JPH066070B2 - Method for quantifying NADH or NADPH by chemiluminescence method - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for quantifying NADH or NADPH by a chemiluminescence method.

(Prior Art) Conventionally, as a method for quantifying NADH or NADPH, a method of directly measuring by an ultraviolet absorption method is very well known. However, it is easily affected by turbidity and its sensitivity is determined as a final concentration. It is about 10 ⁻⁶ M, which is a disadvantage that it is difficult to perform high sensitivity measurement. In order to increase the sensitivity, a method is known in which diaphorase and resazurin are allowed to act on NADH to measure the fluorescence of the produced resorufin, but the effect of turbidity cannot be avoided and the final concentration is 10 ^−7. Since the limit is about M, it cannot be said that the sensitivity is sufficient for detecting a trace component. Further, the measurement of NADH by the currently used diaphorase-resazurin system requires complicated calculation and is not practical. In addition, the fluorescence measuring device is expensive, which is a factor that prevents the spread of the above method. A suitable fluorescence method for NADPH has not been found yet.

(Problems to be Solved by the Invention) From the above circumstances, a method of highly sensitively measuring NADH or NADPH is required.

(Means for Solving Problems) The present inventor has found a method for quantifying hydrogen peroxide with higher sensitivity than a chemiluminescence method, and has already proposed it (JP-A-60-20).
The present invention has found that it is possible to quantify NADH or NADPH with high sensitivity by introducing NADH or NADPH to hydrogen peroxide and measuring hydrogen peroxide by a chemiluminescence method. Reached

That is, the present invention provides samples containing NADH or NADPH with NAD (P) H-FMN oxidoreductase and NADH.
-At least one enzyme selected from the group consisting of FMN oxidoreductase, NADPH-FMN oxidoreductase, diaphorase and old yellow enzyme is allowed to act in the presence of flavins, and the resulting reduced flavins are present in the presence of oxygen, Quantification of NADH or NADPH by chemiluminescence method, characterized in that hydrogen peroxide is produced by auto-oxidation, and the obtained hydrogen peroxide is measured by chemiluminescence method to quantify NADH or NADPH in a sample Is the law.

In the present invention, when quantifying NADH in a sample, first, N
AD (P) H-FMN oxidoreductase, NADH-
FMN oxidoreductase or diaphorase is allowed to act in the presence of flavins.

NAD (P) H-FMN oxidoreductase or NAD
H-FMN oxidoreductase can be obtained from the luminescent bacterium Vibrio fischeri or Vibrio harveyi (Biochemistry Vo16, No. 13, p2932, 197).
7, the same Vol. No. 4, p672, 1978, etc.).
Diaphorase is an enzyme classified as EC1, 6, 4, 3 and may be of animal or microbial origin.

In quantifying NADH, the enzyme is added to a sample containing flavins such as FMN, FAD or riboflavin and NADH to react with each other to produce reduced flavin,
Hydrogen peroxide is produced by autoxidation in the presence of oxygen.

NAD (P) H-FMN oxidoreductase, NADH
-FMN oxidoreductase or diaphorase is usually used at a final concentration of 1 to 50 mu. Reduced flavin is usually used at a concentration of 0.1 to 100 μM.

Further, in the present invention, when quantifying NADPH in a sample,
First, NAD (P) H-FMN oxidoreductase, NA
DPH-FMN oxidoreductase or old yellow enzyme is allowed to act in the presence of flavins.

NAD (P) H-FMN oxidoreductase or NAD
PH-FMN oxidoreductase can be obtained from the luminescent bacterium Vibrio fischeri or Vibrio farveyi (Biochemistry Vol. 16, No. 3, p 2932, 197).
7, the same Vol. 17, No. 4, p672, 1978, etc.). The old yellow enzyme is an enzyme classified as EC1,6,99,1 and is obtained from yeast, marine luminescent bacteria and the like.

In quantifying NADPH, the above enzymes are flavins,
For example, FMN, FAD or riboflavin and NADPH are added to a sample and reacted to produce reduced flavin, and hydrogen peroxide is produced by autoxidation in the presence of oxygen.

NAD (P) H-FMN oxidoreductase, NADP
H-FMN oxidoreductase or old yellow enzyme is usually used at a final concentration of 1 to 50 mu.

In the present invention, the produced hydrogen peroxide is quantified by the chemiluminescence method. As the chemiluminescence method, a chemiluminescence method using luminol or lucigenin or a chemiluminescence method using an oxalic acid diester and a fluorescent substance can be used.

When quantifying the generated hydrogen peroxide by the luminol reaction,
Luminol is used at a concentration of 10 ⁻⁶ to 10 ⁻⁷ M, and as a catalyst, for example, about 10 mM potassium ferricyanide is used.
Used in 00 mM borate buffer (pH 10). When using peroxidase as a catalyst, for example, 100m
M phosphate buffer (pH 7-7.5) or 100 mM Tris-HCl buffer (pH 7-8.5) may be used.

When the generated hydrogen peroxide is subjected to chemiluminescence with an oxalic acid diester and a fluorescent substance for quantification, about 1 to 10 μM fluorescein or 8-anilinonaphthalene-1-sulfonic acid is added to about 1
10 μM is added, and about 0.1 to 50 mM oxalic acid diester such as bis (2,4,6-trichlorophenyl) oxalate (TCPO) or bis (2,4-dinitrophenyl) oxalate (DNPO) is used.

A method of causing the fluorescence reaction by 2 ', 7'-dichlorofluorescein-diacetate developed by the present inventor to emit light with oxalic acid diester and newly added hydrogen peroxide has a sensitivity or accuracy as a chemiluminescence method. It is preferable from the point. Next, the method for quantifying the produced hydrogen peroxide by the chemiluminescence method developed by the present inventor will be described in detail. That is, a sample containing the produced hydrogen peroxide is reacted with an oxidizable non-fluorescent substance through an oxidation catalyst to convert the non-fluorescent substance into the fluorescent substance, and then under the conditions of inhibiting the oxidation catalyst. Hydrogen peroxide in a sample is quantified by reacting a fluorescent substance with oxalic acid diesters and hydrogen peroxide and measuring the amount of luminescence produced.

The reaction scheme of this method is as follows.

Fluorescent substance + oxalic acid diester + H ₂ O ₂ → hν + product ...
(2) That is, the oxidizable non-fluorescent substance is converted to the fluorescent substance in the presence of the oxidation catalyst by the hydrogen peroxide generated by the above reaction (first reaction), and then the oxidation catalyst is inhibited under the inhibiting condition. ,
For example, after adding an inhibitor to inhibit the oxidation catalyst, the generated fluorescent substance is caused to emit light in the presence of hydrogen peroxide added again with oxalic acid diesters (second reaction), and the amount of emitted light is measured. Quantification of hydrogen peroxide in the first stage reaction is performed.

The oxidizable non-fluorescent substance used in the present invention is leucofluorescein (fluorescein), 2 ', 7'-.
Examples include dichlorofluorescein-diacetate. The amount of non-fluorescent substance used is generally 0.001 to 5 mM.

Examples of the oxidation catalyst used in the present invention include peroxidase, microperoxidase, hemin, hematin and the like. The amount of oxidation catalyst used is generally 1 to 100 units.

The oxalic acid diesters used in the present invention include bis (2,4,6, -trichlorophenyl) oxalate (TCPO) and bis (2,4-dinitrophenyl) oxalate (DNPO). The amount of oxalic acid diesters used is generally 0.1 to 50 mM.

Examples of the inhibitor of the oxidation catalyst of the present invention include potassium cyanide, sodium cyanide, sodium azide, sulfide, fluoride, ferrocyanide, hydroxyphenylhydrazine, 2,3-dimercaptopropanol, ethyl acetate, acetonitrile, ethanol and acetone.

In the present invention, it is necessary to carry out the first reaction in an aqueous system. This reaction is usually carried out at about 20-40 ° C and pH 5-9. After the completion of the first reaction, the oxidation catalyst should be inhibited. If the oxidation catalyst which is not deactivated exists in the second reaction, the hydrogen peroxide added anew reacts with the remaining non-fluorescent substance to generate an excessive amount of fluorescent substance. When 10 ⁻⁵ to 10 ⁻¹ M of cyclodextrin is added to the first reaction system, stabilization of the non-fluorescent substance and increase of the luminescent property of the produced fluorescent substance are observed.

Further, in the present invention, in order to prevent the oxidation of the non-fluorescent substance (leucofluorescein) in the presence of an oxidation catalyst (peroxidase), zinc sulfate heptahydrate is preferably about 0.04 mg.
It is preferable to add to the first reaction system so that the final concentration will be / m.

The second reaction of the present invention is carried out in an organic solvent containing water.
Examples of the organic solvent include ethyl acetate, acetone, acetonitrile and the like. The organic solvent may be a single kind or a mixture of two or more kinds. The second reaction is usually carried out at about 20 to 40 ° C. under alkaline conditions at pH 8 to 12.

The amount of hydrogen peroxide added to the second reaction system is generally 0.1 to
It is 50 mM.

The luminescence activity generated in the second reaction system is measured by a usual measuring method. For example, the amount of light emitted at the peak can be measured by the Lumi Photometer-T
Count with D4000 (manufactured by Lab Science).
In luminescence activity, the luminescence value is always expressed per reaction time, and thus the reaction rate is expressed as it is.

With this method, hydrogen peroxide can be quantified with high sensitivity by the chemiluminescence method without being interfered by other substances.

According to the conventional knowledge, the quantification of hydrogen peroxide by bis (2,4,6-trichlorophenyl) oxalate and a fluorescent substance is poor in sensitivity and is usually detected only at 10 ⁻⁷ to 10 ⁻⁶ M or more. However, in the present invention, it becomes possible to measure hydrogen peroxide up to 10 ⁻⁹ M by selecting an oxidizable non-fluorescent substance. Further, in the present invention, when the first reaction is carried out in an aqueous system to produce a fluorescent substance, if oxidation is carried out by an enzyme such as peroxidase, the accompanying hydrogen peroxide producing system reaction, for example, glucose oxidase in glucose determination, etc. may be impaired. However, it is possible to quantify hydrogen peroxide, and thus glucose, under mild conditions.

In the present invention, a trace amount of hydrogen peroxide produced by a coexisting reaction from components in a biological sample is highly sensitive, particularly 10 ^−9.
It is possible to measure in the range of 10 ⁻⁵ M. In addition, in the analysis of biological components, a stable fluorescent substance is generated without inhibiting the reaction of the water system, and hydrogen peroxide generated by the interference of catalase in the biological sample is not reduced, and it is truly quantitative and It is possible to analyze biological components with high sensitivity. In the present invention, since the second reaction is not affected by the interfering substance in the biological sample, a highly sensitive luminescence activity can be obtained.

NADH or NADPH to be quantified in the present invention may be produced from in vivo components such as sodium cholate and glutamic acid.

The present invention can quantify not only NADH or NADPH but also in vivo components such as sodium cholate and glutamic acid.

(Effect) In the present invention, it can be quantified with high sensitivity and accuracy by converting into NADH or NADPH hydrogen peroxide and further by chemiluminescence method.

(Examples) Next, the present invention will be specifically described with reference to Examples.

Example 1 (Quantification of NADH by NADH-FMN oxidoreductase) N in 50 mM Tris-HCl buffer (pH 7.0) 1 m
Add 20 mu of ADH-FMN oxidoreductase and 5u of peroxidase, and further add 2 ', 7'-dichlorofluorescein-diacetate solution (25 μg / m) 3
m and 1 mM FMN (5 μm) were added, then various concentrations of NADH solution (1 m) were added, and the mixture was incubated at 30 ° C. for 1 hour. 0.1 mM per 0.5 m of the fluorescent solution
Bis (2,4,6-trichlorophenyl) oxalate and 0.4 mM hydrogen peroxide solution were added, and luminescence was measured by a Lumiphotometer.

The relationship between the emission intensity and the NADH concentration in the 0.5 m solution is shown in FIG. As is clear from FIG. 1, the concentration of NADH of 5 × 10 ⁻⁹ M to 10 ⁻⁵ M can be quantified.

Example 2 (Determination of sodium cholate by NADH-FMN oxidoreductase) 3α in 50mM Tris-HCl buffer (pH 7.0) 1m.
-Hydroxysteroid dehydrogenase 25u, 1m
M NAD ⁺ , NADH-FMN oxidoreductase 20 mu, peroxidase 5 u were dissolved, and 2 ', 7'-dichlorofluorescein-diacetate solution (25 μg / m) 3 m and 1 mM FMN 5 μ were added, and then various concentrations of Cole were added. Add sodium acidate 3
Incubated at 0 ° C for 1 hour. Fluorescent solution 0.
0.1 mM bis (2,4,6-trichlorophenyl) oxalate and 0.4 mM hydrogen peroxide solution were added to 5 m, and the luminescence was measured by a luminometer.

The relationship between the luminescence intensity and the sodium cholate concentration in a 0.5 m solution is 5 × 10 ⁻⁹ M to 1 sodium cholate concentration.
It correlated seen in 0 ^-5 M. That is, a sodium cholate concentration of 5 × 10 ⁻⁹ M to 10 ⁻⁵ M can be quantified.

Example 3 (Determination of NADPH by NADPH-FMN oxidoreductase) N in 50 mM Tris-HCl buffer (pH 7.0) 1 m
20 mu of ADPH-FMN oxidoreductase and 5u of peroxidase were added, and 2 ', 7'-dichlorofluorescein-diacetate solution (25 µg / m) 3
m and 1 mM FMN (5 μm) were added, and then various concentrations of NADPH solution (1 m) were added and the mixture was incubated at 30 ° C. for 1 hour. Example 1 was added to 0.5 m of the fluorescent solution.
In the same manner as above, oxalic acid diester and hydrogen peroxide were added, and the luminescence was measured by a Lumiphotometer.

The relationship between the light emission speed and the NADPH concentration in the 0.5 m solution is shown in FIG. As is clear from FIG. 2, the NADPH concentration of 5 × 10 ⁻⁹ M to ⁵ × 10 ⁻⁵ M can be quantified.

Example 4 (Quantification of glutamic acid by NADPH-FMN oxidoreductase) Glutamate dehydrogenase 25u, 1 mM NADP was added to 1 mM of 50 mM Tris-HCl buffer (pH 7.0).
⁺ , NADPH-FMN oxidoreductase 20m
u and peroxidase 5u were added, and 2 ', 7'-
Dichlorofluorescein-diacetate solution (25 μg /
m) after adding 3m and 1mM FMN 5μ,
Glutamic acid at various concentrations was added, and the mixture was incubated at 30 ° C for 1 hour. 0.1 mM per 0.5 m of the fluorescent solution
Bis (2,4,6-trichlorophenyl) oxalate and 0.4 mM hydrogen peroxide solution were added, and luminescence was measured by a Lumiphotometer.

The correlation between the emission intensity and the concentration of glutamic acid in a 0.5 m solution is found at a concentration of glutamic acid of 10 ⁻⁹ M to 10 ⁻⁵ M. That is, glutamic acid concentration 10
^-9 M to 10 ^-5 M can be quantified.

[Brief description of drawings]

FIG. 1 shows a quantitative curve of NADH using NADH-FMN oxidoreductase, in which the vertical axis shows the emission intensity (peak value) and the horizontal axis shows the amount of NADH. FIG. 2 shows a quantitative curve of NADPH using NADPH-FMN oxidoreductase, in which the vertical axis shows the emission intensity (peak value) and the horizontal axis shows the amount of NADPH.

Claims (2)

[Claims] 1. NA in a sample containing NADH or NADPH
D (P) H-FMN oxidoreductase, NADH-F
At least one enzyme selected from the group consisting of MN oxidoreductase, NADPH-FMN oxidoreductase, diaphorase and old yellow enzyme is allowed to act in the presence of flavins, and the resulting reduced flavins are auto-oxidized in the presence of oxygen. A method for quantifying NADH or NADPH by a chemiluminescence method, characterized in that hydrogen peroxide is produced by the method, and the obtained hydrogen peroxide is measured by a chemiluminescence method to quantify NADH or NADPH in a sample. 2. The obtained hydrogen peroxide is reacted with an oxidizable non-fluorescent substance through an oxidation catalyst to convert the non-fluorescent substance into the fluorescent substance, and then under the conditions of inhibiting the oxidation catalyst. By reacting the fluorescent substance with oxalic acid diesters and hydrogen peroxide, and measuring the amount of luminescence produced, NADH in the sample is obtained.
Alternatively, the method for quantifying NADH or NADPH by the chemiluminescence method according to claim 1, characterized by quantifying NADPH.