JPH0779714B2 - Method for quantifying NADH and / or NADPH by visible part absorption method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of application] The present invention is capable of highly sensitively measuring NADH and / or NADPH (hereinafter referred to as NAD (P) H when they are collectively described) by a visible light absorption method. It is about how to do it.

[Prior Art] The ultraviolet absorption method is the most general method for quantifying NADH and NADPH.

[Problems to be Solved by the Invention] However, the above-mentioned ultraviolet absorption method is susceptible to turbidity, and cannot obtain sufficient sensitivity because the molecular absorption coefficient of NAD (P) H is small. It had drawbacks.

Therefore, NAD (P) H can be led to a reducing color-forming dye (formazan color-developing method) by a catalyst such as diaphorase or phenazine methoyl sulphate (PMS) to increase the sensitivity or measure in the visible region. Many attempts have been made to develop such technologies.

However, even in such a formazan coloring method, there is another problem that the generated coloring substance contaminates the flow path of the analytical instrument. In addition, in this method, the reduced dye substance may become insoluble and may not show sufficient color development, so it is necessary to add an appropriate dispersant to the reaction solution, which is troublesome both in terms of reaction composition solution and labor. Is the way. Further NAD
When PMS is used as the (P) H oxidation catalyst, other reducing substances mixed in the reaction system may be present because PMS is easily reducing.
It is known to act directly on PMS and show high background absorption. Therefore, no matter which procedure is adopted in the subsequent steps, it is not preferable to use PMS as the oxidation catalyst in view of the intended purpose of realizing a highly sensitive quantitative method.

The present invention has been made in order to solve the above problems of the prior art, and the object thereof is to produce NADH and highly sensitive NADH without causing a problem of contaminating the flow path of an analytical instrument. And / or to provide a method capable of quantifying NADPH.

[Means for Solving Problems] The present invention capable of achieving the above-mentioned object is NADH and / or NADP.
In a sample containing H, in the presence of flavins, NAD (P) H-
Peroxidation by allowing at least one enzyme selected from the group consisting of FMN oxidoreductase, NADH-FMN oxidoreductase, and NADPH-FMN oxidoreductase to act and autoxidizing the resulting reduced flavins in the presence of oxygen. Hydrogen is generated, and the resulting hydrogen peroxide is caused to act on a chromogen and peroxidase to cause oxidative coloring,
NADH and / or
Or NADH and / or N having the gist of quantifying NADPH
This is a quantification method by the visible part absorption method of ADPH.

[Operation] The present invention is configured as described above, but basically, in order to avoid the problem of contaminating an analytical instrument, a method of leading to oxidative coloring by peroxidase through the production of hydrogen peroxide is basically adopted. , As a means for further producing hydrogen peroxide, instead of PMS, in the presence of flavins NADH-FMN oxidoreductase, NADPH-FMN oxidoreductase,
Using at least one enzyme selected from the group consisting of NAD (P) H-FMN oxidoreductase, NADH, NADPH, etc. were quantified. That is, in the presence of flavins in a measurement system containing NAD (P) H, the above various oxidoreductases are allowed to act, and the reduced flavins produced are automatically oxidized by molecular oxygen to produce hydrogen peroxide. (P) H is all converted to hydrogen peroxide. Then, oxidative coloring is performed by causing the chromogen and peroxidase to act on the generated hydrogen peroxide, and the theoretical recovery rate at this time is 100%.
% Was found.

The NADH-FMN oxidoreductase is an enzyme that specifically acts on the NADH-FMN system to promote the redox reaction,
NADPH-FMN oxidoreductase means an enzyme that specifically acts on the NADPH-FMN system to promote a redox reaction. NAD (P) H-FMN oxidoreductase is NA
It means an enzyme that specifically acts on both the DH-FMN system and the NADPH-FMN system to promote the redox reaction.

The flavins used in the present invention include flavin adenine dinucleotide (FAD) and flavin mononucleotide (FAD).
MN), riboflavin, etc., but FMN is most suitable. Various oxidoreductases used in the present invention are generally isolated from luminescent bacteria, and those obtained from Vibrio harveyii are most suitable for the method of the present invention.

In the method of the present invention, NAD is used with FMN as flavins.
Examples of the reaction system for quantifying H include the following (1) to
It is expressed as in equation (3).

FMNH ₂ ＋ O ₂ → H ₂ O ₂ ＋ FMN… (2) However, in the above formula (3), 4-aminoantivirine (4-AA) and N-ethyl-N- (2-hydroxy-) are used as chromogens.
3-Sulfopropyl) m-toluidine sodium (TOO
It shows that hydrogen peroxide (H ₂ O ₂ ) according to the above formula (2) is produced non-enzymatically at an extremely high rate. Since the quinone imine dye, which is a coloring substance produced, has a maximum absorption at a wavelength of 555 nm, the absorbance may be measured at the above wavelength.

Next, in the method of the present invention, when FNA is used as the flavins and NADPH is quantified, the reaction system is represented by the following formulas (4) to (6).
It can be understood in the same manner as in the case of the reaction system of.

FMNH ₂ ＋ O ₂ → H ₂ O ₂ ＋ FMN… (5) As is clear from the above meaning, by using either NADH-FMN oxidoreductase or NADPH-FMN oxidoreductase, each of NADH and NADPH can be individually quantified, NAD (P) H-FMN oxidoreductase. When is used, the mixture of NADH and NADPH can be quantified as the sum.

In carrying out the method of the present invention, the concentration range of the enzyme, substrate, reagent, etc. used may be set according to the case and is not limited at all, but the preferable range is as follows.

Various oxidoreductases… 5 to 1000 mU / ml peroxidase… 0.5 to 10 U / ml FMN… 1 to 10 μM 4-AA… 0.01 to 0.1% TOOS… 0.02 to 0.2% The buffer solution used is not limited at all and can be measured. What is conventionally used may be selected according to the object to be measured and the measurement system, but the pH should be set in the range of 5-9. Further, 4-AA, TOOS and the like are chromogens as hydrogen donors in the peroxidase reaction, and other chromogens can be used depending on the set pH and the selected buffer solution.

[Examples] The present invention will be described in more detail with reference to Examples below, but the following Examples are not intended to limit the present invention.

Example 1 Bile acid (sodium cholate) was quantified using NADH-FMN oxidoreductase. That is, sodium cholate can be quantified by measuring NADH produced by the following formula (7) according to the above formulas (1) to (3).

The reagents used at this time are as follows.

Reagent 1 Phosphate buffer (100 mM), pH 7.0… 0.185 ml 3α-hydroxysteroid dehydrogenase (75 U / ml)… 0.1 ml NAD ⁺ (30 mM)… 0.1 ml NADH-FMN oxidoreductase (0.6 U / ml)… 0.1 ml FMN (1 mM)… 15 μl Reagent 2 Peroxidase (90 U / ml)… 0.1 ml TOOS (0.4%)… 0.3 ml 4-AA (0.2%)… 0.3 ml Phosphate buffer (pH 7,100 mM)… 1.7 ml Reagent 1 above After adding 0.1 ml of sodium cholate to the above, Reagent 2 was mixed after 1 minute or more had elapsed. And 37
The absorbance at a wavelength of 555 nm was measured at a temperature of ° C.

The molecular extinction coefficient of quinone imine dye produced by the action of peroxidase in the above chromogen is 14.66 mM ^-1 from the results of the addition experiment of hydrogen peroxide and uricase (3 U / ml) and uric acid standard solution at existing concentrations. It was shown to be cm ⁻¹ . Although pH 7.0 at the time of measurement was not the optimum pH for 3α-hydroxysteroid dehydrogenase, the reaction was terminated within 5 minutes by increasing the amount of enzyme.

The amount of 3α-hydroxysteroid dehydrogenase can be reduced by substituting phenol compounds for TOOS as a chromogen and allowing the reaction to proceed under alkaline conditions.

The calibration curve prepared according to Example 1 is shown in FIG. That is, Fig. 1 shows sodium cholate concentration and absorbance (555 nm).
It is a graph which shows the relationship with.

According to the calibration curve shown in FIG. 1, sodium cholate could be accurately measured in the final concentration range of 0.2 to 50 μM. From these results, it can be seen that the method of the present invention has a sensitivity capable of measuring a very small amount of cholic acid in serum. Furthermore, the recovery rate at this time is 100% from the value of the molecular extinction coefficient.
Was confirmed.

Example 2 Glucose-6-phosphate (hereinafter referred to as G-6-P) was used as NADP.
It was quantified using H-FMN oxidoreductase. That is, the NADPH generated by the following equation (8) is converted into the above (4)-
G-6-P can be quantified by measuring according to the formula (6).

The reagents used at this time are as follows.

Reagent 1 Phosphate buffer (100 mM), pH 7.0 0.185 ml G-6-P dehydrogenase (30 U / ml) 0.1 ml NADP ⁺ (30 mM) 0.1 ml NADPH-FMN oxidoreductase (0.6 U / ml) 0.1 ml FMN (1mM)… 15μl Reagent 2 Peroxidase (90U / ml)… 0.1ml TOOS (0.4%)… 0.3ml 4-AA (0.2%)… 0.3ml Phosphate buffer (ph7,100mM)… 1.7ml Reagents above After adding 0.1 ml of G-6-P to 1, the reagent 2 was mixed after 1 minute or more had elapsed. Then, the absorbance at a wavelength of 555 nm was measured. Molecular extinction coefficient 14.66 mM _-1 cm _-1
The recovery rate obtained from the above was 100%.

The calibration curve prepared according to Example 2 is shown in FIG. That is, FIG. 2 is a graph showing the relationship between the G-6-P concentration and the absorbance. As is clear from the results of FIG. 2, G-6-P
It can be seen that linearity is exhibited in the concentration range of 0.5 to 40 μM. Thus, the G-6-P concentration can be measured with high sensitivity according to the method of the present invention.

[Effects of the Invention] As is clear from the above results, NADH and / or NADP
By converting H into hydrogen peroxide without excess or deficiency and leading to oxidative color development by peroxidase reaction, NADH can be highly sensitive without the problem of contaminating the flow path of the analytical instrument.
And / or NADPH could be quantified.

[Brief description of drawings]

FIG. 1 and FIG. 2 show the calibration curves obtained in Examples 1 and 2, respectively.

Claims (1)

[Claims] 1. A sample containing NADH and / or NADPH is selected from the group consisting of NAD (P) H-FMN oxidoreductase, NADH-FMN oxidoreductase and NADPH-FMN oxidoreductase in the presence of flavins. At least one enzyme is allowed to act, and the resulting reduced flavins are auto-oxidized in the presence of oxygen to generate hydrogen peroxide, and the resulting hydrogen peroxide is allowed to act with a chromogen and peroxidase to be oxidized. A method of quantifying NADH and / or NADPH in a sample by a visible light absorption method, which comprises quantifying NADH and / or NADPH in a sample by causing a color to develop and measuring its absorbance.