JP2003083864A - Method for analyzing structural change of organism polymer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a means capable of easily analyzing the structural change of an organism polymer fixed on a solid phase in an aqueous medium to predict the function. SOLUTION: This method for analyzing the structure of the organism polymer on the surface of the solid phase comprises (a) fixing the organism polymer to the other electrode of a quartz oscillator comprising a quartz plate having both surface and reverse sides and a side surface, a single coating material for covering one electrode provided on the reverse side with its inner wall being in contact with the side surface of the quartz plate, and a sealant provided between the surface of the quartz plate having the other electrode provided thereon and the inner wall; (b) dipping the quartz oscillator having the organism polymer fixed thereto in the aqueous medium to measure the stable vibration frequency of the quartz oscillator; and (c) measuring the vibration frequency change of the quartz oscillator after changing the structure of the immobilized organism polymer dipped in the aqueous medium.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for analyzing a structural change of a biopolymer immobilized on a solid phase in an aqueous medium using a crystal oscillator.

[0002]

2. Description of the Related Art For example, casein, which is the main component of milk, is a kind of protein which is a biopolymer, which is in a dissolved state near neutrality but is abruptly precipitated under acidic conditions. . In addition, proteins such as insulin dissolve under acidic conditions, but polymerize near neutrality and become insoluble. Such physical properties vary greatly depending on the salt concentration, but it is considered that there are many factors that determine the physical properties in vivo. Searching for such factors is important for elucidating various molecular functions in vivo.

Conventionally, the structural analysis of biopolymers has been carried out by means such as circular dichroism measurement, scattered X-ray diffraction, fluorescence energy transport, and sedimentation constant.

However, with these conventional methods, it is possible to measure the solution state, but it is not possible to analyze immobilized biopolymers such as proteins, or the biopolymers to be analyzed are in microgram order. It was not sufficient because it had to be completely purified, and it was difficult to predict the function of the biopolymer.

As a method for analyzing the structural change of the immobilized protein, a method using surface plasma resonance is known (Anal. Chem., 1988, 70, 2019-2024).

[0006] However, this method can analyze the structural change of the protein with the pH change of the aqueous medium, but cannot analyze the structural change of the protein under the conditions such as addition of a denaturant or salt. .

[0007]

Therefore, the above-mentioned drawbacks can be improved, and under various environmental conditions,
There is a need for a means capable of predicting the function by analyzing the structural change of a biopolymer immobilized on a solid phase in an aqueous medium.

[0008]

A first embodiment of the present invention is a method for analyzing the structure of a biopolymer on a solid surface, which comprises (a) a quartz plate having both front and back surfaces and side surfaces. An electrode provided on the front and back surfaces of the crystal plate, and a single-sided covering material that covers one electrode provided on the back surface and the inner wall of which contacts the side surface of the crystal plate, and the other electrode. Fixing a biopolymer to the other electrode of the crystal oscillator provided with a sealing material provided between the surface of the crystal plate and the inner wall, and (b) a crystal oscillator having the biopolymer fixed thereon. Measurement of the stable frequency of the crystal oscillator by immersing it in an aqueous medium and (c) change of the crystal oscillator frequency after changing the structure of the immobilized biopolymer immersed in the aqueous medium. Is measured.

A second embodiment of the present invention is a method for analyzing the structure of a biopolymer on the surface of a solid phase by changing the association state of the biopolymer, which comprises (a) a quartz plate having front and back surfaces and side surfaces. An electrode provided on the front and back surfaces of the crystal plate, and a single-sided covering material that covers one electrode provided on the back surface and the inner wall of which contacts the side surface of the crystal plate, and the other electrode. Fixing a biopolymer to the other electrode of the crystal oscillator provided with a sealing material provided between the surface of the crystal plate and the inner wall, and (b) a crystal oscillator having the biopolymer fixed thereon. After immersing in an aqueous medium, measuring the stable frequency of the crystal oscillator, and (c) changing the structure of the immobilized biopolymer immersed in the aqueous medium, Add a compound capable of associating and measure the frequency change of the crystal unit. It is a method that includes a.

The crystal oscillator used in the present invention is particularly suitable for use in an aqueous medium.
It is a crystal oscillator described in Japanese Patent No. 53777. Crystal oscillator configured as described above, since the sealing material is provided between the surface and the inner wall of the crystal plate provided with the other electrode, the solution enters one electrode, A short circuit between electrodes can be prevented. Since the encapsulating material is adhered to the inner wall that is in contact with the side surface of the crystal plate, the encapsulating material is necessarily located at the end of the crystal plate. Therefore, the crystal plate can be easily oscillated.

The sealing material of the crystal oscillator is preferably an elastic body such as a silicon resin. If the sealing material is an elastic body,
The edge of the crystal plate can also vibrate, and the crystal plate can easily oscillate.

Further, the crystal oscillator is preferably provided with lead terminals connected to the respective electrodes, and the lead terminals are preferably constructed so as to be covered with the one-side coating material. The oscillator can be easily manufactured by gathering the lead terminals on one side and the opposite side.

The biopolymer which can be used in the present invention is not particularly limited as long as it can be immobilized on a crystal oscillator, and examples thereof include proteins, nucleic acids and polysaccharides, preferably casein, albumin, collagen, gelatin, actin and myosin. , Keratin, fibrin, tubulin, and other proteins.

In the present invention, the biopolymer can be fixed to the crystal oscillator by a conventionally known method. For example, the protein can be fixed to the crystal oscillator by a direct method or an indirect method after the crystal oscillator is washed as described below.

When the crystal oscillator is used for the first time and reused, it is preferable to clean the crystal oscillator as follows.

(1) Hydrogen peroxide water (30% solution) 100
Place μl in a glass test tube. Set the test tube in a beaker with a capacity of about 30 ml to prevent it from falling. (2) Add 300 μl of concentrated sulfuric acid slowly and stir with gentle shaking. Although this mixed solution is called a piranha solution, it is a very dangerous solution that causes holes in clothes, so wear protective glasses and gloves when working. (3) Place the resonator on a glass plate with the electrode side up, and place the piranha solution only on the round gold electrode part in the center (about 5 μm).
l) Let stand for 5 minutes. Be careful not to touch the encapsulant such as silicone resin with this solution. If you touch the encapsulant, wash it immediately with water and try again. (4) Carefully wash with distilled water using a washing bottle and air dry. Most of the constituents of the oscillator are made of hydrophobic material, so it is easy to repel water, and it winds and dries quickly. (5) Repeat the above (3) and (4). The gold electrode is preferably washed immediately before using the crystal oscillator. The protein can be fixed to the crystal oscillator by the following method.

A. Direct method (1) 100 μl glycine solution (0.1 M glycine-HCl (pH 2.4)) was added to the oscillator washed with piranha solution.
Then, let stand for 10 seconds at room temperature. (2) Gently and thoroughly rub the gold electrode surface with a cotton swab (be careful because the oscillator is easily broken). (3) After washing with distilled water, the target protein has an appropriate concentration (depending on the type of protein, 10-100
(5 μl / μg / ml) on the central gold electrode part and leave for 1 hour. At this time, be careful not to evaporate the solution.

B. Indirect method (example of immobilizing avidin) (1) Dissolve 3,3-dithiodipropionic acid in ethanol to a concentration of 21 mg / ml (0.1 M ethanol solution). (2) Place the cleaned crystal oscillator on a table, place 100 μl of the solution of (1) diluted 100 times with distilled water, and spread it over the crystal part of the oscillator.
Soak for 0 minutes or more. (In the case of processing a large amount, use a beaker or the like to raise the oscillators and process them all at once.
Stir the solution from time to time). (3) Wash with distilled water. Be careful not to let the surface dry until the next step. That is, about 100 μl of distilled water is placed on the oscillators arranged on the desk, or placed in a beaker filled with water. In the latter case, be careful that the plug part of the resonator does not get wet with water. If the plug is damp, good oscillation cannot be obtained. (4) 1- (3-Dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) is dissolved in distilled water to 100 mg / ml. (5) N-hydroxysuccinimide (NHS) was added to 100
Dissolve with distilled water to give a mg / ml. (6) EDC and NHS prepared in (4) and (5) above
After mixing equal amounts of the solutions and discarding the liquid of the oscillator prepared in (3), 100 μl of this mixed liquid is immediately put on the oscillator (before the crystal surface is dried) and left at room temperature for 10 minutes. (Be careful not to evaporate the solution). (7) Wash with distilled water, and soak in distilled water until proteins are fixed so that the crystal surface does not dry.
At this stage, the oscillator is in a state where proteins are easily fixed, so be careful of the mixture of other proteins. (8) Avidin 10 mM Tris-at 1 mg / ml
Dissolve in HCI (pH 8.0), dispense into a suitable dose in a unit that can be used up, and store frozen at -20 ° C or below to obtain a preservation solution. (9) Dilute with 10 mM Tris-HCl (pH 8.0) so that the final concentration of avidin is 150 μg / ml,
The oscillator (7) is treated at room temperature for 1 to 2 hours. At this stage, various proteins can be immobilized instead of avidin. The protein concentration and reaction time used in this step differ depending on the characteristics of the protein used.
Generally, a concentration of 100 μg / ml to 200 μg / ml is the most efficient. If the concentration is too high, the risk of impairing the activity of the protein increases, so caution is required. The amount of biopolymer fixed to the crystal oscillator is 0.3 to 300 ng, preferably 3
~ 60 ng.

The crystal oscillator on which the biopolymer such as protein is immobilized as described above is immersed in an aqueous medium, and then the stable frequency of the crystal oscillator is measured. What is an aqueous medium?
Water, or a liquid medium containing water as a main component, may include an aqueous solution, a suspension, an emulsion, and a body fluid.

Further, in the present invention, after changing the structure of the immobilized biopolymer (first embodiment) or after changing the structure of the immobilized biopolymer, the biopolymer is added to an aqueous medium. After adding a compound capable of associating with (second aspect),
The frequency change of the crystal oscillator immersed in the aqueous medium is measured.

In the present invention, the structural change of the biopolymer is caused, for example, by changing the temperature, pH, pressure or salt concentration of the aqueous medium, irradiation with light or sound waves, or various salts and associations. Cofactors and competitors of
Although it is due to the addition of a denaturant, a chemical substance such as an organic solvent, preferably by changing the pH or salt concentration of the aqueous medium, or a denaturant such as urea or guanidine, an organic solvent (for example, Alcohol, ketone, ether, nitrile, amide, hydrocarbon) and the like.

The compound capable of associating with a biopolymer is not limited to a compound capable of associating with a biopolymer, but it is unknown whether or not it is capable of associating with a biopolymer, but may include a compound whose association state is to be tested. Examples of compounds that associate with biopolymers include compounds that are structurally identical or similar to biopolymers.

When the biopolymer adheres to the electrodes of the crystal oscillator, the weight of the adhered substance and the change in frequency are in a proportional relationship.
For example, 27MHz, AT-cut crystal oscillator 30p
When 1g of substance is deposited on the electrode, the frequency is reduced by about 1Hz. Thereby, the mass of a substance having a very small mass and the interaction between substances can be qualitatively or quantitatively measured.

Therefore, in the first aspect of the present invention, the apparent weight of the biopolymer in the aqueous medium is measured by measuring the change in the frequency of the quartz oscillator before and after the structural change of the biopolymer. The change or the structural change can be known, and the structural analysis and the function prediction can be performed.

Further, in the second aspect of the present invention, by measuring the change in frequency of the crystal oscillator, a compound that associates only when the structure of the biopolymer changes can be detected,
It is possible to measure the amount of compound associated. Further, it can be used for screening of compounds that prevent or reversibly restore the structure of biopolymer.

[0026]

BEST MODE FOR CARRYING OUT THE INVENTION An application example of a crystal oscillator according to the present invention to solution measurement will be described below. The crystal oscillator is attached to a crystal oscillator mounting arm and immersed in a solution in a constant temperature heat block. The constant temperature heat block is for keeping the temperature of the solution constant. The solution is stirred by a stirrer. The oscillator circuit oscillates the crystal oscillator by applying an alternating electric field to the electrodes of the crystal oscillator. The oscillation frequency of the oscillation circuit is counted by a universal counter, analyzed by a computer, and the mass and interaction of substances in the sample are displayed.

[0027]

EXAMPLES Examples of the present invention will be shown below, but the present invention is not limited thereto. Example 1 Quartz crystal of casein molecule (sensor chip
The immobilization method 1) 3,3'-dithiodipropionic acid to flop) was dissolved in ethanol so that the 21 mg / ml (0.1 M ethanol solution). 2) The washed oscillator was placed on a desk, 100 μl of the solution of 1) diluted with distilled water 100 times was spread over the entire crystal part of the oscillator, and left for 2 hours. 3) Washed with distilled water. 4) EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) was dissolved in distilled water to 100 mg / ml. 5) 100 mg of NHS (N-hydroxysuccinimide)
It was dissolved in distilled water so that the concentration became / ml. 6) EDC and NHS solution prepared in 4) and 5) are mixed in equal amounts, and 100 μl is placed on the oscillator prepared in 3).
Let stand for 30 minutes at room temperature. 7) Casein was dissolved in PBS so as to be 1 mg / ml, dispensed at an appropriate dose in a usable unit, and stored frozen at -20 ° C or lower to obtain a storage solution. 8) The casein was diluted with PBS so that the final concentration was 10 μg / ml, and the oscillator of 7) was treated at room temperature for 1 hour. The amount of casein immobilized on the chip was 30 ng.

Example 2 A casein-immobilized chip was used.
Detection Example 1 of Change in Molecular Structure ) Quartz oscillator (Affine, Inc.)
The casein-immobilized chip was set in ix Q), 8 ml of PBS was put in the measuring cell of the apparatus, the reaction temperature was set at 25 ° C., and the reaction was waited in PBS until the frequency became stable. 2) The same molecule (casein) immobilized on the sensor chip was added to the measurement cell so that the final concentration was 5 ng / ml (10 μl dose). 3) 100 μl of 1M hydrochloric acid aqueous solution was added to the cell. The frequency has decreased. 4) The amount of change in frequency for 5 minutes was measured, and 100 μl of 1M sodium hydroxide aqueous solution was added. The frequency has returned to the original level. 5) Increase the casein concentration in 2) above (final concentration;
The operations of 3.8 μg / ml), 2) to 4) were repeated. The same analysis was performed using bovine serum albumin (BSA) instead of casein in the measurement cell of 2).

The results obtained by the above experiment are shown in FIG. From FIG. 1, it can be seen that the present invention enables the amount of association with the casein molecule to be measured, and the degree of association between the same molecule (casein) and different molecule (BSA) to be evaluated and classified.

[0030]

INDUSTRIAL APPLICABILITY According to the present invention, the structure of a biopolymer such as an immobilized protein can be analyzed, and the function of the compound can be predicted in addition to the change in the structure of the biopolymer. .

The present invention can be applied to analysis of the influence on the association state due to changes in salt concentration and addition of coexisting amino acids, in addition to changes in pH of the aqueous medium. In addition, it can be used to search for cofactors and competitors.

The present invention is a novel method for functionally analyzing a molecule (protein), and can be a tool for genome analysis or proteome analysis.

[Brief description of drawings]

FIG. 1 is a diagram showing a relationship between a protein concentration in a cell and a change in frequency of a crystal oscillator.

Claims (6)

[Claims] 1. A method for analyzing a structure of a biopolymer on a solid phase surface, comprising: (a) a quartz plate having front and back surfaces and side surfaces; and electrodes provided on the front and back surfaces of the quartz plate. A single-sided covering material that covers one electrode provided on the back surface and whose inner wall contacts the side surface of the crystal plate, and a seal provided between the inner wall and the surface of the crystal plate on which the other electrode is provided. Fixing a biopolymer to the other electrode of the crystal oscillator provided with a stopper, and (b) immersing the crystal oscillator having the biopolymer fixed in an aqueous medium to obtain a stable frequency of the crystal oscillator. And (c) changing the structure of the immobilized biopolymer soaked in an aqueous medium, and then measuring the frequency change of the crystal oscillator. 2. A method for analyzing the structure of a biopolymer on the surface of a solid phase from changes in the association state thereof, comprising: (a) a crystal plate having both front and back surfaces and side surfaces; and a front and back surface of the crystal plate. An electrode provided on the one side provided on the back surface, one side coating material whose inner wall is in contact with the side surface of the crystal plate, and the other side of the surface of the crystal plate provided with the electrode and the inner wall. Fixing a biopolymer to the other electrode of the crystal oscillator provided with a sealing material provided between the two, (b)
Immersing the crystal oscillator with the biopolymer fixed in an aqueous medium,
Measuring the stable frequency of the crystal unit and (c)
A method comprising changing the structure of an immobilized biopolymer immersed in an aqueous medium, adding a compound capable of associating with the biopolymer to the aqueous medium, and measuring a frequency change of a quartz oscillator. . 3. The structure of the immobilized biopolymer is changed by changing the pH or salt concentration of the aqueous medium or by adding a modifier or an organic solvent to the aqueous medium. The method according to 1 or 2. 4. The analysis method according to claim 2, wherein a compound associated with the biopolymer having a structural change is detected and the amount thereof is measured. 5. The method according to claim 2, wherein the compounds are classified using the degree of association associated with the structural change of the biopolymer as an index. 6. The biopolymer is a protein.
5. The method according to any one of 5 to 5.