JPH1175894A - Measurement of reduced nicotinamide adenine dinucleotide(nadh) or reduced nicotinamide dinucleotide phosphate(nadph) - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for measuring reduced nicotinamide adenine dinucleotide(NADH) or reduced nicotinamide dinucleotide phosphate(NADPH), enabling to rapidly and simply measure the NADH or the NADPH in high sensitivity. SOLUTION: This method for measuring NADH or NADPH comprises electrochemically measuring the concentration of the NADH or the NADPH with two electrodes consisting of a working electrode and a counter electrode. Therein, the characteristics comprise applying a constant electric potential to the working electrode within a potential range not causing the electrolysis of the NADH or the NADPH, subsequently applying a pulse electric potential to the working electrode within a potential range causing the electrolysis of the NADH or the NADPH, and subsequently measuring an electric current flowing between the working electrode and the counter electrode at 10-1000 msec after the application of the pulse potential. The method enables to measure the NADH or the NADPH in high sensitivity having a detection limit concentration of <=10 μmmol/L.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to reduced nicotinamide adenine dinucleotide (hereinafter referred to as "NADH") or reduced nicotinamide adenine dinucleotide phosphate (hereinafter referred to as "NADPH") which acts as a coenzyme for many enzymes. The present invention relates to a method for measuring the activity of an enzyme having NADH or NADPH as a coenzyme and a method for measuring the concentration of an enzyme substrate.

[0002]

2. Description of the Related Art It is known that NADH or NADPH works as a coenzyme in various enzyme reactions. For example, NADH and the like have α
It acts as a coenzyme during the oxidation of the hydroxy acid to the corresponding ketone acid. As one of the specific examples, oxidation of lactic acid to pyruvic acid represented by the following reaction formula (Formula 1) can be mentioned.

## STR1 ## CH ₃ —CHOH—COOH + NAD ⁺ → CH ₃ CO
-COOH + NADH

[0004] NADH also acts as a coenzyme in the conversion of 2-oxoglutarate to L-glutamate by ammonium salts in the presence of GLDH (glutamate dehydrogenase).

[0005] In order to examine enzyme activity and enzyme substrate concentration by utilizing such properties as a coenzyme such as NADH,
Measurement of the concentration of NADH and the like is widely performed in the field of clinical tests. For example, in the case of the aforementioned GLDH, by measuring the residual NADH, the amount of ammonia in the reaction medium can be determined. That is, when the substrate is oxidized by an enzymatic reaction in the presence of NAD ⁺ (or NADP ⁺ ), NADH (or NADPH) is generated. Also,
When the substrate is reduced by an enzymatic reaction in the presence of NADH (or NADPH), NAD ⁺ (or NADP
⁺ ) Is generated. Therefore, in the former case, NA
By measuring the amount of increase in DH (or NADPH) and, in the latter case, the amount of decrease in NADH (or NADPH), to measure the change in enzyme substrate concentration and the activity of an enzyme using NADH or the like as a coenzyme. Can be.

A method for measuring NADH or NADPH is as follows:
It can be broadly classified into two types: an optical method and an electrochemical method.

As an optical method, NADH or NA is used.
Ultraviolet light absorption of DPH itself (maximum absorption wavelength: 340n
m), directly measuring the fluorescence of NADH or NADPH itself, NADH in the presence of a catalyst.
Alternatively, a method of measuring fluorescence of resorufin generated by a reaction between NADPH and resazurin and the like are known.

As an electrochemical method, two electrodes, a working electrode and a counter electrode, are used, and NADH or NADPH is electrolytically oxidized by applying the working electrode to the working electrode. At this time, there is a passage between the working electrode and the counter electrode. Methods for measuring current are known.
This electrochemical method has the advantage that it can be measured quickly using a relatively simple and inexpensive measuring device compared to the optical method, but has the disadvantage that its sensitivity is lower than that of the optical method. Having. Therefore, conventionally, NADH
Alternatively, in an electrochemical measurement method of NADPH, various attempts have been made to improve sensitivity.

[0009] For example, Japanese Patent Publication No. 58-16694 discloses an enzyme electrode in which NADH is directly immobilized on the electrode surface by chemical bonding. This technology improves the sensitivity by fixing NADH, which is to be oxidized directly on the electrode surface, on the electrode surface by chemical bonding, thereby always bringing NADH close to the electrode surface and improving the oxidation efficiency. Is intended.

JP-A-56-35050 discloses that, instead of electrolytically oxidizing NADH, Meldra Blue is used as a mediator and the current value when electrolytically oxidizing the reduced form of the mediator is used. NADH
There is disclosed a technique for measuring the temperature. In this technique, the sensitivity is improved by using a redox cycle of a mediator.

Japanese Patent Application Laid-Open Publication No. 1-503409 discloses an active carbon layer or activated graphite particle comprising a porous and heterogeneous resin binding layer in which a binding agent such as a natural resin or a synthetic resin is used to bind the activated carbon particles or the activated graphite particles. An electrochemical measurement method of NADH using a carbonized electrode is disclosed. Although the cause of the improvement in sensitivity in this technique is not clear, it is assumed that the porous and heterogeneous structure of activated carbon or activated graphite increases the effective area of the electrode. Furthermore, it is supposed that activated carbon or activated graphite is excellent in compatibility with NADH and can oxidize NADH more quickly than electrodes made of other materials, which is also supposed to be a factor of the improvement in sensitivity.

[0012]

Among these methods for measuring NADH or NADPH, the optical method requires a complicated and expensive measuring device as compared with the electrochemical method. Among the above optical methods, in the method of measuring ultraviolet light absorption such as NADH, since the wavelength region of light is ultraviolet, both the light source and the detector are inexpensive and have excellent durability. There is a problem that it is difficult to obtain. The N
In the method of measuring the fluorescence of ADH or the like itself or the method of measuring the fluorescence of resorufin generated by the reaction with NADH or the like, for example, when measuring urine, the influence of a fluorescent substance such as a protein or bilirubin contained as a coexisting substance is used. There is a problem that it is difficult to avoid.

As described above, the electrochemical method has an advantage that the measurement can be easily performed using a simple and inexpensive measuring device. However, although the sensitivity has been improved by the above-described various attempts, the lower detection limit concentration is still insufficient at about 0.1 to 1 mmol / L, which is inferior to optical methods in terms of sensitivity. The lower detection limit of NADH or NADPH for enzyme activity measurement is desired to be about 10 μmol / L. However, in a conventional electrochemical measurement method having low sensitivity, it is difficult to measure enzyme activity or enzyme substrate concentration. It is practically difficult.

Therefore, an object of the present invention is to provide a high sensitivity NA
N capable of measuring the concentration of DH or NADPH
It is to provide a method for electrochemical measurement of ADH or NADPH.

[0015]

To achieve the above object, a method for measuring NADH or NADPH according to the present invention comprises:
NA using at least two electrodes, a working electrode and a counter electrode
A method for electrochemically measuring the concentration of DH or NADPH, wherein NADH or NADP is applied to the working electrode.
A constant potential is applied in a potential range in which H electrolysis does not occur, and then a pulse potential is applied to the working electrode in a potential range in which NADH or NADPH electrolysis occurs, and 10 to 1000 milliseconds after applying the pulse potential This is a method of measuring a current flowing between the working electrode and the counter electrode later.

In the measuring method of the present invention, it is preferable that a series of operations in the measuring method is one cycle, and this operation is repeated for two or more cycles, and an average value of the current measured in each cycle is calculated.

At this time, it is preferable to repeat the operation for two cycles or more continuously in time. This allows
At the end of one cycle and the beginning of the next cycle,
From the pulse potential in the potential range where electrolysis such as NADH occurs, to N
This is because the potential fluctuates substantially instantaneously to a constant potential within a potential range where electrolysis such as ADH does not occur, and is maintained at the constant potential, thereby stabilizing the electrode.

That is, in FIG. 2, T is the time of one cycle, τ is the pulse potential application time, and (T−τ) is the constant potential application time. As described above, the electrode may be stabilized by fluctuating the potential substantially instantaneously from the pulse potential to the constant potential.Therefore, the cycle is repeated continuously in time, and the average value of the measured current is calculated. In the case of calculating and converting to a concentration, (T−τ) is desirably a time required for the electrode to stabilize. In FIG. 2, E
i indicates the constant potential, ΔE indicates a difference between the pulse potential and the constant potential, and I indicates a current flowing between the working electrode and the counter electrode.

The constant potential is -500 to 200 mV
(Vs. Ag / AgCl), and the application time of the constant potential is preferably a time during which the electrode is stabilized.
Specifically, the range is preferably from 1 to 120 seconds, particularly preferably from 2 to 10 seconds. The pulse potential is 10
It is desirable to be 0-1000 mV (vs. Ag / AgCl). The constant potential, the constant potential application time and the pulse potential, the temperature of the measurement sample, pH, since a suitable value varies depending on the material and type of the electrode to be used,
It is preferable to select an optimal combination from necessary sensitivity, S / N ratio, and the like. The time from the application of the pulse potential to the measurement of the current passing between the working electrode and the counter electrode is the time in the above-mentioned predetermined range.
Since a suitable value varies depending on the pH, the material and type of the electrode used, and the like, it is preferable to select an optimal value from the required sensitivity, S / N ratio, and the like. The material of the working electrode is not particularly limited, and if necessary, an electrode of a conventionally known material such as plastic formed carbon, glassy carbon, gold, platinum, or silver can be used. Among these, plastic-formed carbon is preferred. The same applies to the form of the working electrode,
It may be a probe-type solid electrode, an electrode using a printing technique, or a paste electrode such as a carbon paste electrode. On the other hand, the counter electrode is not particularly limited, and a conventionally known one can be used. For example, a platinum electrode, glassy carbon, gold, silver, printed carbon, carbon paste, or the like can be used. In the present invention, it is preferable to use a reference electrode, for example, silver / silver chloride (Ag / AgCl).
Electrodes and the like can be used.

When it is desired to avoid the influence of various interfering substances, it is preferable to cover the working electrode with an interference removing film. The interference removal film refers to electrode contamination, NA
This is a membrane for reducing the influence of electrochemically active species other than DH or NADPH. For example, when a bodily fluid such as blood, urine, or any other fluid is used as a measurement sample, it is necessary to prevent protein from adsorbing to the electrode surface. Examples include a dialysis membrane, a cellulose acetate membrane, and a Nafion membrane for blocking.

NADH or NAD of the measuring method of the present invention
In the electrochemical concentration measurement of PH, the lower detection limit is 10 μmo
1 / L is possible. Therefore, by measuring NADH or NADPH by the measurement method of the present invention, it becomes possible to measure the activity of an enzyme having NADH or NADPH as a coenzyme and the concentration of an enzyme substrate.

[0022]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Embodiments of the present invention will be described with reference to examples and comparative examples.

The measurement conditions in the following examples and comparative examples are as follows. First, an ordinary three-electrode system (probe-type solid electrode) was used as an electrode. The working electrode is plastic formed carbon (PFC)
An electrode (φ3 mm: manufactured by BAS Co., Ltd.) was used. A platinum electrode (φ1 mm: manufactured by Ichimura Metal Co., Ltd.) was used as a counter electrode, and silver / silver chloride (Ag / A) was used as a reference electrode.
gCl) electrodes (manufactured by BAS). The working electrode surface is provided with a dialysis membrane (Seamless Cellulose Tub) to remove interference such as proteins.
ing, an import distributor: Sanko Junyaku). As a measurement solution, a solution prepared by using a 0.1 mol / L phosphate buffer as a base solution and adjusting the NADH concentration to 5 to 200 μmol / L was used. The temperature of the measurement solution is 2 during the measurement.
It was adjusted to be constant at 5 ° C.

(Example 1) Under the above conditions, NADH was measured using a pulse potential. The initial potential applied to the working electrode before application of the pulse potential is -250 mV (v
s. (Ag / AgCl). The pulse potential is a potential when the applied potential is increased in a pulsed manner (substantially instantaneously) from an initial potential to a potential that can oxidize NADH. In this embodiment, the pulse potential is 500 mV (vs. Ag / AgCl).
And Then, the current value 40 ms after the application of the pulse potential was measured. The time width for applying the pulse potential was 40 milliseconds. These operations are defined as one cycle, and the time of one cycle is set to 3 seconds (that is, the constant potential application time is set to 3000-40 = 2960 milliseconds).
The operation of 0 cycle was continuously performed temporally, and the average value of the current of 10 measurements was calculated. The average value of this current and NA
The relationship with the DH concentration is shown in the graph of FIG.

From the above results, in this embodiment, NADH
Good linearity (correlation coefficient: r = 0.999) was obtained up to a concentration of 1.0 mmol / L. Then, the lower detection limit concentration of NADH was determined by calculation.
1 / L. The lower detection limit concentration was calculated on the calibration curve (regression line of the current-concentration plot) as the value of X (NADH concentration) which gives the following equation (Equation 1).

(Equation 1) Y = b + 3Sy / x X: NADH concentration Y: Current value b: Intercept of calibration curve Sy / x: Statistics Sy / x = ｛Σ (yi-ya) ² /
n-2｝ ² yy: i-th measured value (current value) ya: average value of yi n: number of measurement points (number of yi)

(Comparative Example 1) As shown below, the NADH concentration was measured by a method of measuring a steady-state current using a constant potential without using a pulse potential.

The working electrode has a potential of 650 mV (vs. Ag / AgC).
l) is applied, and the NADH concentration is measured.
The relationship between ADH concentration and current was examined. Conditions other than the applied potential were the same as those in Example 1 using the pulse potential. The results are shown in the graph of FIG. As shown in FIG.
good linearity up to mol / L (correlation coefficient: r = 0.9
99) was obtained, but the lower detection limit concentration of NADH calculated by the same method as in Example 1 was 37.7 μmol / L.
Met.

From the results of Example 1 and Comparative Example 1, according to the measurement method of the present invention using the pulse potential, compared with the lower detection limit concentration, the steady-state current measurement using the constant potential is about It can be said that a four-fold improvement in sensitivity was obtained.

(Example 2) D-3-hydroxybutyric acid was measured using D-3-hydroxybutyrate dehydrogenase (HBDH). This enzymatic reaction is represented by the following formula (Formula 2). As can be seen from this equation, the D-3-hydroxybutyric acid concentration (substrate concentration) can be determined by measuring the amount of NADH produced.

(Formula 2)

Under the same conditions as in Example 1, the current value after the application of the pulse potential was measured, and the relationship between the measured current value and the D-3-hydroxybutyric acid (substrate) concentration was examined. The results are shown in the graph of FIG. As can be seen from the graph in FIG.
Good linearity up to 70 μmol / L (correlation coefficient: r =
0.990). The lower limit of detection of the substrate concentration calculated by the same method as in Example 1 was 9.23 μmol.
/ L.

[0033]

As described above, the measuring method of the present invention is a quick and simple electrochemical measuring method,
H or NADPH at a lower detection limit concentration of 10 μmol /
It is a method that can be measured with a high sensitivity of L or less. That is,
By applying the measurement method of the present invention, a trace amount of NADH or the like can be measured quickly and simply, and the activity of an enzyme using NADH or the like as a coenzyme and the concentration of its substrate can be measured quickly and simply.

[Brief description of the drawings]

FIG. 1 is a graph showing the relationship between current and NADH concentration in one embodiment of the present invention.

FIG. 2 is an explanatory diagram of an example of the measuring method of the present invention.

FIG. 3 is a graph showing the relationship between current and NADH concentration in a comparative example.

FIG. 4 is a graph showing the relationship between current and substrate concentration in another example of the present invention.

Claims (12)

[Claims] 1. A method for electrochemically measuring the concentration of reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH) using at least two electrodes, a working electrode and a counter electrode. And the working electrode is N
A constant potential is applied within a potential range where electrolysis of ADH or NADPH does not occur, and then NADH is applied to the working electrode.
Alternatively, a pulse potential is applied within a potential range in which NADPH electrolysis occurs, and 10 to 100 after applying the pulse potential.
A method for measuring NADH or NADPH, which measures a current flowing between the working electrode and the counter electrode after 0 millisecond. 2. A series of operations in the measurement method according to claim 1 is defined as one cycle, and this operation is repeated for two or more cycles, and an average value of current measured in each cycle is calculated and converted into a NADH concentration or a NADPH concentration. Measurement method to be performed. 3. The method according to claim 2, wherein a series of operations of two or more cycles are performed continuously in time. 4. The measuring method according to claim 1, wherein the time for applying a constant potential within a range in which electrolysis of NADH or NADPH does not occur is a time required for the electrodes to stabilize. 5. A constant potential within a range in which electrolysis of NADH or NADPH does not occur is -500 to 200 mV (v
s. (Ag / AgCl) is the measurement method according to any one of claims 1 to 4. 6. A time for applying a constant potential within a range in which electrolysis of NADH or NADPH does not occur is 1 second to 120 seconds.
The measuring method according to any one of claims 1 to 5, which is in a range of seconds. 7. The measuring method according to claim 1, wherein the time for applying a constant potential within a range in which electrolysis of NADH or NADPH does not occur is in a range of 2 seconds to 10 seconds. 8. A pulse potential of 100 to 1000 mV (v
s. (Ag / AgCl). The method according to claim 1. 9. The measuring method according to claim 1, wherein the working electrode is a plastic formed carbon electrode. 10. The measuring method according to claim 1, wherein the working electrode is covered with an interference removing film. 11. An enzyme activity for measuring the activity of an enzyme having NADH or NADPH as a coenzyme by measuring the concentration of NADH or NADPH using the method according to any one of claims 1 to 10. Measuring method. 12. An enzyme substrate for measuring the concentration of a substrate of an enzyme having NADH or NADPH as a coenzyme by measuring the concentration of NADH or NADPH by using the method according to any one of claims 1 to 10. How to measure the concentration.