JPH0365490B2 - - Google Patents

Description

[Detailed Description of the Invention] This invention relates to NADH oxidase or NADPH oxidase (hereinafter referred to as "NAD(P)H oxidase").
This invention relates to a novel quantitative device for measuring various biochemical substances using an immobilized enzyme carrier on which is immobilized.

Quantification of various biochemical substances within a living body is extremely important as a means of obtaining biochemical information within that living body. Particularly in the field of clinical testing, it is essential to quantify biochemical substances in body fluids in order to understand pathological conditions. Biological components that are important for diagnosis and treatment are complex and diverse, ranging from low-molecular components to high-molecular components, so enzymes with high substrate specificity must be used to selectively measure specific components. Enzyme analysis methods are widely used.

One of the substances frequently measured in enzyme analysis methods is NADH and NADPH (hereinafter referred to as "NAD(P)"), which are reduced forms of the coenzymes NAD and NADP.
(denoted as "H"). Since these have a maximum absorption wavelength of 340 nm, the increase or decrease in NAD(P)H is determined by ultraviolet absorption measurement, and the substrate or enzyme activity is determined thereby. However, in this method, it is difficult to quantify if the sample is cloudy or absorbs not only the ultraviolet region but also the visible region. Therefore, pretreatment such as decolorization, centrifugation, or protein removal may be necessary in some cases.

In order to overcome the drawbacks of the colorimetric method as mentioned above,
Recently, electrical measuring devices using enzyme electrodes using immobilized enzymes have been proposed. However, the enzyme electrode method that is currently in practical use does not cover all the substrates that can be quantified by colorimetric methods, and only some of the substrates and reaction products become electrode active substances. It could only be used in enzymatic reaction systems. For this reason, the current enzyme electrode method can only measure three or four types of measurement items, such as coenzyme NAD or
It has not been used at all in enzymatic reaction systems that require NADP.

Therefore, the present inventors overcame the above-mentioned conventional problems and used an enzyme-immobilized carrier on which NAD(P)H oxidase was immobilized to quickly and accurately produce NAD(P)H and other substrates. The purpose is to provide a device for measuring or quantifying.

This invention, in short, at least NAD
(P)H oxidase immobilized enzyme immobilization carrier;
This is a quantitative device that stoichiometrically quantifies NAD(P)H by contacting a solution containing NAD(P)H and measuring the decrease in oxygen due to the enzymatic reaction using an electrode.

In another embodiment of this invention, the coenzyme NAD(P)
NAD(P)H is produced by an enzymatic reaction requiring By measuring H using an electrode, biochemical substances involved in enzymatic reactions other than NAD(P)H oxidase can be measured or quantified.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Below, the principles and some embodiments of the present invention will be described in detail with reference to the accompanying drawings.

The NAD(P)H oxidase used in this invention is
It is an enzyme that donates oxygen to NAD(P)H to produce NAD(P), and the following two types are known. 1
One is an enzyme that exhibits the reaction shown in the following formula (1), and was developed by Mr. Sakuzo Fukui in Lactobacillus
plantarum No. 11 (Agricultural Biological Chemistry Vol. 25, No. 11)
No. 870 to 878 Tribute: 1961).

2NAD(P)H+2H ⁺ +O ₂ NAD(P)H oxidase――――――――――――――→ 2NAD(P)+2H ₂ O …(1) The other one is by Mr. Rudi Reinards By et al.
It is known to exist in Acholeplasma Iaidlawaii. This gives the enzyme and results in NAD (P) and hydrogen peroxide H ₂ O ₂ as shown in the following equation (2).
(European Journal of Biochemistry, Vol. 120, No. 329-337)
Mitsugu: 1981).

NAD(P)H+H ₂ O+O ₂ NAD(P)H oxidase――――――――――――――→ NAD(P)+H ₂ O ₂ …(2) This invention relates to the above-mentioned NAD (P)H oxidase,
For example, it is immobilized on an immobilized enzyme membrane to be attached to an electrode as an enzyme electrode, or an immobilized enzyme column or an immobilized enzyme tube (coil) to be introduced into a sample flow path. As an immobilized enzyme membrane, a porous polymer thin film such as a cellulose derivative such as acetylcellulose or nitrocellulose is generally used, but the immobilized enzyme membrane used in this invention is limited to these materials. It's not a thing. The film thickness is 100μm or less, especially 20μm.
The following are preferred. On the other hand, in the case of an immobilized enzyme column, glass beads or cellulose derivatives of polysaccharide particles used in column gels or polymer fine particles can be used as carriers for immobilizing enzymes. In the case of immobilized enzyme tubes (coils),
As the carrier, nylon can generally be used, but it is needless to say that the carrier is not limited thereto.
The NAD(P)H oxidase may be immobilized by any known method, ie, chemical method (covalent bond method, cross-linking method, etc.) or physical method (entrapment method, adsorption method, etc.). I can do it.

This invention relates to NAD(P)H of the above formula (1) or (2).
Although NAD(P)H is quantified by the reaction of oxidase, this NAD(P)H also includes those produced by the following enzymatic reaction.

(i) a (substrate) + NAD (P) NAD (P) H oxidase――――――――――――――→ b (reaction product) + NAD (P) H (ii) c ( Substrate) + d (substrate) enzyme to be conjugated――――――――――→ e (substrate) + f (reaction product) e (substrate) + NAD(P)NAD(P)H oxidase―― ――――――――――――→ g (reaction product) + NAD(P)H For example, to quantify triglyceride, quantify NAD(P)H produced by the following reaction. This is done by

Triglyceride + H ₂ O lipoprotein lipase――――――――――――――→ Glycerol + fatty acid ion Glycerol + NAD(P) glycerol dehydrogenase―――――――――――――― ---→ Dihydroxyacetone+NAD(P)H The present invention will be explained in more detail below using an example using an enzyme electrode.

FIG. 1 is an illustrative cross-sectional view showing an example of an enzyme electrode that can be used in the present invention, and FIG. 2 is an enlarged cross-sectional view of an immobilized enzyme membrane. The enzyme electrode 1 includes a housing 2 in which a working electrode 3 is disposed.
and an associated reference electrode 4 are housed therein. housing 2
A lid 5 is attached to the upper end, for example, by screwing.

The working electrode 3 is made of gold, platinum, glassy carbon, etc. when measuring the decrease in oxygen as an electrode active substance, and the reference electrode 4 is made of silver, lead, silver chloride, or a saturated calomel electrode. can be used. The working electrode 3 is cylindrical, and the reference electrode 4 is configured as a ring surrounding it. These two electrodes 3 and 4
are molded with resin layers 6, such as epoxy resin, at predetermined intervals.
The end faces of the working electrodes 3 and 4 are formed at the lower end of this resin layer 6 so as to be exposed therefrom. Lead wires are drawn out from the working electrode 3 and the reference electrode 4 through the resin layer 6 and the lid 5, and these lead wires are connected to a current detector 11, which will be described later. The lower end surface of the resin layer 6 is covered with an immobilized enzyme membrane 8 attached by an O-ring 7. Therefore, the immobilized enzyme membrane 8 and the end surfaces of the two electrodes 3 and 4 are brought into contact.

The immobilized enzyme membrane 8 includes, for example, an oxygen-permeable Teflon (trade name) membrane 81, an immobilized enzyme membrane 82 in which NAD(P)H oxidase is adsorbed and immobilized on a cellulose filter, and a polymer substance in the sample. Polycarbonate membrane 8 as a porous membrane to prevent the permeation of
3.

Such an enzyme electrode 1 is immersed in a reaction tank 10 as shown in FIG. 3, and this enzyme electrode 1 is connected to a current detector 11. That is, a voltage of about -0.5V to -0.7V is normally applied between the working electrode 3 and the reference electrode 4 through the lead wire by the current detector 11, and this applied voltage is within the stable range in polarography. is set to The current flowing between the working electrode 3 and the reference electrode 4 (FIG. 1) changes depending on the amount of oxygen reduced by the enzyme reaction, and the current change is detected by the current detector 1.
Detected by 1. The current detector 11 is connected to a recording device 12, and the recording device 12 records the differential value of the current change as a calibration curve.
Note that a magnetic stirrer (not shown) is placed in the reaction tank 10, and the buffer solution stored therein is constantly stirred.

Example 1 NADH oxidase is immobilized on the immobilized enzyme membrane 8. Then, 0.1M phosphate buffer (PH7.0) is put into the reaction tank 10, and then the enzyme electrode 1 is immersed in the reaction tank 10 so that the immobilized enzyme membrane 8 and the buffer are in contact with each other. The phosphate buffer is constantly stirred by a magnetic stirrer (not shown). When the dissolved oxygen (concentration) becomes stable, NADH solutions of various known concentrations are added to the reaction tank 10. As a result of the enzymatic reaction between NADH oxidase immobilized on the enzyme electrode and NADH, oxygen in the reaction tank 10 decreases according to the above equation (1), and this decrease in oxygen is detected by the current detector 11. is detected and further recorded by the recording device 12. As a result, a calibration curve shown in FIG. 4 was obtained.

Next, a sample containing NADH of unknown concentration was added to reaction tank 1.
Current detector 1 is injected into current detector 1 by the same operation.
The NADH concentration in this sample was determined from the current change value detected by 1 with reference to the above-mentioned calibration curve.

FIG. 5 is a flow diagram showing another embodiment of the present invention. This example specifically applies to NAD
A biochemical substance that participates in enzymatic reactions other than the reaction of NAD(P)H oxidase by conjugating the reaction of (P)H oxidase with an enzyme reaction requiring the coenzyme NAD(P) or other enzyme reactions This is an example suitable for quantifying.

In this FIG. 5 embodiment, the enzyme electrode 1 is similar to that shown in FIGS. 1 and 2. A flow cell communicating with the channel 13 is integrally attached to the lower end of the enzyme electrode 1, so that the solution flowing through the channel 13 comes into contact with the immobilized enzyme membrane 9 (FIG. 1) of the enzyme electrode 1. It is like that. A buffer tank 15 is connected to one end of the flow path 13 . A sample tank 16 is connected to the buffer flow path 13 by a three-way valve 17. A pump 18 is provided on the downstream side of the flow path 13, and a waste liquid tank 19 is connected to the downstream end of the flow path 13. Therefore, pump 1
8 suctions the buffer flow path 13, and the three-way valve 1
7, the buffer solution in the buffer solution tank 15 and the sample tank 1
6 flows into the waste liquid tank 19 through the flow path 13. An immobilized enzyme column 20 is inserted into the sample 13 on the upstream side of the enzyme electrode 1, and an immobilized enzyme column 20 is coated with a well-known method using an immobilized carrier such as glass beads. , an enzyme that causes a reaction to be coupled to the enzymatic reaction of NAD(P)H oxidase is immobilized. The enzyme electrode 1 is connected to a current detector 11, from which a predetermined voltage is applied, and current changes in response to a decrease in electrode active substances, such as oxygen, accompanying the enzymatic reaction of NAD(P)H oxidase. be read. The current change read by the current detector 11 is recorded in the recording device 12 as its differential value (calibration curve).

Example 2 (Quantification of lactic acid) The immobilized enzyme membrane 8 of the enzyme electrode 1 (Fig. 2) contains
Immobilize NADH oxidase. Lactate dehydrogenase LDH is immobilized on the enzyme carrier of the immobilized enzyme column 20 by a well-known method. In the buffer tank 15,
0.1M phosphate buffer (PH
7.0) at 30℃. This is flowed into the buffer channel 13 by the pump 18 so as to come into contact with the immobilized enzyme column 20 and the immobilized enzyme membrane of the enzyme electrode 1. A sample containing lactic acid is injected into the buffer channel 13 from the sample tank 16 via the three-way valve 17 . The injected sample, ie, lactic acid, reacts with LDH in the immobilized enzyme column 20, and NADH
is generated. generated in this way
NADH flows through the channel 13 and comes into contact with the immobilized enzyme membrane of the enzyme electrode 1.

Using samples containing lactic acid of various known concentrations, the same procedure as described above was repeated to obtain the calibration curve shown in FIG. 6.

Next, a sample containing lactic acid of unknown concentration was injected,
Through the same operation, the lactic acid concentration in this sample was determined from the current change value detected by the current detector 11 with reference to a calibration curve as shown in FIG.

In addition, when measuring NAD(P)H contained in a sample or NAD(P)H generated by the enzymatic reaction of NAD(P)H oxidase, immobilization can be performed without using an enzyme electrode. An enzyme column and electrode combination or an immobilized enzyme coil or tube and electrode combination can also be used.

Note that the enzyme for the enzymatic reaction to be coupled with the enzymatic reaction of NAD(P)H oxidase may be immobilized on separate carriers as described above, or may be immobilized on the same carrier, for example, an immobilized enzyme membrane. may be converted into

As described above, according to the present invention, NAD(P)
NAD(P)H can be quantified by a combination of an enzyme-immobilized carrier formed by immobilizing H-oxidase and an electrode, and can be used alone or in combination with other enzyme reactions to perform various reactions. biochemical substances can be quantified.

[Brief explanation of drawings]

FIG. 1 is an illustrative cross-sectional view showing an example of an enzyme electrode that can be used in the present invention. FIG. 2 is an enlarged sectional view showing an example of an immobilized enzyme membrane. FIG. 3 is a flow diagram showing one embodiment of the present invention. Figure 4 was created according to the example in Figure 3.
It is a graph showing an example of a calibration curve of NAD(P)H. FIG. 5 is a flow diagram showing another embodiment of the present invention. FIG. 6 is a graph showing an example of a lactic acid calibration curve obtained according to the example shown in FIG. In the figure, 1 is the enzyme electrode, 3 is the working electrode, and 4 is the enzyme electrode.
8 is a control electrode, 8 is an immobilized enzyme membrane, 11 is a current detector, and 20 is an immobilized enzyme column.

Claims (1)

[Claims] 1. An immobilized enzyme carrier on which NADH oxidase or NADPH oxidase is immobilized, and NADH oxidase or NADPH oxidase immobilized on the immobilized enzyme carrier and NADH or
A quantitative device using immobilized NAD(P)H oxidase, comprising: an electrode for causing an enzymatic reaction with a solution containing NADPH and measuring a decrease in oxygen accompanying the enzymatic reaction. 2. The immobilized enzyme carrier includes an immobilized enzyme membrane,
Claim 1, wherein the immobilized enzyme membrane is attached to the electrode surface, thereby forming an enzyme electrode, and the enzyme electrode is immersed in the solution containing the NADH or NADPH.
Quantification device as described in section. 3. The quantitative device according to claim 1, wherein the immobilized enzyme carrier is an immobilized enzyme column. 4. The quantitative determination device according to claim 1, wherein the immobilized enzyme carrier is an immobilized enzyme tube.