻⁵ and 10 ⁻⁶ M were prepared.

2. Measurement of fluorescence intensity 300 μl of 250 nM concentration OBPb solution prepared as described above and concentrations of 10 ⁻⁶ , 10 ⁻⁵ , 10 ⁻⁴ and 10
^-3 M of each DMO solution was mixed with 0.3 μl to give final concentrations of DMO of 10 ⁻⁹ , 10 ⁻⁸ , 10 respectively.
Sample solutions that were ^-7 and ^10-6 M were prepared. Further, by the same preparation method as in the case of DMO, sample solutions having final concentrations of BPA of 10 ⁻⁹ , 10 ⁻⁸ , 10 ⁻⁷ and 10 ⁻⁶ M were prepared.

After the sample solutions were prepared as described above, the fluorescence intensity of each sample solution was measured by the same method as in Example 1. Furthermore, among the results obtained by measuring the fluorescence intensity, based on the result at the time point 20 minutes after the start of measurement and the result at the time point 30 minutes after the start of measurement, the concentration of DMO and BPA and the fluorescence intensity were measured. I investigated the relationship. The result is shown in FIG. 7.

Also in this case, as in the case of Example 1, the measurement start time was set to the time of 0 minute elapsed time, and the fluorescence intensity at this time was set to 100%,
Using this as a reference value, the relative value of the fluorescence intensity at each time point was calculated based on this reference value.

As shown in FIG. 7, in each of the sample solutions containing DMO and BPA, the higher the concentration of DMO and BPA was, the smaller the fluorescence intensity was attenuated, and also after 20 minutes and 30 minutes. It was revealed that there was a correlation between the concentration of DMO and BPA and the fluorescence intensity even at the time point.

Thus, at each time point, there is a correlation between the concentration of DMO and BPA and the fluorescence intensity, so that within the range up to 10 ⁻⁹ M (about 200 ppt), DMO and BPA were observed. It was clarified that the quantification based on the fluorescence intensity of is possible.

Example 3 In this example, OBPb in which no mutation was introduced and OB in which a mutation was introduced were introduced.
Changes in tryptophan fluorescence intensity were examined for various odorants using Pb. Hereinafter, OBPb in which a mutation has not been introduced is referred to as OBPb-norm, and OBPb in which a mutation has been introduced is referred to as OBPb-mut.

Here, in order to improve the odorant selectivity, the binding ability of OBPb is reduced,
OBPb-mut was designed so that it will not bind unless it is an odorant having a certain degree or more of hydrophobicity.

1. 8 shown in Table 6 as the material OBPb-mut
The seed OBPb was used.

[0131]

[Table 6]

In Table 6, OBPb-mut (Glu122) is
It is OBPb in which the 122nd Leu (leucine) is replaced with Glu (glutamic acid). OBPb-mut (Asp15
6), OBPb-mut (Lys156), OBPb-mut (His156), O
BPb-mut (Gly156), OBPb-mut (Gln156) and OB
Pb-mut (Ser156) is the 156th Pro (proline)
Are Asp (aspartic acid), Lys (lysine), His (histidine), Gly (glycine), G
OBPb substituted with In (glutamine) and Ser (serine).

As an odorant, DMO (dimethy
loctanol), citronellal, geraniol, nonanal, nonanal, heptanal, cineol, methylmercaptan, isobutylaldehyde, decylaldeh
yde), methylamine, ethylamine and dimethylamin
Twelve odorants from e) were used.

2. Preparation of OBPb solution and odor solution OBPb was dissolved in PBS and the concentration was 10 μM.
Solution b was prepared. Further, each of the above 12 kinds of odorants was dissolved in 10% ethanol solution to prepare an odorant solution having a concentration of 10 ⁻³ M for each odorant.

3. Procedure for measuring fluorescence intensity First, 10 μl of an OBPb solution was added to 990 μl of PBS to prepare two OBPb solutions having a concentration of 100 nM,
It was left for 1 hour in a circulator adjusted to 25 ° C for temperature equilibration.

Next, the first OBPb having a concentration of 100 nM was used.
10 μl of an odorant solution was added to the solution to obtain a sample solution containing an odorant. Immediately after that, 20
0 μl was taken out and the fluorescence intensity of tryptophan was measured. Here, the excitation wavelength of tryptophan is 295 nm, the bandpass (pass band) is 5 nm, the fluorescence wavelength is 340 nm, and the bandpass is 5 nm.

Subsequently, the second OB having a concentration of 100 nM was used.
10 ml of 10% ethanol solution was added to the Pb solution,
A sample solution containing no odor substance was used. Similarly, 200 μl of the sample solution was taken out, and the fluorescence intensity of tryptophan was measured.

These fluorescence intensities were taken as values at time 0, and the rest of the sample solution was incubated in the circulator (incubation at constant temperature).

At 60 minutes after the fluorescence intensity was measured, the fluorescence intensity of tryptophan was measured again under the same conditions, and the value at the time of 60 minutes was taken.

At the time of 60 minutes, the difference between the fluorescence intensity of the sample solution containing the odorant and the fluorescence intensity of the sample solution containing no odorant (added with the ethanol solution) was determined, and this difference was excluded. The value obtained by dividing the fluorescence intensity of the sample solution (added with the ethanol substance) at time 0 was taken as the relative fluorescence intensity (%) of the odor substance. The relative fluorescence intensity of the odorant is expressed by the following equation.

Relative fluorescence intensity (%) of odorant = [odorant (60) -EtOH (60)] / [EtOH (0)] ×
100 Here, the odorant (60) is the fluorescence intensity of the sample solution containing the odorant after 60 minutes. Also, EtO
H (0) and EtOH (60) are the fluorescence intensities of the sample solution containing no odor substance (added with the ethanol solution) at time points 0 and 60 minutes, respectively.

4. Results FIGS. 8 to 19 show the response of OBPb-norm and OBPb-mut to each odorant (relative fluorescence intensity (%) described above).
3 is a radar chart showing the measurement result of FIG. 8 to 1
9, norm represents the relative fluorescence intensity of OBPb-norm, and Glu122, Glu122, Asp156, Lys156, His156, Gly1.
56, Gln156 and Ser156 are OBs shown in Table 6, respectively.
The relative fluorescence intensity of Pb-mut is shown. The characteristics of the results of FIGS. 8 to 19 are listed below.

(1) As is clear from the results shown in FIG.
High affinity (k for DMO-like OBPb-norm
In the case of the odorant showing D-0.3 μM), the response of all mutants (OBPb-mut) is lower than that of OBPb-norm. This is considered to be the result of replacing the 122th or 156th hydrophobic amino acid among the amino acids forming the central pocket CP, which is the site where the odorant is first bound, with a hydrophilic amino acid. That is,
The results showed that the effect of the mutation introduced to improve the odorant selectivity was as designed (the mutation was introduced into OBPb so that it would not bind unless it is an odorant having a certain degree of hydrophobicity). I can say.

(2) As designed, in OBPb-mut, the odorant binding ability is reduced due to the hydrophilicity of the central pocket CP, but not all OBPb-muts are similarly reduced in binding ability. Absent. As shown in FIGS. 9 to 13, while OBPb-norm showed similar responses to odorants of citronellal, geraniol, nonanal, heptanal and cineol, each OBPb-mut was different. Shows the response (the pattern connected by the line is different for each type of odorant). That is, it is understood that not only the polarity of the amino acid at the 122nd or 156th position, but also the structure or the like influences the binding of the odorant.

(3) FIGS. 14 to 19 show OBPb-nor.
O for an odorant with a m response of 0 or very small
The response of BPb-mut is shown. It can be seen that there is OBPb-mut showing a relatively large response to these odorants, although the response of each OBPb-mut is small.

For example, as shown in FIG. 14, for isobutyraldehyde, OBPb-mut (Gln122) and OBPb-mut (Gln122)
The response of BPb-mut (His156) was large, and as shown in FIG. 15, it was found that OBPb-mut (Ser15
6), OBPb-mut (His156) and OBPb-mut (Lys15)
The response of 6) is large. Further, as shown in FIG. 16, for methylamine, OBPb-mut (Ser156), OBPb-m
ut (Gly156), OBPb-mut (Lys156) and OBPb-mut
The response of (Gln122) is slightly large, and as shown in FIG. 17, OBPb-mut (Ser156) and OBP were found to be more sensitive to ethylamine.
The responses of b-mut (Lys156) and OBPb-mut (Asp156) are large. Further, as shown in FIG. 18, for dimethylamine, OBPb-mut (His156) and OBPb-mut (G
ln122) has a large response, and as shown in FIG. 19, OBPb-mut (Asp156) has a large response to methyl mercaptan.

From the above characteristic results, the following is clarified. Introducing a mutation into the central pocket CP, which is the site where the odorant binds first, is an effective means for changing the selectivity of the odorant. Also,
Since the binding characteristics greatly change depending on the type of the substituted amino acid to be introduced, various sites (especially central pocket C)
OBPb-m in which the amino acid of the site that constitutes P) is substituted
By creating a library using ut, it is possible to identify odorants by the fluorescence intensity pattern.

(Binding Mechanism Model) The binding mechanism of the odorant to OBPb and the substitution mechanism of the odorant were estimated from the results of the experiment described later as shown in FIG.

1. The odorant M1 first binds to the central pocket CP. Then, the internal cavity (internal cavity) IC is opened, and the odorant M1 is ready for bonding (FIGS. 20 (a) (1) to (4)).

2. In this state, when the odorant M1 binds to the internal cavity IC, the affinity of the odorant M1 for OBPb is relatively high (at least KD <1.3 μM), that is, the internal cavity IC and the odorant M1. Is relatively stronger than the interaction between the central pocket CP and the odorant M1, the odorant M1 bound to the central pocket CP is dissociated (FIG. 20 (a) (5)-
(7)). Here, KD is a dissociation constant.

On the other hand, when the affinity of the odorant M1 for OBPb is relatively low (at least when KD> 3 μM), that is, the central pocket CP and the odorant M
If the interaction with 1 is stronger than the interaction between the internal cavity IC and the odorant M1, the odorant M1
Is separated from the internal cavity IC, and as a result, the odorous substance M1 remains in the central pocket CP (FIGS. 20 (a) (1) to (4)).

3. After the odorant M1 is bound, there are the following two types of substitution with the other odorant M2.

3-1 When the affinity of the other odorant M2 for OBPb is relatively low (at least KD> 3 μm).
M), when another odorant M2 is bound to the central pocket CP, the odorant M1 bound to the internal cavity IC is dissociated, and the odorants M1 and M2 are replaced (FIG. 20 (b) (1 )-(4)).

3-2 When the affinity of the other odorant substance M2 for OBPb is relatively high, the other odorant substance M2 is stored in the internal cavity IC to which the odorant substance M1 is not bound in advance among the two internal cavity ICs. The odorant M1 that has been bound and previously bound is dissociated from the internal cavity IC (FIG. 20 (c) (1)-
(4)).

(Demonstration of Binding Mechanism Model) In order to confirm whether the central pocket CP functions as a binding site, OBPb was prepared by removing the C-terminal side amino acid constituting the central pocket CP.

The OBPb-norm shown in FIG. 21 (a) is normally
It is (normal) OBPb (without mutation introduced).
The OBPb-del shown in FIG. 21 (b) excludes Phe (phenylalanine) 149 to Glu (glutamic acid) 159 (dele
ted) OBPb.

First, with respect to OBPb-norm and OBP-del, the relationship between the elapsed time after addition and the change in relative fluorescence intensity in the case of adding odorant dimethyloctanol (DMO) to the OBPb solution was examined.

The concentration of OBPb in PBS was 250 nM and the concentration of DMO solution was 10 μM. After adding the DMO solution to the OBPb solution, incubating at 25 ° C. (incubating at a constant temperature), taking out a part at each time and measuring the fluorescence of tryptophan existing in the central pocket CP (excitation wavelength: 295 nm, fluorescence wavelength: 340 nm). It was

FIG. 22 is a diagram showing the results of measurement of changes in relative fluorescence intensity. In FIG. 22, the horizontal axis is the elapsed time after addition, and the vertical axis is the relative fluorescence intensity difference. The relative fluorescence intensity difference was calculated by dividing the difference between the fluorescence intensity at time 0 and the fluorescence intensity at each time by the fluorescence intensity at time 0.

As shown in FIG. 22, in OBPb-norm, the fluorescence intensity once flattened in 20 minutes, and then increased by another level. Thus, in OBPb-norm,
The fluorescence intensity increases with time.

On the other hand, in OBP-del, although the fluorescence intensity is also increased, the fluorescence intensity finally disappears. That is, in OBP-del, there was no difference in the presence or absence of the odorant.

From these results, it can be seen that the odorant first binds to the central pocket CP.

Next, regarding DMO and geraniol, which are odorants, the central pocket C
The time course of the peak of the fluorescence spectrum of tryptophan in the central pocket CP upon binding to P was examined.

The odorant is the central pocket C.
When bound to P, the central pocket CP becomes a hydrophobic environment, and when the odorant leaves the central pocket CP, the central pocket CP becomes a hydrophilic environment.

When tryptophan is in OBPb, the peak wavelength of the fluorescence spectrum of tryptophan is 3
It is 45 nm. Generally, the fluorescence wavelength of tryptophan shifts to a long wavelength in a hydrophilic environment and shifts to a short wavelength in a hydrophobic environment. Here, the fluorescence spectrum at the time of binding of the odorant was measured, the difference from the spectrum at 0 minutes was calculated, and the relationship between the peak intensity and the elapsed time was examined.

FIG. 23 is a diagram showing a difference spectrum between the fluorescence spectrum at each time and the fluorescence spectrum at 0 minutes when DMO and geraniol were bound to OBPb-norm. Further, FIG. 24 is a diagram showing a difference spectrum between the fluorescence spectrum at each time and the fluorescence spectrum at 0 minute in the case where DMO and geraniol were bound to OBP-del.

The difference spectrum was calculated by subtracting the fluorescence spectrum at each time from the fluorescence spectrum at 0 minutes. Therefore, when the peak of the fluorescence spectrum at a certain time is shifted to a wavelength shorter than 345 nm (that is, made hydrophobic), the peak of the difference spectrum is conversely 345.
The wavelength will be shifted to a wavelength longer than nm.

23 and 24, d0-1
0, d0-20, d0-40 and d0-60 are DM
Fluorescence spectra at 0 minutes for O and 10 each
The difference spectra from the fluorescence spectra at minutes, 20 minutes, 40 minutes and 60 minutes are shown. Also, g0-10, g0-2
0, g0-40 and g0-60 are the fluorescence spectra at 0 min for geraniol and 10 min, 20 respectively.
The difference spectra from the fluorescence spectra at minutes, 40 minutes and 60 minutes are shown.

FIG. 25 shows OBPb-norm and OBP-del.
It is a figure which shows the time-dependent change of the peak of the difference spectrum of a fluorescence spectrum when DMO and geraniol are combined with. In FIG. 25, the vertical axis represents the peak wavelength of the difference spectrum of the fluorescence spectrum, and the horizontal axis represents the elapsed time immediately after the addition of the odorant.

FIG. 25 shows OBP at 0, 20, 40 and 60 minutes after the addition of DMO and geraniol.
The change of the peak of the difference spectrum in b-norm and OBP-del is shown.

In OBP-del, DMO which is an odor substance
There was no change in the peak wavelength of the difference spectrum of tryptophan during the binding reaction of geraniol and geraniol. That is, it can be seen that the environment of the central pocket CP hardly changes depending on the presence or absence of the odor substance, and the hydrophobic interaction between the odor molecule and the central pocket CP is low.

In OBP-norm, it can be seen that the environment in the central pocket CP becomes hydrophobic due to the increase in the peak wavelength of the tryptophan difference spectrum.
Further, this change to hydrophobicity was maintained during the binding reaction with geraniol, whereas in DMO, the hydrophobicity gradually decreased, and after 40 minutes, it turned hydrophilic and after 60 minutes. It can be seen that it has become hydrophobic again.

In other words, geraniol remains bound to the central pocket CP, and DMO dissociates after binding to the central pocket CP. Since one odorant molecule binds to the dimer of OBPb, it is considered that another DMO binds to the internal cavity IC when the DMO dissociates from the central pocket CP.

This reaction is based on the internal cavity IC
It is considered that the DMO in the central pocket CP can no longer bind and is dissociated due to the structural change caused by the binding of the odorant molecule to the compound.

Further, it was investigated how an odorant once bound to OBPb behaves when another odorant is bound later. Aminoanthracene (AMO), which is a fluorescent odorant, was used as the odorant to be bound in advance, and DMO and geraniol were used as the odorant added to the AMA bond.

An OBPb solution having a concentration of 10 μM was prepared.
In addition, AMA, DMO and geraniol were each dissolved in ethanol to give a concentration of 10 ^-2 M AMA solution and DM.
O solution and geraniol solution were prepared.

As a preliminary step of the experiment for investigating the substitution of the odorant substance with another odorant substance after binding to OBPb, first, the product of the fluorescence spectrum of tryptophan and tyrosine and the absorption spectrum of AMA was measured.

FIG. 26 is a diagram showing the results of measurement of the product of the fluorescence spectra of tryptophan and tyrosine and the fluorescence spectrum of AMA. In FIG. 26, the vertical axis represents the product of the fluorescence intensity of tryptophan or tyrosine and the absorption rate of AMA, and the horizontal axis represents the wavelength.

From the measurement results shown in FIG. 26, it can be seen that AMA has a property of absorbing fluorescence from tryptophan or tyrosine. This means that the presence of AMA near tryptophan or tyrosine reduces the fluorescence intensity from tryptophan or tyrosine.

Next, changes with time of the fluorescence from AMA and the fluorescence from tryptophan when AMA was added were measured.
FIG. 27 is a diagram showing measurement results of changes in fluorescence intensity of tryptophan at the time of AMA binding. The excitation wavelength of tryptophan is 295 nm and the fluorescence wavelength is 340 nm. Further, FIG. 28 is a diagram showing the measurement results of changes in the fluorescence intensity of AMA upon AMA binding. Excitation wavelength of AMA is 295 nm
And the fluorescence wavelength is 480 nm. 27 and 28, the horizontal axis represents the elapsed time after addition and the vertical axis represents the relative fluorescence intensity difference. The relative fluorescence intensity difference was calculated by dividing the difference between the fluorescence intensity at time 0 and the fluorescence intensity at each time by the fluorescence intensity at time 0.

In the measurement result of FIG. 27, the fluorescence intensity of tryptophan is once decreased after the addition of AMA and then increased. In the measurement result of FIG. 28, AM
The fluorescence intensity of A increases after the addition of AMA, takes a constant value, and then decreases.

From these results, the following is considered.
Timing of increase in fluorescence intensity of tryptophan and AMA
Coincides with the timing of the decrease of the fluorescence intensity. Further, as described above, the fluorescence from tryptophan is AMA.
Absorbed by. From these, the following can be considered.

AMA once enters the central pocket CP. Fluorescence from tryptophan in the central pocket CP is absorbed by AMA and reduced. At the same time, the fluorescence from AMA that absorbed the fluorescence from tryptophan was 0.
Increase for ~ 15 minutes. After that, AMA is released from the central pocket CP. Thereby, the fluorescence from tryptophan increases and the fluorescence from AMA decreases.

As described above, in OBPb, 1 is included in the dimer.
Since it has been known that two odorants bind, it is considered that when the odorant is released from the central pocket CP, the odorant binds to one internal cavity IC. The binding of geraniol shown by the change in the hydrophobicity of the central pocket CP in FIG. 25 is the case where there is no binding to the internal cavity IC.

The odorant is an internal cavity IC
Go to or stay in the central pocket CP
It is considered to be determined by the affinity of the odorant for BPb. The dissociation constant KD of geraniol is about 3 μM, and DMO
Has a dissociation constant KD of about 0.3 μM and AMA has a dissociation constant of about 1.3 μM. Therefore, at least the binding to the internal cavity IC and the central pocket C
The boundary with the binding to P is 1.3 μM <KD <3 μM
It is believed to be in.

Next, the substitution of another odorant after binding the odorant to OBPb was examined.

First, 0.1 μl of AMA solution was added to 100 μl of OBPb solution, and the mixture was allowed to stand at 25 ° C. for 1 hour.
AMA was attached to Pb. Phosphate buffer (PBS) was added to this solution to make 4 ml (40-fold dilution), and 4 μl of ethanol, DMO or geraniol was added, and then 0, 5, 10, 25, 20, 25, 30, 40 and 60. When 300 minutes passed, 300 μl was taken out and the fluorescence intensity of AMA (excitation wavelength: 295 nm, measurement wavelength: 481 n
m) and tryptophan fluorescence intensity was measured.

FIG. 29 shows A when each odorant was added.
It is a figure which shows the measurement result of the fluorescence intensity change of MA.

It can be seen that when ethanol is added, the fluorescence intensity of AMA gradually decreases and AMA is dissociated from OBPb over time. In contrast,
It is understood that when the odorants DMO and geraniol are added, the fluorescence intensity of AMA decreases and the AMA dissociates immediately after the addition (0 minutes) in both odorants (DMO and geraniol).

FIG. 30 is a diagram showing the measurement results of the fluorescence intensity change of tyrosine when each odorant was added.

In measuring the fluorescence intensity of tyrosine, the excitation wavelength is 250 nm and the measurement wavelength is 300 nm. Tyrosine is a monomer internal cavity I of OBPb.
C. Therefore, the dissociation of the odorous substance from the internal cavity IC can be measured by the change in the fluorescence intensity of tyrosine (the fluorescence intensity decreases when it becomes hydrophilic).

When ethanol and DMO were added, no change in fluorescence intensity was observed immediately after the addition (0 minutes). On the other hand, when geraniol was added, the fluorescence intensity was decreased immediately after the addition (0 minutes).

As a result, the low-affinity odorant typified by geraniol (at least KD> 3 μM) enters the central pocket CP to dissociate the previously bound odorant, and the high affinity typified by DMO. It can be seen that the odorous odor substance binds to the internal cavity IC to dissociate the odorant substance previously bound to the other internal cavity IC.

The results of FIGS. 29 and 30 are obtained by the AMA being coupled to the internal cavity IC. On the contrary from this result, AMA or D
It can be concluded that the odorant that dissociates from the central pocket CP, such as MO, eventually binds to the internal cavity IC.

[Brief description of drawings]

FIG. 1 is a schematic diagram showing the structure of an odorant binding protein in one embodiment of the present invention.

FIG. 2 is a schematic diagram showing an example of a method for detecting an odorant using a library composed of a plurality of types of OBPb.

FIG. 3 is a schematic configuration diagram showing an example of an odorant detection method according to an embodiment of the present invention.

FIG. 4 is a diagram schematically showing changes in fluorescence intensity of tryptophan of OBPb contained in the reaction solution.

FIG. 5: OBPb by binding of odorant to OBPb
FIG. 3 is a diagram for explaining the principle of recovery of the tryptophan strength.

FIG. 6 is a diagram showing the measurement results of Example 1.

FIG. 7 is a diagram showing the measurement results of Example 2.

FIG. 8: OBPb-norm and OB for odorants
It is a radar chart which shows the measurement result of the response of Pb-mut.

FIG. 9: OBPb-norm and OB for odorants
It is a radar chart which shows the measurement result of the response of Pb-mut.

FIG. 10: OBPb-norm and O for odorants
It is a radar chart which shows the measurement result of the response of BPb-mut.

FIG. 11: OBPb-norm and O for odorants
It is a radar chart which shows the measurement result of the response of BPb-mut.

FIG. 12: OBPb-norm and O for odorants
It is a radar chart which shows the measurement result of the response of BPb-mut.

FIG. 13: OBPb-norm and O for odorants
It is a radar chart which shows the measurement result of the response of BPb-mut.

FIG. 14: OBPb-norm and O for odorants
It is a radar chart which shows the measurement result of the response of BPb-mut.

FIG. 15: OBPb-norm and O for odorants
It is a radar chart which shows the measurement result of the response of BPb-mut.

FIG. 16: OBPb-norm and O for odorants
It is a radar chart which shows the measurement result of the response of BPb-mut.

FIG. 17: OBPb-norm and O for odorants
It is a radar chart which shows the measurement result of the response of BPb-mut.

FIG. 18: OBPb-norm and O for odorants
It is a radar chart which shows the measurement result of the response of BPb-mut.

FIG. 19: OBPb-norm and O for odorants
It is a radar chart which shows the measurement result of the response of BPb-mut.

FIG. 20 is a diagram showing the mechanism of odorant binding to OBPb and the mechanism of odorant substitution.

FIG. 21 is a diagram showing ordinary OBPb and OBPb excluding the C-terminal amino acid.

FIG. 22 is a diagram showing measurement results of changes in relative fluorescence intensity.

FIG. 23 is a diagram showing a difference spectrum between a fluorescence spectrum at each time and a fluorescence spectrum at 0 minute when DMO and geraniol were bound to OBPb-norm.

FIG. 24 is a diagram showing a difference spectrum between a fluorescence spectrum at each time and a fluorescence spectrum at 0 minute in the case where DMO and geraniol were bound to OBP-del.

FIG. 25 is a diagram showing a change with time of a peak of a difference spectrum of a fluorescence spectrum when DMO and geraniol were bound to OBPb-norm and OBP-del.

FIG. 26 shows the measurement results of the product of the fluorescence spectra of tryptophan and tyrosine and the fluorescence spectrum of AMA.

FIG. 27 is a diagram showing the measurement results of changes in the fluorescence intensity of tryptophan at the time of AMA binding.

FIG. 28 is a diagram showing the measurement results of AMA fluorescence intensity changes upon AMA binding.

FIG. 29 is a diagram showing measurement results of changes in fluorescence intensity of AMA when each odorant is added.

FIG. 30 is a diagram showing measurement results of changes in fluorescence intensity of tyrosine when each odorant is added.

[Explanation of symbols]

1 microplate 2 holes 3 Reaction solution 4 Outside air introduction section 5 light sources 6 First wavelength selector 7 Second wavelength selector 8 Fluorescence detector 9 Data processing unit 30 Smell-binding protein 31 protein 32 Tryptophan 40 Odorant 100 chemical substance sensor CP central pocket IC internal cavity M1 and M2 odorants

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 33/53 G01N 33/566 33/566 C12N 15/00 AF term (reference) 2G043 AA01 BA16 CA03 DA02 EA01 FA03 GA07 GB21 JA03 KA03 KA05 LA01 NA01 NA06 NA11 2G045 AA40 BB41 DA35 FB12 GC15 2G054 AA10 CA30 EA03 GA04 4B024 AA11 BA80 HA06 4H045 AA10 AA30 CA40 EA50

Claims (17)

[Claims] 1. A method for modifying the binding property of an odorant binding protein, which comprises substituting at least one amino acid in the binding site of said odorant binding protein for a chemical substance with another amino acid. How to modify properties. 2. The odorant binding protein has a dimer structure in which two monomeric proteins are bound by a non-covalent bond, and a central pocket is formed by the portions of the two monomeric proteins. 2. The method for modifying the binding property of an odorant binding protein according to claim 1, wherein at least one amino acid in the two monomeric protein moieties constituting the protein is replaced with another amino acid. 3. A plurality of types of odorant binding proteins, and a measuring means for measuring the amount of binding of chemical substances respectively bound to the plurality of types of odorant binding proteins, wherein each of the plurality of types of odorant binding proteins comprises 2 One monomer protein has a dimer structure in which it is bound by a non-covalent bond, and a central pocket is constituted by the two monomer protein portions. A chemical substance sensor, wherein at least one amino acid of a portion is different in said plurality of types of odorant binding proteins. 4. The identification means for identifying the chemical substance based on the amount of the chemical substance bound to the plurality of types of odorant binding proteins measured by the measuring means, further comprising: Chemical substance sensor. 5. The quantification means for quantifying the chemical substance based on the binding amount of the chemical substance to the plurality of types of odorant binding proteins measured by the measurement means, further comprising: a quantification means. The chemical substance sensor described. 6. The light source for irradiating the plurality of types of odorant binding proteins with excitation light, a fluorescence detection unit for detecting fluorescence obtained from the plurality of types of odorant binding proteins, and the fluorescence detection unit. The data processing part which processes the data regarding the intensity | strength of the detected fluorescence is included, The chemical substance sensor in any one of Claims 3-5 characterized by the above-mentioned. 7. A plurality of storage parts for storing a plurality of types of odorant binding proteins having a dimer structure, and a sample introduction part for introducing a sample into the plurality of types of odorant binding proteins stored in the plurality of storage parts. A light source that irradiates the plurality of types of odorant binding proteins with excitation light, a fluorescence detection unit that detects fluorescence obtained from the plurality of types of odorant binding proteins, and a fluorescence detected by the fluorescence detection unit. A data processing unit that processes data regarding strength, each of the plurality of types of odorant binding proteins has a dimer structure in which two monomeric proteins are bound by non-covalent bonds, and A central pocket is constituted by a body protein portion, and a small amount of the two monomer protein portions constituting the central pocket is contained. Both chemical sensors one amino acid are different from each other in the plurality of types of odorant binding proteins. 8. A first wavelength selection unit that selectively transmits light of a specific wavelength of the excitation light emitted from the light source,
8. The chemical substance sensor according to claim 7, further comprising a second wavelength selection unit that selectively transmits light of a specific wavelength among the fluorescence obtained from the plurality of types of odorant binding proteins. 9. The odorant binding protein is a bovine-derived odorant binding protein.
Alternatively, the chemical substance sensor described in 8. 10. The data processing unit stores a relationship between a chemical substance and fluorescence intensity obtained from the plurality of types of odorant binding proteins in advance, and stores the relationship between the plurality of types of odorant binding proteins detected by the fluorescence detection unit. The chemical substance sensor according to claim 7, wherein the chemical substance contained in the sample is detected based on the fluorescence intensity and the stored relationship. 11. A method comprising: bringing a chemical substance into contact with a plurality of types of odorant binding proteins; and measuring the amount of chemical substances bound to each of the plurality of types of odorant binding proteins. Each of the proteins has a dimer structure in which two monomeric proteins are bound by a non-covalent bond, and a central pocket is constituted by the portions of the two monomeric proteins. A method for detecting a chemical substance, wherein at least one amino acid of one monomer protein portion is different in the plurality of types of odorant binding proteins. 12. The detection of a chemical substance according to claim 11, further comprising the step of specifying the chemical substance based on the measured binding amount of the chemical substance to the plurality of kinds of odorant binding proteins. Method. 13. The chemical substance according to claim 11, further comprising a step of quantifying the chemical substance based on the measured binding amount of the chemical substance to the plurality of types of odorant binding proteins. Detection method. 14. The measuring step comprises the step of irradiating the plurality of types of odorant binding proteins with excitation light, the step of detecting fluorescence obtained from the plurality of types of odorant binding proteins, and the step of detecting the detected fluorescence. Processing data relating to intensity.
14. The method for detecting a chemical substance according to any one of 13 to 13. 15. A step of bringing a sample into contact with a plurality of types of odorant binding proteins having a dimer structure, and irradiating the plurality of types of odorant binding proteins with which the sample has contacted with excitation light to cause the plurality of types of odorant binding to occur. A step of obtaining fluorescence from a protein, a step of detecting fluorescence obtained from the plurality of types of odorant binding proteins, and a step of analyzing a chemical substance contained in the sample based on the intensity of the detected fluorescence Each of the plurality of types of odorant binding proteins has a dimer structure in which two monomer proteins are bound by a non-covalent bond, and a central pocket is formed by the two monomer protein portions, At least one amino acid in the portions of the two monomeric proteins forming the central pocket has the plurality of types of amino acids. Detection method of chemicals being different from each other in the y-binding protein. 16. The analyzing step stores a relationship between a chemical substance and fluorescence intensities obtained from the plurality of types of odorant binding proteins in advance, and the detected fluorescence intensities of the plurality of types of odorant binding proteins. And a step of detecting a chemical substance contained in the sample based on the stored relationship, and the method for detecting a chemical substance according to claim 15. 17. The method for detecting a chemical substance according to claim 15, wherein the odorant binding protein is a bovine odorant binding protein.