JPS6043398A - Determination of nad(p)h - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION BACKGROUND OF THE INVENTION Technical Field The present invention relates to the production of NAnH or NAD by a novel enzymatic method.
PH (hereinafter referred to as rNAD(P)H,J) relates to the Yodosode method.

In living organisms, there are many dehydrogenase reactions that use NAD (cotinamide adenine dinucleotide) or NADP (cotinamide adenine dinucleotide phosphate) as coenzymes, and many enzymes that react with these reactions have also been isolated and purified. There is. Therefore, using NAD(P)H (reduced nicotinamide adenine dinucleotide P or reduced nicotinamide adenine dinucleotide phosphorus 1~) as a signal and 1-, the reactive group J- involved in the dehydrogenase reaction can be quantified. Alternatively, dehydrogenase activity measurement, as well as quantitative determination of reaction substrates or enzyme activity measurements of other enzyme reaction systems that can be linked to this dehydrogenase reaction system, are widely performed in the field of clinical chemistry analysis in relation to pathological conditions. It is being said.

Table 1 shows an example of clinical chemistry analysis using NAD(P)H as the final signal.

Table 1 Prior art A typical method for determining NAD(P)I is the colorimetric method (λ = :MOnm), which measures the increase or decrease in NAD(P)H by oxidoreductase reaction. How to measure with (
Uv method), also the Keiku method (λ.X-365nm, λe
m-460 nm), NAD (P
) methods using diaphorase or phenazine methosulfate (PMS) and a tetrazolium salt for the reaction and measuring in the visible region are known. The TJV law is
For example, NAD(P))I is treated with glutamate dehydrogenase (hereinafter referred to as [GLDI
The absorbance of NAD(P)H at 340 nm was reduced by allowing lactate dehydrogenase to act in the presence of pyruvic acid (called pyruvic acid) or lactate dehydrogenase in the presence of pyruvic acid. However, this method has the disadvantage of poor sensitivity due to the low molar extinction coefficient of NAD(P)Tl. Although the light beam method has higher sensitivity than the IJv method, it has the disadvantage of requiring the use of a less widely used fluorescence photometer. In addition, the visible part measurement method also
S has problems such as being easily autooxidized and being unstable to light.

On the other hand, as a method for converting into a signal of hydrogen peroxide (NAD(P)H) and quantifying it, there is a method of reducing oxygen through methylene sol and leading to hydrogen peroxide, but the M element reaction of this oxygen is slow. It has the following drawbacks.

SUMMARY OF THE INVENTION The purpose of the present invention is to solve the above-mentioned problems, and attempts to achieve this purpose by enzymatically treating a sample and regularly testing the signal from the contained NAD(P)H hydrogen peroxide. It is something to do.

The NAD(P))I electric resistance stitching method according to the method of the present invention includes the steps (A) to (DJ) below.

(A) A sample to be passed through NAD(P)11 at a constant rate is treated with GLDH in the presence of ammonia and α-I (G to generate elm glutamic acid corresponding to NAD(P)Hl in the sample) (I) A step of causing glutamate oxidase (hereinafter referred to as "GLXD") to act on the sample in the presence of oxygen to generate hydrogen peroxide in response to glutamate in the sample, (C) hydrogen peroxide. Q)) Calculating NAD(p')T(t) from the measured production amount or production rate of hydrogen peroxide.

The oxygen reaction formula in the method of the present invention is as follows.

L-glutamic acid + NA, D (P) ”NHa −
) (1-KG+H202 Effect) Thus, the method of the present invention is effective for NAD(P)H wholesale T, DH
The method is characterized in that NAD(P)H is quantified by the signal of hydrogen peroxide by converting L-glutamic acid into hydrogen peroxide using GLXT).

By using hydrogen peroxide as a signal, detection systems such as chemiluminescence method, spectroscopic method, and electrode method that are easy to operate and capable of highly sensitive quantification can be easily used to quantify NAD(P)H. It became possible to apply it.

Conventionally, a method for quantifying NAD(P)H by converting it into a hydrogen peroxide signal through a specific enzyme reaction has not been established, and such a method has been established for the first time with the present invention.

The method of the present invention can be carried out by employing a continuous automatic analysis system using a reactor comprising an immobilized enzyme. Therefore, according to the present invention, it has become possible to quantify NAD (P) H with high sensitivity and in a short time using such an automatic analysis system.

3. Detailed Description of the Invention Sample to be analyzed The sample to be analyzed in the method of the present invention is an aqueous sample containing NAD(P))T to be quantified, and the NAD(P)H in the sample is In addition to what is originally contained therein, it also includes NAD(P)H produced by the redemption of NAD(p) by a dehydrogenase reaction. Therefore, the purpose of the present invention is to quantify NAD(P))T in a sample.
It also includes measuring the enzyme activity of the enzyme.

substance, glucose-6-phosphorus #/glucose-6-phosphate dehydrogenase, glucose/glucose dehydrogenase, UD
PG/TJr) PG dehydrogenase, phosphogluconate/phosphoglyconate dehydrogenase, galactose/galactose dehydrogenase, alcohol/alcohol dehydrogenase, glycerol/glycerol dehydrogenase, glycerol-
Enzyme numbers such as 3-phosphate/glycerol-3-phosphate dehydrogenase, incitric acid/isocitrate dehydrogenase, steroid r/steroid dehydrogenase, aldehyde/aldehyde dehydrogenase [1,1,1,' 1, [1,2゜1]
, [1,3,1], [1,4,1] or [1,5,1
Various dehydrogenase systems belonging to 3 are exemplified.

Furthermore, the target samples of the present invention include those for the purpose of measuring reaction substrates or reaction reagents in a reaction system consisting of a single or multiple steps that can be linked to the dehydrogenase system as described above. An example of such a reaction system is, for example, glucose and ATP/hexokinase for the glucose-6-phosphate membrane hydrogen enzyme system;
and g) vco-,
Examples of the 3-phosphate membrane hydrogen enzyme system include glycerol and ATP/glycerose←+-etriazylglycerol/lipase, and creatinine,
Creatinine kinase, α-amylase, etc. can also be measured (see Japanese Patent Publication No. 57-31439).
.

The enzymatic reaction of the dehydrogenase system or the process operation of the reaction system connected thereto before the measurement operation of NAD (P) (according to the present invention) should be carried out by employing methods or conditions suitable for each reaction system. Furthermore, if these reaction systems do not interfere with the reaction of GLD)I in the first step of the present invention, a configuration in which the reaction proceeds simultaneously with the first step can be adopted.

GLDT (reaction The first step in the present invention is NAD (
The reaction for producing L-glutamic acid corresponding to P)H is a known reaction per se, and in application to the present invention, the following G
No special methods or conditions are required other than adding technical considerations to the LXD reaction. For this reaction,
Descriptions of known books and reviews, for example, HoIT
, l (by Ergmeyer, [Methods of
&zy+natlc Analysis j 2nd
, EnglishFJItlon, Vol. 4.
p, 2054-2055, Academic P
ress.

Inc., (1974), etc. can be referred to.

In other words, there are no particular restrictions on the origin or origin of GLDH, and the strain name L-glutamine #: NAD + oxidoreductase (deaminating) (EC1, 4, 1
, 2), L-glutamic acid: NAD (P) oxy-P reductase (deaminating) (EC1, 4,
1,3), L-glumemic acid: NADP+oxidoreductase (deaminating) (EC1,4,1,
The enzyme 4) is selected and used according to the measurement purpose.

Ammonia and α-KG as reaction substrates are used in molar excess, but the optimum amounts can be easily determined experimentally depending on the reaction conditions, enzyme size, etc. When adding ammonia to reaction β), considering the influence on p)I in the reaction system, add water-soluble neutral groups that do not interfere with the enzymatic reaction, such as (ammonium IIiC acid, ammonium l'!\ acid,
This may be carried out using ammonium carbonate or the like in a solid form or in a solution form.

The reaction may be carried out in the presence of ADP for phosphorylation as a buffer, usually at pH 7-9.5.

GL, XD reaction The reaction for producing hydrogen peroxide corresponding to glutamic acid by GLXT, which is the second step in the present invention, is known (Agric, I(iol, Chem, V
ol, 47, N001, p, 179-182 (198
3) ), and a method for quantifying glutamic acid using this reaction, March 10, 1980, published by the Japan Agricultural Chemistry Society, Collection of Lecture Abstracts (1983 Convention), p. 184, 2L- 1, and more details in the patent application 1982.
-145346 specification can be referred to.

That is, as GLXD, one having high substrate specificity for L-glutamic acid is more preferably used.

G LXD is conventionally produced by Strezotomyces violetscens (see Japanese Unexamined Patent Publication No. 57-43685), and Strezotomyces sp.
19-6 (Microtechnology Research Center No. 6560, ATCC'393
43) What is produced by (Agric, Biol, Ch.
Em,.

Vol, 47, No. 6, p1323-1328 (
1983)) is known. The latter has better substrate specificity and stability, especially the optimal pH of GT and DH, than the former.
The latter enzyme is more preferably used in the present invention because of its excellent pH stability and the like, and hereinafter the latter enzyme will be referred to as GLXD. A method for preparing this enzyme is shown in the Reference Examples below.

The #elementary reaction of GLXD is usually at pTI5-10 (
1]) Performed in an appropriate buffer.

Measurement of Hydrogen Peroxide Chemiluminescence analysis, electrochemical analysis, optical analysis, spectroscopic analysis, etc. are known as methods for measuring the rate of formation of hydrogen peroxide. (For example, Journal of the Society of Organic Synthetic Chemistry, Volume 39, No. 7, p659-666 (
(1981)). In the present invention, any of these methods can be applied, but from the purpose of the present invention, it is preferable to adopt a system that detects hydrogen peroxide quickly and with high sensitivity.

Typical methods for measuring hydrogen peroxide are listed below.

First, as a chemiluminescence analysis method, there is a method in which luminol is reacted with hydrogen peroxide in the presence of red blood salt and the amount of luminescence accompanying oxidation is measured. Isoluminol, pyrogallol, bis(2,4,6-) instead of luminol! j
dichloroenyl) oxalate instead of red blood salt.ξ
- Methods using oxidases, hematin, hemin, and chloride are also known.

(Is) (12) As an electrochemical analysis method, a method using a Clark type hydrogen peroxide electrode is generally used. A method in which hydrogen peroxide is reacted with iodine ions in the presence of a catalyst such as peroxidase or molybdate and measured using an iodine ion electrode can also be used.

As a fluorescence analysis method, homovanillic acid is reacted with hydrogen peroxide in the presence of noso oxidase to produce 2,
A method for measuring the fluorescence intensity of 2'-dihydroxy-3,3'-dimethoxybiphenyl-5,5'-diacetic acid can be mentioned. 4-Monone Neeleen-[-1L-Kikishi≠-
In the presence of the 4 gongs, ℃ peroxide, hydrogen and ashes, - Hiiragi Kaido←
ruichi 2-cho 2′-11 夛一Hea r-low key sea-4cho 3
! -Ichiri 4-Tosaki Tsuze-) -Nie product Lou 5. A method to increase the fluorescence intensity of -5'-diacetic acid is as follows. fluorescin, etc.)
etc. can also be used.

Common spectroscopic analysis methods include the so-oxidase method and the catalase method. In the baroxidase method, the oxidizable coloring agent is treated with flea oxidase (po-oxidase).
This is a method for colorimetric determination of the dye produced by reacting it with hydrogen peroxide in the presence of n). Color formers and 1-sniffing reagents such as 0-dianisidine, 4-methoxy-1-naphthol, 2,2'-azinobis(3-ethylpenzothiazoline)-6-sulfonic acid (ABTS), etc. , 4-aminoantipyrine (4AA) and phenolic compounds (e.g., phenol, p-chlorophenol, 2,4.6-dribromophenol, etc.), aniline-based compounds (e.g., N,N-dimethylaniline (D
3-methyl-2-benzothiazolinone hydrazone and N , a combination reagent with N-dimethylaniline, etc. can be used. Examples of the catalase method include a method in which an aqueous peroxide system is reacted with an alcohol (such as methanol) in the presence of catalase, the resulting aldehyde is introduced into a coloring system, and the absorbance of the dye is measured.

All of these hydrogen peroxide detection systems are known methods, and their respective known operations and conditions may be referred to in their implementation.

Quantification system The present invention basically consists of GLDH reaction, GLXD reaction,
It consists of three steps: hydrogen peroxide detection. Although these process operations can be performed independently for each step in a batch manner, it is more efficient and can be quantified in a short time if performed using a continuous automatic analysis system using a reactor consisting of immobilized enzymes. It can be performed.

Enzyme immobilization methods are broadly classified into adsorption methods, covalent bonding methods, matrix methods, and microcapsule methods, but the selection of immobilization methods, immobilization phases, etc. depends on the automated analysis employed. Depending on the system, a book (for example, edited by Chibata, Chiku, Showa), March 2015, Canada/Japan, @) Published by Kodansha, "Immobilized Enzyme"~'bsbachK, author, J Method
in Enzy+nology J Vol, XL
TV.

Immobilzed hEnzymes, Acad
emic Press, New York 1976, etc.) or review articles (e.g., April 1980, published by Sonankodo, [Biomaterial Science, Volume 1,
Chemistry Special Edition No. 134) p55-67, p69-79
etc.).

We also pledged our automated analysis system.
t.

Written by G.G. [HANDB00KoFENzYMATIc
METH6DsOF ANALYSISJ p445~
612. Marcel T)ekker.

Inc., New York, 1976, [Met
Hods In Enzymology J (supra), etc.).

As an automatic analysis system suitable for carrying out the present invention,
An example of this method is the method disclosed in Publication No. 6-47484.

According to this system, it is possible to quantify NAD(P)H, fix reaction substrates in a dehydrogenase reaction system or a reaction system connected thereto, or measure enzyme activity in a very short time of only a few seconds.

This system supplies the test sample to the immobilized enzyme column,
A luminol-red blood salt-based luminescent agent is mixed with the column-passing liquid containing hydrogen peroxide generated by an enzyme reaction, causing chemiluminescence and guiding it to a flow cell. Alternatively, there is a method of integrating using a photomulti-scillator. The basic equipment configuration is shown in FIG. In FIG. 1, a test sample supply channel 1 is composed of a substrate solution supply channel 2, a sample introduction 1 attached to the supply channel 2, and an immobilized enzyme column 4.

On the other hand, the luminescent agent supply channel 5 is composed of a luminol solution supply channel 6 and a red blood salt supply channel 7 arranged in parallel, and a screw-shaped mixing coil 8 that joins both channels 6 and 7. The buffer solution supply channel 2, the luminol bath solution supply channel 6, and the red blood salt supply channel 7 are connected to the basic ponzo 9.
It is adjusted so that it can be supplied in a predetermined amount n, respectively.

The test sample supply channel 1 and the luminescent agent supply channel 5 are connected to the flow cell 1 ( ) under a means that allows both liquids to mix quickly and uniformly.

The flow cell is connected to the photomultiscillator-1 through the light-receiving surface.
Connected to 1. In the present invention, the Okiya Kayase column is a coupled enzyme column containing at least GLDH and GLXD171^1 quantified enzyme column, and further contains NAD(P).
When the dehydrogenase reaction to produce H or the reaction substrate of the reaction system that can be linked to it is determined, an immobilized enzyme column of the dehydrogenase or the enzyme of the reaction system to be further linked is connected. The amount of each immobilized enzyme at this time may be determined experimentally.

If a reaction substrate or a coenzyme is required for the dehydrogenase reaction or a reaction connected thereto, a supply path for these may be provided as appropriate.

Examples Hereinafter, an example of a more specific embodiment of the present invention will be given,
This is a more detailed explanation. However, these illustrate embodiments and do not limit the technical idea of the present invention.

Example 1 (Quantification of NADPH) An automatic analyzer having the configuration shown in Fig. 1 was prepared, and NADPH
ADPH was quantified.

(]) Preparation of immobilized #prime column Enzyme immobilization was performed according to Weetall's method.

That is, porous alkylamine glass particles (pore size 5o
oX, particle size 80-120 mesh, Pierce Co.)
200 mg of 2.5% glutaraldehye P solution 3
ml, and after degassing, the reaction is allowed to proceed for 30 to 60 minutes at room temperature and atmospheric pressure. Remove the glutaraldehyde solution and wash several times with distilled water. To this, GLD) I% base solution (850
Add 1.0 ml of 0U, Toyobo) or 2.0ml of GLXD enzyme solution (130U, see reference side below), and
Incubate overnight at °C. The reaction solution is removed, washed three times or more with an appropriate buffer solution, and washed with a 1M aqueous sodium chloride solution.

Each of the obtained immobilized enzymes was transferred to an acrylic resin column (
inner diameter] dll) to prepare an immobilized enzyme column. The length of each column is 20 +++a, and G
LDH-GLXD were connected in this order and introduced into device d.

(2) Preparation of reagents Luminol solution Luminol Rua
2.10mM phosphate solution, α-K G 1.4 m
M N ammonium sulfate 3. OmM.

EDTAφ2Na containing 0.53 mM gold 3) Quantitative operation Measurement was performed by injecting 1 μl of an aqueous solution of NADPH with a known concentration using a microsyringe at 1° with the sample solution. The luminol solution, potassium ferricyanide solution, and substrate buffer were fed at a rate of 0.6 ml/min using a peristaltic pump.

The relationship between NAD P H enrichment and chemiluminescent sound was as shown in the calibration curve shown in FIG.

Example 2 (Quantification of NADH) pH 7,'l, 100 ml of 10 mM phosphate buffer
A sample of an NADH solution with a known concentration was quantitated in the same manner as in Example 1 using the solution dissolved in NADH.

The calibration curve showing the relationship between NAD)f concentration and chemiluminescence gate is the third one.
It was as shown in the figure.

Example 3 (Determination of NADPH 1: Colorimetric method) NADPHIO μl of a known concentration was added to the following enzyme coloring solution, reacted for 5 minutes at 27° C., and then colorimetrically measured at a measurement wavelength of 640 nm. The needle reading line showing the relationship between NAP) PH concentration and absorbance is shown in Figure 4.

Oxygen coloring solution 50mM pH 7,5) Liss-HCl buffer 1.4ml11
0mM α-KG, 10mM ammonium sulfate solution 0.
2m1GLDH (1030U/ml) 50 μIGL
XD (6.5 U/ml) 50 μm meter 3.3 ml Example 4 (Glucose quantification) A continuous flow automatic analysis system for glucose and NAD PH quantification as shown in Figure 5 was installed. Created. In Fig. 5, 12 is a sample supply path;
3 is a substrate solution supply path, 14 is a single mixing coil, 15 is a 6-inch dialyzer, and 16 is a hexokinase (HK
)・Glucose-6-phosphate membrane hydrogenase (G6PDH)
co-immobilized enzyme column, 17 is 0LDH immobilized enzyme column, 18 is GLXD immobilized enzyme column, 19 is 4AA
- DMA supply path, 21 is a flow cell (15mn5.550nm colorimeter), and 22 is a recorder.

)IK-06PDH co-immobilized enzyme column is HK2
mg @G6PDH1mg/l ml, pH7.0,
50 mM phosphate buffer was simultaneously immobilized on 200 mg of glass particles using the glutaraldehyde method in the same manner as in Example 1 (e.g.
Q-immohlization). An immobilized enzyme column for GLDH and GLXI was also prepared in the same manner as in Example 1. Column size is HK-
G6PI) H Column 1.5 X 20mm5GLI) H Column 1.5 X 2 (1mm.

GT, XI) column 1.5×In mm.

Reagent 111 is as follows.

Substrate solution: ATP 62.6 mg, NADP 2
6.6 mg, α-KG 5 mg X ammonium sulfate 4.4 mg.

Dissolve 48 mg of magnesium chloride in 100 mg of 50 mM, pH 7.5 phosphate buffer 4-AA-DMA: 42.5 mg of 4-AA,
Dissolve 130 μm of DMA in 0.3 N acetic acid. For regular operation, add the sample (containing a known concentration of D-glucose) from the sample supply path at a rate of 0.23 ml/min, and add physiological saline (
Supply 4-AA-DMA by supplying 1.2 ml/min of substrate solution and 0.6 ml/min of air from the substrate solution supply path by supplying 1.6 ml/min of air and 0.6 ml/min of air. 4AA-DMA was fed from the channel to the analysis system at a feed rate of 0.16 m1/min.

The calibration curve for glucose emplacement thus obtained is the sixth
It was as shown in the figure.

Example 5 (Ij1 of pyruvate) 0-8mM pyruvate-0.1M phosphate buffer (pH 7)
, 4) Solution 1 ml K llOmMNAD) I Solution 1
00 μl and lactate dehydrogenase (500 U/ml)
c) μm was added and left at room temperature for 3 minutes to equilibrate, and then 1 μl of the obtained reaction solution was used as a sample for measurement using the apparatus of Example 2. As a substrate buffer,
α-KG 1.4mM, ammonium sulfate 1.5mM, A
10mM phosphate buffer containing 1.8mM DP・2Na (p
H7,1) was used.

A calibration curve showing the relationship between pyruvic acid concentration and chemiluminescent sound is shown in FIG.

Reference example: 500ml 20g of wheat bran and 16+nl of water were placed in a J Erlenmeyer flask, sterilized under pressure at 120°C for 130 minutes, and Streptomyces sp. X-119
-6 (Tetsukoken Closing No. 6560) was inoculated, and the temperature was 2.8”C.
After culturing for 17 days, glaze locks were prepared.

20 bran in each of 6 5 liter Erlenmeyer flasks (
1 g of water and 160 ml of water were added and sterilized at 120° C., then the above-mentioned inserts were aseptically inoculated, and 2.
After culturing at 8°C for 2 days, the temperature was raised to 20°C and cultured for another 2 weeks.

The obtained culture was immersed in 37.51 J of water, filtered and filtered through diatomaceous earth to obtain about 34 liters of crude enzyme solution. @Lr1li for crude enzyme solution! Ammonium was added to 504 saturation, and the resulting precipitate was collected, dissolved in 3.9 liters of 0.02 M acetate buffer (Pil 5.5), and heated at 57°C for a minute. After cooling to below 5°C, add twice the amount of cold ethanol, collect the generated precipitate, dissolve in 2 liters of 0.02 M phosphate buffer (P)I7.4), and incubate overnight in the same buffer. Dialyzed.

The precipitate generated during dialysis was removed, and the supernatant was loaded onto a DEAE-cellulose column (3.5 x 50 cm) equilibrated with 0.02M phosphate buffer (pH 7.4),
Elution was performed with 0.02M phosphoric acid buffer containing M sodium chloride. The eluted active fractions were collected and dialyzed overnight against a 0.05 M acetate buffer (pH 5.5) containing 1.05 M sodium chloride.
-Sepharose CI, -6B column (2X10 cm) was loaded and bathed with 0.05-0.75M sodium chloride solution by linear gradient method. Collect the eluted active fraction,
After dialysis and concentration, Sephadex G-200 column (25
Gel filtration was performed using X 120 am). After collecting and concentrating the active fractions, o, 02M potassium phosphate buffer (
The dialyzed solution was centrifuged, the supernatant was microfiltered, and the supernatant was lyophilized to obtain GLXT) purified specimen (specific activity 55, IU//rng protein, yield 18). .4
%) 30 mg was obtained.

[Brief explanation of drawings]

FIG. 1 shows an example of the equipment configuration of a continuous automatic analysis system for suitably implementing the present invention. Figure 2 shows the calibration curves of NADPH obtained in Examples of the present invention.
The figure shows the same < NADH test tLt line, and the figure 4 shows the same <N
The calibration curve of Ar)PH is shown. FIG. 5 is a flow diagram of the continuous flow automatic analysis system for glucose determination used in the examples of the present invention, and FIG. 6 is a calibration curve of glucose obtained by the system. Stripe 7 is a calibration curve for pyruvic acid obtained in an example of the present invention. Applicant's agent Moromi Inomata (27.) Figure 2 NADPH (Milk M) Figure 3 ADH

Claims (1)

[Scope of Claims] A standard I method for NAD(P)H consisting of the following steps (A) to (Dl). Bottom, NAD(P)H in the sample is removed by the action of glutamate dehydrogenase.
(B) causing glutamate oxidase to act on the sample in the presence of oxygen to generate hydrogen peroxide in response to glutamine V- in the sample; (C) ) A step of measuring the production amount or production rate of hydrogen peroxide; (D) A step of calculating NAD(P)Hi from the measured production amount or production rate of hydrogen peroxide.