JPH01257500A - Measurement of enzyme or substrate - 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 [Industrial Application Field] The present invention relates to a method for measuring enzymes or substrates involved in dehydrogenation reactions, particularly enzymes or substrates present in biological samples.

[Conventional technology]

There are broadly two methods for measuring enzymes or substrates involved in dehydrogenation reactions, particularly enzymes or substrates present in biological samples in the field of clinical testing. The first method uses a chemical reaction, and the second method uses an enzymatic reaction.

Since the present invention is a measurement method that utilizes an enzymatic reaction, this will be explained below.

Measurement methods using enzymatic reactions include methods using oxidase,
The present invention is a measurement method related to a method using dehydrogenase. Regarding methods using dehydrogenase, the following three methods are known.

These all measure reduced coenzymes produced by dehydrogenase reactions, typically reduced nicotinamide adenine dinucleotide (NADH) and reduced nicotinamide adenine dinucleotide phosphate (NADPH).

The reaction formula and measurement means are shown below when nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) are used as coenzymes.

Dehydrogenase 1) NADH and NADPH produced in the above reaction are
Measured by ultraviolet absorbance at nm.

2) NADH and NADPH produced in the above reaction are treated with an electron carrier, formazan, and the produced diformazan dye is measured by absorbance in the visible region.

3) An electron carrier is made to act on NADH and NADPH produced in the above reaction in the presence of oxygen, and the produced hydrogen peroxide is measured.

However, method 1) above cannot measure in the visible region, and is therefore limited to measurement in the ultraviolet region.

Moreover, not only do biological samples contain many substances that absorb in the ultraviolet region, but also the turbidity of the biological sample greatly affects the measurement, resulting in poor measurement accuracy.

Method 2) has the disadvantage that the diformazane dye adsorbs to the reaction tube or cell and contaminates the spectrophotometer and other equipment.

In method 3), PMS (phenacine methosulfate) used as an electron carrier easily deteriorates at room temperature,
Problems such as decreased activity remain.

Furthermore, in any of the above methods, the amount of coenzyme added to the measurement system is large, so they tend to be economically inferior.

Furthermore, there is a long-awaited method for more efficiently quantifying the enzyme or substrate by shortening the measurement time.

(Problems to be Solved by the Present Invention) The purpose of the present invention is to eliminate the drawbacks or problems of the above-mentioned conventional methods, and to accurately extract enzymes or substrates involved in the dehydrogenation reaction without contaminating equipment. The object of the present invention is to provide a simple, time-consuming, and economical method of measurement.

[Means for Solving the Problems] The present invention provides a method for measuring an enzyme or substrate involved in a dehydrogenation reaction, in which a reduced coenzyme is produced by allowing a dehydrogenase to act on a substrate in the presence of a coenzyme. This is a method for measuring enzymes or substrates, which is characterized by reacting pyrroloquinoline 1-one and quantifying the hydrogen peroxide produced.

A typical example of the reaction in the method of the present invention using NAD as a coenzyme and glucose-6-phosphate dehydrogenase as a dehydrogenase is as follows.

G-6-P dehydrogenase/NAD” +G-6-P NADH+glucono-δ-lactone-6-phosphate/NA
DH0PQQ→NAD” 10pQQH!・PQQHz
+0g -PQQ+Ht Ox [in the above formula, G-6-P
represents glussu-6-phosphate, PQQ represents pyrroloquinoline quinone, and PQQHg represents the reduced form of pyrroloquinoline quinone] In the present invention, regarding the enzymes and substrates involved in the dehydrogenation reaction to be measured, for example, as substrates, glucose, glucose-6-phosphate, maleide,
3α-hydroxysteroids, lactic acid, etc. Therefore, examples of enzymes include glucose dehydrogenase, glucose-6-phosphate dehydrogenase, maleic dehydrogenase, 3α-hydroxysteroid dehydrogenase, lactate dehydrogenase, etc. In the present invention, these substrates and enzymes contained in biological samples are particularly targeted for measurement.

In the method of the present invention, the steps up to producing the reduced coenzyme, that is, the step of allowing the dehydrogenase to act on the enzyme or substrate involved in the dehydrogenation reaction in the presence of the coenzyme, can be carried out by means known per se. 'It's enough.

Next, the reduced coenzyme thus produced is reacted with pyrroloquinoline quinone to generate hydrogen peroxide, and the hydrogen peroxide is quantified.

Pyrroloquinoline quinone is a recently discovered coenzyme of dehydrogenase, and its chemical name is 4,5-dihydro-4,5-dioxy-IH-pyrrolo(2,3-f)quinoline-2,7,
The origin of the pyrroloquinoline quinone used in the present invention is not particularly limited, and it is known to exist in bacterial cell membranes, mitochondria, etc.
hylotroρh) and acetobacter (^cetob)
(acter) or a synthetic material is used.

The reaction between the reduced coenzyme of the present invention and pyrroloquinoline quinone is usually carried out in an appropriate buffer (eg, Tris-HCl buffer, phosphate buffer).

The pH of the reaction solution is usually in the range of 4 to 10, preferably 5 to 8. The reaction temperature is also not particularly limited, but is usually in the range of 20 to 4.
It is in the range of 0°C.

The concentration of pyrroloquinoline quinone used in the method of the present invention is not particularly limited, and is appropriately set depending on the reaction conditions and measurement time, and is usually 1 minute to 20 minutes, particularly preferably 3 minutes to 8 minutes.

Examples of coenzymes used in the present invention include NAD,
Examples include NADP, and reduced coenzymes produced from this include NADH, NADPH, and the like.

The amount of coenzyme used in the method of the present invention is usually 0.01-2.
The amount used is approximately 000 mM, preferably approximately 0°21 to 20+l'1, but the object of the present invention can be sufficiently achieved even with approximately 20% of the amount used in the conventional method. This is due to the fact that NAD can be used cyclically in the present invention, as will be described later.

Hydrogen peroxide produced in the method of the present invention may be measured by any method as long as the object of the present invention can be achieved, including the electrode method using a hydrogen peroxide electrode, 4-aminoantipyrine and a chromogen in the presence of peroxidase. It is sufficient to use conventional methods such as the oxidative coloring method in which a quinone imine dye is produced by reacting the body, or the catalase coloring method in which catalase is used.

[Action/Effect]

In the present invention, one molecule of pyrroloquinoline quinone is non-enzymatically converted into a reduced coenzyme, such as NADH or NADP.
Reacts with a HI molecule to produce one molecule of hydrogen peroxide.

At this time, since the reaction between pyrroloquinoline quinone and the coenzyme is irreversible, the apparent reaction rate of the dehydrogenase reaction itself becomes faster, and therefore the reaction time required for the measurement in the present invention is shortened.

Furthermore, in the measurement method of the present invention, since NAD can be used cyclically, the amount of NAD added can be reduced compared to conventional methods.

Therefore, according to the method of the present invention, when measuring dehydrogenases or substrates, the drawbacks or problems of conventional methods can be solved, and the method can be easily, quickly, and economically done without contaminating the equipment. This has the effect that the measurement can be carried out at any time.

(Example) The present invention will be described below in more detail using Examples.

Example 1 (Quantification of NAD (P)H) Reagent mixture 1 Pyrroloquinoline quinone (2+*M) 0.21 dT
ris-HCI Ll buffer solution pH 75 (20 m°-1 main mouse-1, 5 m reagent mixture 2 4-aminoantipyrine (10-monitor) 0.1jd
TOO3 (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-totoluidine) (10+wM) 0.1 dl Peroxidase (10011/ml) 0.1
mTris-HCj! Buffer pH 7,5 (20+s °-
R1 Reagent mixture 1 was incubated at 25°C, and after 5 minutes, N'AD (P)H (0,1-2.0mM each concentration) was added and incubated at 25°C for exactly 10 minutes. Make it react.

After 10 minutes, reagent mixture 2 was added, and the absorbance at 550 nm was measured after 3 minutes. The results are shown in FIG.

From the results shown in Figure 1, an increase in absorbance at 550 nm occurs depending on the concentration of NAD (P)H, and at 2.0 m
It can be seen that it is linear up to the M concentration. From this,
It can be seen that NAD (P)H can be quantified by the method of the present invention.

Example 2 [Quantification of glucose-6-phosphate] Reagent mixture 1 Glucose-6-phosphate dehydrogenase (1000 U/ml) 0.1 rd Pyrrolokihanquinone <fil> 0.1 adtT
ria-HCj! Buffer pH 7.5 (20mM)
0.7 rrdlNAD” (2n+
M) mR-1, 〇- Reagent mixture 2 4-aminoantipyrine (10mM) 1.0
dT OOS (1h+M) 1.
0 d peroxidase (10 hM) 1.
0 dlTris-HCJ! Buffer pH 7,5 (20+
mM) 10 d4, Om The above reagent mixture 1 was incubated at 25°C, and 5 minutes later, glucose-6-phosphate (10-100 mg/dt
Add 0.2d of each concentration) and react at 25°C for exactly 15 minutes. Thereafter, reagent mixture 2 was added for 0.8 d, and absorbance at 550 nm was measured 3 minutes later. The results are shown in FIG.

From the results shown in FIG. 2, the absorbance at 550 nm increased linearly depending on the concentration of glucose-6-phosphate.

This shows that the concentration of glucose 6-phosphate can be determined by the method of the present invention.

Example 3 [Quantification of glucose-6-phosphate dehydrogenase] Reagent mixture l Glucose-6-phosphate (IM) 0.1 mPQQ
(2mM) 0.1rdTris
-HCIl buffer pH 7.5 (2 (1 wM) 0.
7 rrrlNAD” (2mM) −
m-'2-1,0m Reagent mixture 2 4-aminoantipyrine (10mM) 1. O
rxlT OOS (lo+wM)
1.0 dl peroxidase (100mM)
1. OWii! Tris-HC/! C/method pH
7,5 (2抛肚--1Od4, Od The above reagent mixture 1 was incubated at 25°C, 5
minutes later glucose-6-phosphate dehydrogenase (1-20
Add 0.2d (U/ml each concentration) and react for exactly 15 minutes at 25°C. After 15 minutes, the reaction was stopped with a 2% 5DS (sodium dodecyl sulfate) solution, 0.8 d of reagent mixture 2 was added, and the absorbance at 550 nm was measured after 3 minutes. The results are shown in FIG.

From the results shown in FIG. 3, it can be seen that the amount of change in absorbance per minute (Δ550 rv/min) increases linearly depending on the concentration of glucose-6-phosphate dehydrogenase. This shows that glucose-6-phosphate dehydrogenase can be quantified by the method of the present invention.

[Brief explanation of the drawing]

FIGS. 1 to 3 are graphs showing measurement results by the measurement method of the present invention. 1○-: NADH -・-: NADPH

Claims (1)

[Claims] In a method for measuring enzymes or substrates involved in dehydrogenation reactions, pyrroloquinoline quinone is reacted with the reduced coenzyme produced by allowing dehydrogenase to act on the substrate in the presence of a coenzyme, and the produced hydrogen peroxide is measured. A method for measuring an enzyme or a substrate, characterized by quantitative determination.