JP2507033B2 - Enzyme derivative and method for controlling enzyme reaction - Google Patents

Description

TECHNICAL FIELD The present invention relates to an enzyme derivative and a method for controlling an enzyme reaction.

Conventional technology Enzymatic reactions are currently used in the biochemical industry, mainly in the detection of specific substances, genetic engineering, and other industries.The method of controlling this reaction is to adjust the reaction temperature, pH, or enzymatic reaction. It is common to stop the enzymatic reaction by adding an inhibitor.

For example, peroxidase (POD) is an enzyme often used for high-sensitivity detection with an enzyme such as enzyme immunoassay, and most efficiently decomposes hydrogen peroxide in the vicinity of neutral to weak alkali. Usually, in this reaction, an appropriate reaction time and the like are determined by preliminary experiments, and after the lapse of such time, the reaction is irreversibly stopped by making the enzyme inactive at a stretch with 4 N sulfuric acid or the like to inactivate the enzyme.

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention However, since the reaction control method by the above method involves irreversible enzyme inactivation, after examining the reaction once stopped, the reaction is restarted to reach the optimal reaction end. It was impossible.

The present invention aims to solve such problems of the conventional technology.

Means for Solving the Problem A photochromic substance that chemically reversibly changes its chemical structure upon irradiation with light is chemically bound to an enzyme.

Action In the above structure, the anti-photochromic antibody is usually bound to the photochromic substance bound to the enzyme.
In this state, contact of the substrate with the reaction site of the enzyme is inhibited by the steric hindrance effect of the antibody, and the enzyme reaction does not proceed. However, when light is irradiated from the outside, the photochromic substance is isomerized and the antibody is desorbed. As a result, the inhibition of the enzymatic reaction due to the steric hindrance of the antibody disappears and the enzymatic reaction starts. That is, the enzyme reaction is turned on by turning on / off the light irradiation.
-OFF control is possible.

Example In the present invention, a photochromic substance that reversibly changes its chemical structure upon irradiation with light is chemically bonded to an enzyme. On the other hand, an antibody (antiserum or monoclonal antibody) that specifically binds to the photochromic substance is prepared by injecting an animal with the conjugate of the photochromic substance and the protein. This photochromic substance-enzyme conjugate and the anti-photochromic antibody coexist in a solution,
Irradiate ultraviolet rays or visible rays from the outside.

 Hereinafter, examples of the present invention will be described.

Examples of photochromic substances include photochromism by Glenn Etch Brown (Glenn H, Brown, Tech.
niques of Chemistry, Vol 3, Photochrromism, Wiley-In
terscience), many kinds of substances are known, including spiropyran, azobenzene, fulgide, indigo, and thioindigo. Among them, spiropyrans show particularly stable photochromism. In carrying out the present invention, the present inventors have previously developed a spiropyran-type photochromic substance (SPMI) capable of introducing a maleimide group and easily binding to a protein or the like.

The maleimide group of this SPMI easily binds to the thiol group of protein in aqueous solution. Utilizing this property, a photochromic substance can be introduced into the enzyme. Representative examples will be described below.

a) Preparation of spiropyran-bound alcohol dehydrogenase 1.23 μmol alcohol dehydrogenase (ADH: yeast origin) was added to PBS buffer (0.1 M sodium phosphate, 1.4 M Na).
Cl, pH 7.0) dissolved in 10 ml. Add 10 mg / ml AMSA to the enzyme solution so that the number of moles of acetylmercaptosuccinic anhydride (AMSA) is 30 times that of yeast.
119 μl of the solution (dissolved in N, N-dimethylformamide (DMF)) was added.

The ADH solution was left at 30 ° C. for 3 hours. Then, gel filtration column chromatography (Sephadex G-25 column, manufactured by Pharmacia) equilibrated with PBS buffer.
Size: 2.6 x 40 cm) flow rate 4.0 ml / min and free AMSA
Was removed. Fractions having an absorption maximum of 280 nm light (A _{280 nm} ) of 0.3 or more were collected.

To this ADH solution was added 8.4 mM tris (hydroxymethyl) aminomethane (Tris pH7.0), 0.84 mM ethylenediaminetetraacetic acid (EDTA) and 84 mM hydroxylamine,
It was deacetylated by leaving it at 10 ° C for 10 minutes to generate a free thiol group. Then, gel filtration column chromatography equilibrated with PBS buffer (Sephadex G-25, Pharmacia column size: 2.6 × 40)
cm) at a flow rate of 4.0 ml / min to remove free Tris, EDTA and hydroxylamine. Fractions with A _{280 nm} of 0.3 or more were collected and the following proteins and free thiol groups were quantified.

To this ADH solution, a spiropyran maleimide (SPMI) / dimethylsulfoxide (DMSO) solution was added so that the molar amount was 10 times that of the free thiol group. That is, 7.8 mg / ml
0.85 ml of SPMI / DMSO solution of was added. Also, this SPMI / DM
The SO solution was adjusted to have a DMSO concentration of 5% after being added to the enzyme solution.

After the addition, the mixture was allowed to stand at 25 ° C for 10 to 12 hours and subjected to ultrafiltration (Amicon, manufactured by DIAFLO: YM10,43mm). After centrifugation (18,000 rpm, 0 ° C, 30 minutes) of this concentrated solution, free SPMI and D
Gel filtration column chromatography (Sephadex G-25, equilibrated with PBS buffer to remove MSO)
Pharmacia column size: 2.6 x 40 cm) and flow rate 5.
It was applied at 0 ml / min. Fractions having A _{280 nm} of 0.3 or more were collected, and the following protein quantification, free thiol group quantification and enzyme activity measurement were carried out.

The number of spiropyran bound to each alcohol dehydrogenase subunit was 8.3, which was calculated by subtracting the number of free thiol groups after introduction of spiropyran from the number of free thiol groups after addition of AMSA. It was

 There was also enzyme activity.

Irradiate the enzyme solution after spiropyran introduction with 365 nm light, and
It was confirmed that spiropyran was bound to each enzyme by increasing the absorption maximum of 50 nm light. Furthermore, when the enzyme solution into which spiropyran has been introduced is subjected to gel filtration column chromatography (Sephadex G-25, manufactured by Pharmacia), this enzyme is eluted in the excluded volume, indicating that spiropyran is bound to the enzyme. confirmed.

Determination of free thiol group 4,4'-in 1.0 ml of enzyme solution of appropriate concentration (2-5 μM)
Add 40 μl of dithiopyridine (4-PDS) and incubate at room temperature for 10
After standing for a minute, the increase in absorption maximum of 324 nm light was measured.

Enzyme activity measurement Alcohol dehydrogenase activity is β-NAD ⁺ at 25 ℃
It was measured by the increase of the absorption maximum of 340 nm light due to the reduction of. The activity measurement system is 100 mM phosphate buffer (pH 8.3), 2
It contained 00 mM ethanol, 2 mM β-NAD ⁺ and 10-200 μg alcohol dehydrogenase.

 The reaction was started by adding the enzyme.

Protein quantification was performed using a BCA protein quantification kit from Pierce.

b) Preparation of immunogen spiropyran-bound γ-globulin 1.67 μmol γ-globulin (CGG: chicken origin) in PBS
It was dissolved in 250 ml of buffer. CGG in this protein solution
2.4 ml of a 10 mg / ml SPDP (dissolved in DMSO) solution was added so that the molar number of N-succinimidyl 3- (2-pyridyldithio) propinate (SPDP) was 20 times the molar amount.

The CGG solution was left at 30 ° C. for 3 hours. Then, it was applied to gel filtration column chromatography (Sephadex G-25, Pharmacia column size: 2.6 × 40 cm) equilibrated with PBS buffer at a flow rate of 4.0 ml / min to release free SPDP.
Was removed. Fractions with an A _{280 nm} of 0.3 or higher were collected.

To this CGG solution, 2.0 mM dithiothreitol (DTT) was added and left at room temperature for 30 minutes to generate free thiol groups. Then, gel filtration column chromatography (Sephadex G-
25, Pharmacia column size: 2.6 × 40 cm) at a flow rate of 4.0 ml / min to remove free DTT. A _280nm ,
Fractions of 0.3 or more were collected and the above proteins and free thiol groups were quantified.

To this CGG solution, a spiropyran maleimide (SPMI) / dimethylsulfoxide (DMSO) solution was added so that the molar amount was 10 times that of the free thiol group. In other words, 7.9m / ml
10.5 ml of SPMI / DMSO solution of was added. Also, this SPMI / DM
The SO solution was adjusted to have a DMSO concentration of 5% after being added to the CGG solution.

After the addition, the mixture was left at room temperature for 36 to 40 hours and subjected to ultrafiltration (Amicon, manufactured by DIAFLO: YM10, 43 mm). After centrifugation (18,000 rpm, 0 ° C, 30 minutes) of this concentrated solution, free SPMI and D
Gel filtration column chromatography (Sephadex G-25, equilibrated with PBS buffer to remove MSO)
Pharmacia column size: 2.6 x 40 cm) and flow rate 5.
It was applied at 0 ml / min. Fractions with A _{280 nm} of 0.3 or more were collected, and the following protein quantification and free thiol group quantification were performed.

The number of spiropyran bound to one CGG protein was determined by subtracting the number of free thiol groups after introduction of spiropyran from the number of free thiol groups after addition of SPDP, and was 11.5. On the other hand, the CGG solution after introducing spiropyran was irradiated with 365 nm light, and it was confirmed that spiropyran was bound to CGG by the increase of the absorption maximum of 550 nm light. Furthermore, CGG with spiropyran introduced
When the solution was subjected to gel filtration column chromatography (Sephadex G-25, manufactured by Pharmacia), this CGG was eluted in the excluded volume, which confirmed that spiropyran was bound.

c) Preparation of anti-spiropyran antibody Spiropyran-bound CGG obtained by the method described in b) above
Antiserum containing anti-spiropyran antibody was obtained by immunizing mice with the above as an immunogen. This antiserum was subjected to protein A affinity column chromatography (column size: 1.0 × 15 cm) to purify γ-globulin. This γ-globulin is an anti-spiropyran antibody, and spiropyran-conjugated bovine serum albumin (binding molar ratio: 1
/ 1) was confirmed by enzyme-linked immunosorbent assay (ELISA) using solid-phase antigen.

d) Control of alcohol dehydrogenase (ADH) activity by light irradiation In 1.0 ml of spiropyran-bound alcohol dehydrgenase (SP-ADH: specific activity 250 U / mg) prepared in the above a), c-purified γ-globulin was added 10 ⁴ ADH activity was measured by the above-mentioned activity measuring method after adding 1.0 ml of a solution diluted twice.

As a result, the specific activity of this solution was 30 U / mg, and the anti-SP
ADH activity was suppressed by the addition of antibody.

After irradiating this solution with 365 nm light at 4 ° C. for 2 hours, the ADH activity was measured again, and the specific activity was restored to 220 U / mg.

Furthermore, the above operation was repeated after leaving the remaining solution at 4 ° C. in a dark room for 3 days, and the activity was restored by irradiation with 365 nm light (25 U / mg 220 U / mg).

From the above, the alcohol dehydrogenase activity could be controlled by light irradiation by the anti-spiropyran antibody and the spiropyran-bound alcohol dehydrogenase.

EFFECTS OF THE INVENTION As described above, according to the present invention, it is possible to study the reaction once stopped and then start the reaction again to reach the optimum end of the reaction.

Claims (3)

(57) [Claims] 1. An enzyme derivative characterized in that a photochromic substance is chemically bound to an enzyme. 2. The enzyme derivative according to claim 1, wherein the photochromic substance is spiropyran. 3. A method for controlling an enzyme reaction, which comprises allowing the enzyme derivative according to claim 1 and an antibody against a photochromic substance to coexist in a solution.