JPS60119459A - Method for measuring concentration of adenosine 5'-diphosphate and enzyme activity of enzyme forming adenosine 5'-diphosphate - Google Patents

Abstract

PURPOSE:To measure the concn. of adenosine 5'-diphosphate (ADP) and the enzyme activity of enzyme forming ADP by using an enzyme electrode having two kinds of immobilized enzyme; immobilized pyruvate oxidase and immobilized pyruvate kinase. CONSTITUTION:An immobilized pyruvate oxidase film 2 and an immobilized pyruvate kinase film 3 are fixed on the surface of an oxygen electrode or hydrogen peroxide electrode 1 to constitute an enzyme electrode. A prescribed amt. of sample is added from a supply pipe 9 to a buffer soln. 3 in the case of measuring the concn. of ADP in the sample. ADP and phosphenol pyruvic acid are diffused in the film 3 to form a pyruvic acid. The pyruvic acid is diffused and oxidized in the film 2 and in this stage said acid consumes oxygen and generates hydrogen peroxide. The hydrogen peroxide is detected by the change in the current of the electrode 1. The intended enzyme substrate is added preliminarily to the buffer soln. and the similar measurement is performed in the case of measuring the enzyme activity of the enzyme forming the ADP in the sample.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a novel method for quickly and easily measuring the concentration of adenosine 5'-diphosphate (hereinafter sometimes abbreviated as ADP) and the enzyme activity of an enzyme whose product is ADP. It is about the method.

In recent years, with advances in biochemistry and clinical medicine, there has been a demand for rapid, simple, and accurate measurement of substance concentrations and enzyme activities in living organisms.

ADP is a substance that occupies an important position in the metabolic system as a high-energy phosphoric acid compound. In addition, examples of enzymes that generate ADP include 6-phosphofructokinase (EC2, 7, Lll), hexokinase (EC2,
7,,1,1), creatine kinase (EC2,7,3
,,2) are important enzymes in the metabolic system. In particular, measurement of taleatin kinase activity in serum
It is also important in clinical medicine because it serves as an indicator for the diagnosis of metabolic diseases.

Thus, there is a strong need in the fields of biochemistry and clinical medicine to develop methods for measuring ADP concentration and enzyme activity of enzymes that use ADP as a product.

Conventionally, methods for measuring ADP concentration and enzymatic activity of enzymes that use ADP as a product include pyruvate kinase and lactate dehydrogenase (ECI).

1.1.27) or myokinase (
EC2°7.4.3), phosphoglycerate kinase (
A spectroscopic method using a combination of EC2,7,2°3) and glyceraldehyde phosphate dehydrogenase (EC1,2,1,1,2) has been proposed and put into practical use. However, this method has drawbacks such as the enzyme being disposable and cannot be used repeatedly and requiring complicated operations.

The present inventors have conducted various studies in order to improve the shortcomings of conventional methods for measuring ADP concentration and enzyme activity of enzymes that use ADP as a product, and to develop a method that is quick, simple, accurate, and can be used repeatedly. As a result, we discovered that the purpose can be achieved very conveniently when using an enzyme electrode that combines two types of immobilized enzymes, immobilized pyruvate oxidase and immobilized pyruvate kinase, and an enzyme electrode or a hydrogen peroxide electrode. He came up with an invention.

That is, the present invention provides a method for measuring the ADP concentration contained in a sample and the enzymatic activity of an enzyme that provides ADP as a product, in which two types of immobilized enzymes, immobilized pyruvate oxidase and immobilized pyruvate kinase, and the enzyme ADP concentration and ADP generation characterized by measuring the concentration and enzyme activity by measuring the current change in the enzyme electrode after adding a sample using an enzyme electrode combined with an electrode or a hydrogen peroxide electrode. The present invention provides a method for measuring enzyme activity of a target enzyme.

The pyruvate oxidase used in the method of the present invention is derived from Pediococcus spp.
Those derived from P. p.) can be used, and are available from Toyo Jozo ■, for example. Pyruvate kinase derived from rabbit or dog muscle can be used, and is available from Sigma, Boehringer Mannheim, etc., for example. As the immobilized enzyme, those made by immobilizing these enzymes separately or in a mixture can be used, and the immobilization method is the carrier binding method that is usually used for enzyme immobilization, or Either comprehensive method may be adopted.

The shape and arrangement of the immobilized enzyme may be those used in ordinary enzyme electrodes. For example, tightly immobilizing a membrane-like immobilized enzyme on the surface of an oxygen electrode or hydrogen peroxide electrode,
Possible options include placing a column-shaped immobilized enzyme near the surface of an oxygen electrode or a hydrogen peroxide electrode. Alternatively, a method may be used in which the immobilized pyruvate oxidase is tightly immobilized in the form of a membrane on an oxygen electrode or a hydrogen peroxide electrode, and the immobilized pyruvate kinase is arranged in the form of a column nearby. As the oxygen electrode used in the method of the present invention, a commonly used one such as a Clark type polarographic type or a battery type which utilizes the oxygen permeability of a porous polymer membrane can be used.

Further, as the hydrogen peroxide electrode, a platinum electrode, a silver electrode, or a commonly used polarographic type electrode in which a porous polymer film is coated on the surface of these electrodes can be used.

In the method for measuring ADP concentration of the present invention, the enzyme electrode prepared as described above is inserted into a buffer solution. There are no particular restrictions on the concentration or type of the buffer solution as long as its pH is in the range of 5 to 10, but it may contain a small amount of phosphoenolpyruvate, which is one of the substrates of pyruvate kinase. There is no particular limit to the concentration of phosphoenolpyruvate, but it is 0.1 to 1Qmmol.
Preferably, the concentration is in the range 1/1. In addition, in order to increase the activity of pyruvate oxidase, at least about 1 mmol/l of phosphate ions, Q, l m
It is preferable to contain cocarboxylase in an amount of about mol/73, and in order to increase the activity of pyruvate kinase, it should contain at least about 1 mmol/l of magnesium ions and a sodium ion concentration of 1 molar.
It is preferable that it is about mol/l or less. Thus, for example l mmol/l phosphoenolpyruvate, l
Qmmol/l potassium phosphate, l mmol/l
l of cocarboxylase and 5 mmol/J of magnesium chloride.
A buffer solution consisting of glycine and potassium hydroxide can be conveniently used. Furthermore, in the method of measuring the enzymatic activity of an enzyme that produces ADP as a product of the present invention, it is necessary to add an appropriate concentration of the substrate of the enzyme to the buffer solution in order to initiate the enzymatic reaction of the target enzyme. It is.

The principle of the method for measuring A and DP concentrations of the present invention is as follows. That is, ADP acts as a catalytic agent of pyruvate kinase.
As a result of its action, it reacts with phosphoenolpyruvate to produce adenosine 5'-3 phosphate (hereinafter sometimes abbreviated as ATP) and pyruvic acid.

The generated pyruvate is oxidized by the catalytic action of pyruvate oxidase, but in this process dissolved oxygen in the buffer solution is consumed and hydrogen peroxide is generated. Consumption of oxygen or production of hydrogen peroxide is immediately detected as a decrease in current at the oxygen electrode or an increase in current at the hydrogen peroxide electrode, respectively.

In this way, the ADP concentration in the sample can be measured from the degree of change in the current at the enzyme electrode. Furthermore, the principle of the method for enzymatic activity of an enzyme that produces ADP as a product according to the present invention is as follows. In the presence of the target enzyme's substrate, the enzyme produces ADP at a rate proportional to its enzymatic activity. The ADP production rate or the ADP concentration after a certain period of time from the start of the enzyme reaction is detected as a change in the current at the enzyme electrode using the same principle as the method for measuring ADP concentration described above. In this way, the enzyme activity of the target enzyme in the sample can be measured from the degree of change in the current at the enzyme electrode.

Next, the method of the present invention will be explained in more detail with reference to additional drawings.

FIG. 1 is a schematic cross-sectional view showing an example of an apparatus used in the method of the present invention, in which a pyruvate oxidase-immobilized membrane 2 and a pyruvate kinase-immobilized membrane 3 are formed on the surface of an enzyme electrode or a hydrogen peroxide electrode 1. It is fixed using an O-ring or the like to form an enzyme electrode. A buffer solution 4 containing phosphoenolpyruvate is poured into the container 10, and the enzyme electrode is inserted into this solution. It also increases the amount of dissolved oxygen in the solution,
It is also preferable that an air or oxygen inlet tube 5 is provided to maintain a constant amount of air or oxygen, and that the solution is further stirred using a magnetic stirrer 6 or the like. The output current of the enzyme electrode is converted to voltage via an ammeter or resistor 7 and recorded on a recorder 8. If the electrode 1 is not battery-powered, an appropriate voltage is applied to the electrode 1 using an external power source 11. The current value at electrode 1 before sample addition shows a constant value proportional to the amount of dissolved oxygen in buffer solution 3 when an oxygen electrode is used as electrode 1, and a constant value proportional to the amount of dissolved oxygen in buffer solution 3 when a hydrogen peroxide electrode is used. It is zero because there is no hydrogen peroxide in solution 3.

When measuring the ADP concentration in a sample, when a predetermined amount of a sample containing ADP is added to the buffer solution 3 using, for example, the supply tube 9, ADP and phosphoenolpyruvate are immobilized with pyruvate kinase. It diffuses into the membrane 3 and pyruvic acid is produced.

The generated pyruvate further diffuses into the pyruvate oxidase-immobilized membrane 2 and is oxidized, consuming oxygen and generating hydrogen peroxide. Accordingly, the current of the electrode 1 decreases when an oxygen electrode is used as the electrode 1, and increases when a hydrogen peroxide electrode is used as the electrode 1. Moreover, the degree of decrease or increase in this current is proportional to the ADP concentration, so if we consider the degree of decrease or increase in current, we can determine the amount of ADP in the sample.
DP concentration can be measured.

When measuring the enzymatic activity of an enzyme that generates ADP in a sample, a substrate for the target enzyme is added to the buffer solution 3 in advance, and a predetermined amount of the sample containing the target enzyme is passed through the supply tube 9, for example.
or by adding a predetermined amount of a sample containing the target enzyme to the buffer solution 3 using, for example, the supply tube 9, and then further adding the substrate of the target enzyme. Initiate the enzyme reaction of the target enzyme. Then, the ADP concentration increases linearly with time in proportion to the activity of the target enzyme. This change is caused by a reaction in the pyruvate kinase-immobilized membrane 3 and the pyruvate oxidase-immobilized membrane 2, resulting in an immediate linear change in the current at the electrode 1, that is, when an oxygen electrode is used as the electrode 1, the current A linear decrease in current causes a linear increase in current when using a hydrogen peroxide electrode. Moreover,
Since the ratio of current change in this linear portion to time is proportional to the enzyme activity of the enzyme, the enzyme activity of the target enzyme in the sample can be measured by subtracting the ratio.

Next, the present invention will be explained in more detail with reference to Examples.

Example I Measurement of ADP concentration Pyruvate oxidase (Toyo Jozo Co., Ltd., 13 U/rn9
) 10 ■ p) 16.7 phosphate buffer solution (0.1 mO
1/l) of photosensitive polyvinyl alcohol 1
It was mixed with 1 ml of 0% solution i, spread on an area of about 10 cnf, air-dried, and further irradiated with light to obtain a water-insoluble pyruvate oxidase-immobilized membrane. Pyruvate kinase (Sigma, 400 IU/■) 5■ pr-r 6
The above photosensitive polyvinyl alcohol 10% solution 1 was dissolved in a phosphate buffered solution (Q, l mol/l) of
A pyruvate kinase-immobilized membrane having an area of approximately ]OcJ was obtained by the same operation as above. Both immobilized membranes were cut out to a diameter of Icm, and the area was 0.1 cf.
The pyruvate oxidase-immobilized membrane and the pyruvate kinase-immobilized membrane were placed in this order on the diaphragm of a battery-operated oxygen electrode with a platinum electrode as the working electrode and a porous polytetrafluoroethylene membrane as the diaphragm, and the entire OIJ song tightened. The enzyme electrode prepared in this way was prepared by: 1. mmol/1 phosphoenolpyruvate, ]Q mmol/l potassium phosphate, 1 mmol/l cocarboxylase,
and 0.4 containing 5 mmol/e magnesium chloride.
mol/l glycine-potassium hydroxide buffer solution (p
H9,0) was inserted into 13 ml. Approximately 200+q/min from the solution surface! The solution was stirred using a magnetic stirrer. The temperature of the solution is 30
It was kept at °C.

Figure 2 shows that the above solution has a concentration of 0. l mmol /1
1 is a graph showing the time change in current at the enzyme electrode when ADP 1 was added, and the current reached a steady value about 30 seconds after the addition of ADP. FIG. 3 is a graph showing the relationship between the decrease in current in a steady state after addition of an ADP-containing sample and the ADP concentration. ADP concentration 0.5 mmol/l
Since a completely proportional relationship holds true below, using the graph shown in Figure 3, we can calculate the unknown A in the sample below this concentration.
The DP concentration can be calculated from the current reduction value.

Example 2 Measurement of creatine kinase activity The enzyme electrode used in Example 1 was heated with l mmol/l phosphoenolpyruvate, lQmmol/l potassium phosphate, 1 mm
ol/l cocarboxylase and 5 mmol/l magnesium chloride, plus 5 mmol/l as substrate for the title enzyme.
It was inserted into 10 ml of Q, 4 mol/Δglycine-potassium hydroxide buffer solution (pEJ 9.0) containing o1/71 ATP and 33 mmol/1 creatine.

Air is blown at a rate of approximately 200 ml per minute from the surface of the solution.
The solution was stirred using a magnetic stirrer. The temperature of the solution was kept at 30°C.

When talleatin kinase is added to this solution, the ADP concentration increases linearly with time according to the following reaction.

+ADP Figure 4 is a graph showing the time change of the current at the enzyme electrode when taleatine kinase with an activity of 100 TJ/l is added to the buffer solution. decreases linearly over time. FIG. 5 is a graph showing the relationship between the time rate of current decrease in the linear portion after addition of a creatine-containing sample and creatine kinase activity. Since a completely proportional relationship holds true when the creatine kinase activity is below 200 U#, we can use the graph shown in Figure 5 to determine the unknown creatine kinase activity in the sample with this activity or below from the time rate of current decrease. I can do it.

[Brief explanation of the drawing]

FIG. 1 shows the ADP igL degree and AT) of the present invention.
FIG. 2 is a schematic cross-sectional view showing an example of an enzyme activity measuring device for an enzyme that uses P as a product, and shows the relationship between the current when an ADP-containing sample is added and the time after sample addition using the method of the present invention. 3 is a graph showing the relationship between the current reduction value and ADP concentration according to the method of measuring ADP concentration of the present invention, and FIG. FIG. 5 is a graph showing the relationship between the current and the time after sample addition in this case, and shows the relationship between the time rate of current decrease and creatine ginase activity according to the method of measuring enzyme activity of an enzyme whose product is ADP according to the present invention. It is a graph showing a relationship. The code 1 in Figure 1 is an enzyme electrode or a hydrogen peroxide electrode, and 2
is an immobilized pyruvate oxidase membrane, 3 is an immobilized pyruvate kinase membrane, 4 is a buffer solution, 5 is an air blowing tube,
6 is a magnetic stirrer, 7 is an ammeter or resistor, 8
9 is a recorder, 9 is a sample supply tube, 10 is a container, and 11 is an external power source. 15- Figure 1 Figure 2 Time after sample addition (minutes) Figure 3 AD141 degrees (mmol/7) Figure 4 Time after sample addition (minutes) Figure 5 Taleatin kinase activity (u71)

Claims (2)

[Claims] (1) In a method for measuring the concentration of adenosine 5'-diphosphate contained in a sample and the enzymatic activity of an enzyme that produces adenosine 5'-diphosphate as a product, immobilized pyruvate oxidase (ECI) is used. 2.3.3) and immobilized pyruvate kinase (EC2
, 7, 1, 40) using an enzyme electrode that combines two types of immobilized enzymes and an oxygen electrode or a hydrogen peroxide electrode, and by measuring the current change at the enzyme electrode after sample addition, the concentration and Adenosine 5'-diphosphate concentration and adenosine 5'-diphosphate characterized by increasing enzyme activity
A method for measuring enzyme activity of an enzyme that uses diphosphate as a product.