JPS60218070A - Quantitative determination of hydrogen peroxide by bacterial luciferase - Google Patents

Abstract

PURPOSE:To measure the coloring intensity of a sample liquid and to determine quantitatively H2O2 with high sensitivity without receiving the influence of the disturbing material in an H2O2-contg. sample by acting lower alcohol, NAD, higher alcohol, etc. as well as specific enzyme and bacterial luciferase to said sample. CONSTITUTION:The lower alcohol such as methanol, ethanol or the like and peroxidase are active to the H2O2-contg. sample to produce aldehyde and aldehyde hydrogenase is acted to said aldehyde in the presence of water and nicotineamide adenine dinucleotide (NAD) or the phosphate thereof (NADH). NAD(P)H-FMN oxidoreductase or diaphrase is acted to the formed NADH or NADPH in the presence of flavine mononucleotide (FMN). The bacterial luciferase is acted to the formed FMNH2 in the presence of straight chain aldehyde of 8-14C and O2. The luminous activity formed in such a way is measured by measuring the increasing speed of luminous intensity and detecting the peak thereof. The exact measurement of the H2O2 even in serum, urine, etc. is made possible with high sensitivity by making use of the higher peak as the concn. of the H2O2 is higher.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a method for quantifying hydrogen peroxide using bacterial luciferase.

(Reference 1. List of techniques) Generally, the quantitative method of biological components that is widely used in clinical testing methods is as follows:
Hydrogen peroxide is generated using various oxidases, peroxidase is allowed to act on the hydrogen peroxide in the presence of a coloring agent, and the intensity of the coloring is measured to quantify biological components. However, in this coloring method (first determined by v!), the concentration of hydrogen peroxide generated from the sample is limited to 10'M, and it is difficult to measure concentrations lower than this.

Although the fluorescence method can increase the sensitivity more than the chromogenic method, it has drawbacks such as being affected by blue discoloration.

In addition, chemiluminescence methods such as luminol reaction and lucigenin reaction can quantify approximately 10"M hydrogen peroxide, but when applied to clinical tests, interfering substances in serum, urine, etc. It is known that sensitivity is significantly reduced due to the influence of

Conventionally, in bioluminescence, bacterial luciferase is used to quantify NADH or NADPH, and NAD-
Although it is known to quantify NADP and further to quantify ATP using firefly luciferase, it has not been known to quantify hydrogen peroxide.

(Purpose of the Invention) Taking these circumstances into consideration, the present inventors have developed various methods for quantifying hydrogen peroxide in order to increase the detection sensitivity, reduce the amount of sample, and measure blood substances that could not be measured conventionally. After extensive research, we have discovered that the bioluminescence method using bacterial luciferase is relatively unaffected by interfering substances in serum and urine, and have arrived at the present invention.

(Structure of the Invention) That is, the present invention (1) allows catalase to act on a sample containing hydrogen peroxide in the presence of an alkyl alcohol, and
) The produced aldehyde is treated with formaldehyde dehydrogenase or aldehyde dehydrogenase in the presence of water and NAD or NADP, and (III) the produced NADH or NADPH is treated with NADH in the presence of FMN.
- Let FMN oxidoreductase, NADPH-FMN oxidoreductase or diaphorase act, (l
v) Allow bacterial luciferase to act on the generated F M NHz in the presence of a linear aldehyde having 8 to 14 carbon atoms and oxygen, and (V) measure the generated luminescent activity to determine hydrogen peroxide in the sample. This is a method for quantifying hydrogen peroxide using bacterial luciferase.

Next, in the method of the present invention, when methanol is used as the alkyl alcohol (4), and when ethanol is used (ω)
is explained using a chemical formula.

H*Ow +CHaOHHCHO+ 2HiO-(1)
NAD枦)H...(2) FMNH2+RCHO+ Oz Misaki pν! -45 and 2y→
h F1FMN 10RCOOH+H20...(4) (B) H202+C2H50HcH3CHO+2H20-(5
) NAD(PIH・(6) Bacterial luciferase P F'MNH2+RCHO+Ohl+FMN+RCOOH
10H20 River (8) (In the formula, RCHO represents a linear aldehyde having 8 to 14 carbon atoms, and RCOOH represents the corresponding carboxylic acid produced from the aldehyde by an oxidation reaction.) That is, the present invention #1 (A ), first, hydrogen peroxide is reacted with catalase in the presence of methanol to produce pomaldehyde. Water and NA are added to the formaldehyde produced.
In the presence of D or NADP, formaldehyde dehydrogenase is allowed to act, and NADN oxidoreductase or NADPH-FMN oxidoreductase or diaphorase is made to act to produce FMNH2. The generated F
MNHs act on bacterial luciferase to emit light in the presence of a linear aldehyde with carbon atoms WI8 to 14 and oxygen. Next, luminescent activity is measured according to a conventional method.

In the present invention (B), first, catalase is allowed to act on hydrogen peroxide in the presence of ethanol to produce acetaldehyde. Add water and NAD or NA to the acetaldehyde produced.
In the presence of DP, aldehyde dehydrogenase is activated to produce NADH or NADPH. generated NAD
In the presence of FMN in H or NADPH, NADH-FMN
Oxidoreductase or NADPH-FMN oxidoreductase is activated to generate FMN H2. The generated F M N Hm is activated by bacterial luciferase in the presence of a linear aldehyde having 8 to 14 carbon atoms and oxygen to emit light. Next, the luminescence activity is determined by a conventional method, for example, by measuring the luminescence peak value using a luminescence measuring device.

In the present invention, the sample containing hydrogen peroxide refers to hydrogen peroxide itself, or a biological sample such as blood pyruvate,
A sample containing hydrogen peroxide produced from lactic acid, glycerol, amino acids, etc.

The alkyl alcohol used in the present invention includes:
Examples include alkyl alcohols having 1 to 3 carbon atoms, such as methanol, ethanol, and propatool. Usually 0.1~IO
This is the concentration of M.

The catalase used in the present invention includes animal liver,
There are those derived from kidneys, red blood cells, and microorganisms.

NAD used in the present invention is an abbreviation for nicotinamide adenine dinucleotide, and its concentration is usually 0.1 to 10 mM.

NADP used in the present invention is an abbreviation for nicotinamide adenine dinucleotide phosphate,
The concentration used is usually 0.1-10mM.

Formaldehyde dehydrogenases used in the present invention include those derived from bovine liver, yeast, and bacteria Pseudomonas, and usually have a final concentration of 100 to 1 unit/-.

Aldehyde dehydrogenase used in the present invention,
Those derived from animal liver and yeast are selected as
D. It is desirable to have specificity for NADP. The concentration used is usually a final concentration of 100 to 1 unit/-.

FMN used in the present invention is an abbreviation for flavin mononucleotide, and the concentration used is usually 1-1.
It is 0 μM.

The NhD)t-FMN oxidoreductase used in the present invention is obtained from the luminescent bacterium Vibrio harveyi and has high specificity for NADH and FMN. The enzyme was developed by Jablonski and Deluca.
(Biochem, 162932-2936.19
77) and Watanaba and Hastlngs (
MOL, C@11°Blochem, 44.181
-187.1e 82).

The amount used is usually 10-0°001 units/-.

The N,ADPH-FMN4 oxidoreductase used in the present invention is also obtained from the luminescent bacterium Vibrio harveyi, and the production method follows the method described in the above-mentioned literature. The amount used is usually 10 to 0.001 units/-.

Examples of linear aldehydes having 8 to 14 carbon atoms used in the present invention include octylaldehyde, nonylaldehyde, decylaldehyde, undecylaldehyde,
Examples include dodecylaldehyde, tridecylaldehyde, and tetradecylaldehyde.

The bacterial luciferase used in the present invention can be used by purifying the crude enzyme obtained from the cells of the luminescent bacteria Vibrio harveyi, Photobacterium fischalei, Photobacterium 7 osphorium, and Photobacterium leiosperum. . The concentration range used is usually 100 to 5 μf/-. The diaphorase used in the present invention includes Escherichia coli,
Some are obtained from yeast and motile organs.

The concentration range used is usually a final concentration of 10 to 1 unit/-.

In the present invention, the buffer used has a pH in the range of 6.5 to 8.0. In particular, 0.05 to 0.2M phosphate buffer or Gudbuff 1- is preferred. When using diaphorase, it is also possible to use a pyrophosphate buffer.

In the method of the present invention, the four enzymatic reactions may be carried out in one step or divided into two or more steps. For example, the method of the present invention (4)
), reaction formulas (1) to (4) may be performed in one step, or reaction formulas (1) and (2) and reaction formulas (3) and (4) may be separated and performed in two steps. .

A conventional method is used to measure the luminescence intensity.

For example, hydrogen peroxide is finally added to the reaction system as a reaction initiation reagent to start the reaction, and the luminescence intensity is measured using a Lumiphotometer TD4000 (manufactured by Labo Science) to determine the change in the increase (rate) of the luminescence intensity. There is a way to read against time. Alternatively, the peak of luminescence intensity may be recorded.

(Effects of the Invention) In the present invention, hydrogen peroxide can be stably quantified using bacterial luciferase without being interfered with by blood substances. In particular, it is possible to measure hydrogen peroxide concentrations as low as 10'M, and is more sensitive than the chemiluminescence method. Moreover, hydrogen peroxide can be measured using near-neutral and non-hazardous chemicals. Furthermore, in the method of the present invention, NADH or NADPH can be determined simultaneously with hydrogen peroxide using the same system.

That is, NADH or NADPH can be measured with a high sensitivity that could not be achieved by conventional absorption methods or fluorescence methods.

(Example) Next, the present invention will be explained in detail with reference to examples.

Example 1 The following reagents were prepared in which the final concentrations of the reagents in the reaction were as follows.

1000 units/- Catalase 0.6M Methanol 0.3 units/lnt Formaldehyde dehydrogenase 0.01164 units/sg NADH-1MN oxidoreductase 300 μM NAD 2.5 μM FMN o, 002% Decylaldehyde (*1) 96.5 μt
/-Luciferase 0.1 M phosphate buffer (pH 7.0) (*1) 0.
It was prepared by adding 0.02% decylaldehyde and 0.5% bovine serum and 0.05% Triton 1-100 as solubilizers to 1M phosphate buffer 1-100.

Add 0.1- of hydrogen peroxide with a known concentration to the above reagent 0.9-, react at 23°C for 60 seconds to emit light, and use Lumi7 Otometer TI) 4000 (manufactured by Labo Science) to emit light. The strength was measured. In FIG. 1, the increase in luminescence intensity is plotted against the hydrogen peroxide concentration as the maximum slope per hour (corresponding to the differential of the luminescence rate).

As is clear from Figure 1, the hydrogen peroxide concentration lO-''~
At a low concentration of 10-'M, the emission intensity is direct! 1! quantified in relation to

Reference Example 1 This shows that bacterial luciferase is less affected by serum than the chemiluminescent method (luminol-peroxidase system).

(1) The following reagents were prepared in which the final concentrations of the reagents in the reaction were as follows.

11.64 milliunits/-NADH-1MN oxidoreductase 96.5 μV/-bacterial luciferase 0.002% Decylaldehyde 2.5 μM FMN OoIM Phosphate buffer (pH 7,0) Control serum diluted to various concentrations in the above reagents and then 2.5X1
0-'MNADH was added and the luminescence intensity was measured. The results are shown in FIG.

As is clear from No. 211, the NADH measurement system using bacterial luciferase is not affected by serum up to a concentration of 1/100 volume.

(2) The following reagents were prepared in which the final concentrations of reaction reagents in luminol chemiluminescence were as follows.

2X10'M Luminol Chemiluminescence/-Peroxidase 0.1 M Hof) Acid Buffer (PH8,0) Control serum diluted to various concentrations in the above reagents and final concentration 10'
M hydrogen peroxide was added and the luminescence intensity was measured. The results are shown in FIG.

As is clear from Figure 3, the luminol-peroxidase system is strongly influenced by serum.

[Brief explanation of drawings]

FIG. 1 shows the relationship between the hydrogen peroxide concentration (M) and the maximum slope of luminescence intensity increase per time (luminescence intensity Io). FIG. 3 shows the relationship between the dilution ratio of added serum and the chemiluminescence intensity due to the luminol-peroxidase system. Figure 1 Peroxyacid L-y'iden (M) 2nd @ fEL blue (it tough)

Claims (1)

[Claims] (1) Catalase is applied to a sample containing hydrogen peroxide in the presence of an alkyl alcohol, (11) formaldehyde dehydrogenase or aldehyde dehydrogenase is applied to the generated aldehyde in the presence of water and NAD or NADP, and (li+) NADH-FMN oxidoredalitase, NADP) (-FMN oxidoredalitase, NADP) (-FMN oxidoredalitase or diaphorase is allowed to act on the generated NADH or NADPH in the presence of FMN, and (1v) the generated F M N H2 has 8 to 14 carbon atoms. Hydrogen peroxide by bacterial luciferase is quantified by causing bacterial luciferase to act in the presence of a linear aldehyde and oxygen, and (V) measuring the luminescent activity produced. Quantitative method.