JPH06245798A - Got assay and reagent therefor - Google Patents

Abstract

PURPOSE:To provide a reagent for assaying glutamate oxaloacetate transaminase(GOT), stable when preserved in a liquid state. CONSTITUTION:The assay is characterized by that both L-aspartic acid and alpha-ketoglutaric acid are made to act on the glutamate oxaloacetate transaminase(GOT) in a specimen and the resultant oxaloacetic acid is made to react with lactate dehydrogenase and maleate dehydrogenase which belongs to Bacillus in the presence of NADH, and the resultant NAD is assayed. The other objective reagent for GOT assay contains NADH, L-aspartic acid, alpha-ketoglutaric acid lactate dehydrogenase and maleate dehydrogenase which belongs to Bacillus.

Description

Detailed Description of the Invention

[0001]

The present invention relates to glutamate / oxaloacetate transaminase (GOT) EC in biological samples.
It relates to the measuring method of 2.6.1.1 and a reagent for the measurement.

[0002]

2. Description of the Related Art GOT, along with GPT (glutamate / pyruvate transaminase),
It is known to exist in large numbers in the liver, kidney, etc., and the activity value in serum fluctuates due to damage to these organs. An ultraviolet measurement method utilizing the following reaction has been adopted as a standard method for measuring the activity of GOT.

[0003]

[Chemical 1]

[0004]

[Chemical 2]

That is, the activity of GOT is measured by converting oxaloacetic acid, which is produced from L-aspartic acid and α-ketoglutarate by its catalytic action, into malic acid by malate dehydrogenase and NADH, which coexist as a coupling enzyme. The rate at which NADH is converted to NAD ⁺ is 34
It is obtained from the rate of decrease in absorbance at 0 nm.

The GOT activity unit per liter of sample is determined as follows. GOT1U (international unit) is 1 at 30 ℃
It is defined as the amount of enzyme that consumes 1 μmol NADH per minute.

[Chemical 3]

Here, the minimum required reagent is NA
DH, L-aspartic acid, α-ketoglutarate, and malate dehydrogenase, but usually it is essential to add lactate dehydrogenase. Oxaloacetic acid produced by the GOT reaction is unstable and decarboxylates to produce pyruvic acid, so that NADH is not stoichiometrically reduced. To avoid this it is necessary to add lactate dehydrogenase.

[0008]

The GOT measuring reagent is usually composed of a two-reagent system, and usually a first reagent containing malate dehydrogenase, lactate dehydrogenase, α-ketoglutarate, or L-aspartate,
And a second reagent containing α-ketoglutarate or L-aspartate. A problem in storing the above reagent in a liquid state after dissolution is the stability of malate dehydrogenase. NADH is known to be stable at pH 9 to 10, and α-ketoglutarate,
There is no problem with L-aspartic acid. Furthermore, a stable enzyme is also commercially available for lactate dehydrogenase.

A commonly used malate dehydrogenase is derived from pig heart, which is well known to have low stability. Therefore, recently, enzymes of thermophilic bacteria have been used. For example, the Malate dehydrogenase of the genus Thermus has excellent thermostability and can be stored for a long time in a buffer.
However, in the GOT measurement reagent, it was found that the stability was not as excellent as expected from the thermal stability.

[0010]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have found that the use of malate dehydrogenase derived from a bacterium belonging to the genus Bacillus
It was found that the activity was stably retained even in the GOT reagent.

That is, according to the present invention, glutamic acid
Oxaloacetate transaminase (GOT) was allowed to act on L-aspartic acid and α-ketoglutarate, and oxaloacetate produced was reacted with lactate dehydrogenase and malate dehydrogenase derived from Bacillus in the presence of NADH to measure the produced NAD ⁺ . Is a method for measuring GOT.

Further, the present invention is a reagent for measuring GOT, which contains NADH, L-aspartic acid, α-ketoglutarate, lactate dehydrogenase and malate dehydrogenase derived from the genus Bacillus.

The malate dehydrogenase used in the present invention may be of any origin as long as it is a bacterium belonging to the genus Bacillus. Particularly preferred is Bacillus stearothermophilus,
It is derived from Bacillus subtilis and Bacillus caldotenax. Among these, Bacillus stearothermophilus IFO12550,
Those derived from IFO12983, IFO13737 and ATCC12016 are preferred.

The malate dehydrogenase used in the present invention is a suitable Bacillus stearothermophilus cultivated,
Purify from the culture. As such a method, for example, J. Biol. Chem., 242, 1548 (1967) can be used. As a medium used for culturing the malate dehydrogenase-producing bacterium, a medium containing an appropriate amount of a carbon source, a nitrogen source, an inorganic substance and other necessary nutrients that can be assimilated by the strain to be used, is either a synthetic medium or a natural medium. Can also be used. Culturing is usually carried out by shaking culture or aeration stirring culture, the culture temperature is 40 to 60 ° C., and the culture pH is 5 to 5.
Perform within the range of 9. The cultivation period is 1 to 5 days, and the malate dehydrogenase is produced and accumulated in the cells. The method for purifying the enzyme used in the present invention can be performed, for example, as follows. Examples of the extraction method from the bacterial cells include ultrasonic disruption, mechanical disruption using glass beads, French press, and surfactant treatment. For the extract,
Salting out methods such as ammonium sulfate and sodium sulfate, metal coagulation methods such as magnesium chloride and calcium chloride, coagulation methods such as protamine and polyethyleneimine, and further DEAE (diethylaminomethyl) -sepharose, CM (carboxymethyl)
-It can be purified by an ion exchange chromatography such as Sepharose. The malate dehydrogenase thus obtained can usually be obtained with a specific activity of 50 U / mg or more in a yield of 25% or more.

The lactate dehydrogenase used in the present invention may be of any origin, but for example, those derived from lactic acid bacteria are excellent in stability and the like.

The GOT measuring reagent of the present invention is NADH,
It contains L-aspartic acid, α-ketoglutarate, lactate dehydrogenase and malate dehydrogenase from the genus Bacillus. If necessary, pyridoxal phosphate may be contained.

As the reagent composition of the present invention, for example, the following are suitable. The following is the composition after mixing the first reagent and the second reagent. Lactate dehydrogenase 500-2000 U / l Malate dehydrogenase 500-5000 U / l L-aspartic acid 150-250 mmol / l α-ketoglutarate 10-40 mmol / l NADH 0.1-0.25 mmol / l pyridoxal phosphate 0.05-0 0.2 mmol / l buffer substance (pH 7.0-8.0) 0.05-0.1 mol / l

The buffer substance is preferably Tris-hydrochloric acid, sodium phosphate, potassium phosphate and GOOD buffer substance.

[0019]

According to the present invention, G having excellent long-term storage stability
A reagent for OT measurement is obtained. Now that liquefied reagents are becoming widespread, such liquid and stable reagents are very useful.

[0020]

EXAMPLES The present invention will be specifically described below with reference to examples. Example 1 The following solution was prepared as a GOT measurement reagent. Lactate dehydrogenase 500 U / l Malate dehydrogenase 1000 U / l L-aspartic acid 175 mmol / l α-ketoglutarate 30 mmol / l NADH 0.22 mmol / l Pyridoxal phosphate 0.1 mmol / l PIPES (pH 7.4) buffer 50 mmol / l

Malate dehydrogenase is a Bacillus stearothermophilus.
It was prepared by ATCC12016 by a known method. After preheating 3 ml of the above reagent at 30 ° C for 5 minutes, the standard GOT1
0.1 ml of a sample in which 500 U / l was serially diluted was added, and the mixture was reacted at 30 ° C. for 3 minutes, and the decrease in absorbance at 340 nm was measured. The results are shown in FIG. 1, and the sample had linearity up to a GOT amount of 1500 U / l.

Example 2 The following solution was prepared as a GOT measuring reagent. Lactate dehydrogenase 500 U / l Malate dehydrogenase 5000 U / l L-aspartic acid 175 mmol / l α-ketoglutarate 30 mmol / l NADH 0.22 mmol / l Pyridoxal phosphate 0.1 mmol / l PIPES (pH 7.4) buffer 50 mmol / l

Malate dehydrogenase is a Bacillus stearothermophilus.
It was prepared by ATCC12016 by a known method. Store the above reagents at 4 ° C, 25 ° C and 40 ° C, respectively,
Residual malate dehydrogenase activity was measured on days 4, 5, 6, and 7. The malate dehydrogenase was measured by the following method. Add the following reaction mixture to a cuvette (d =
1.0 cm) and preheated at 25 ° C. for about 5 minutes. 2.8 ml 0.1 M K-phosphate buffer (pH 7.
5) 0.1 ml 15 mM oxaloacetate solution (dissolved in K-phosphate buffer) 0.1 ml 6.0 mM NADH aqueous solution

After adding 0.05 ml of the enzyme solution and gently mixing, water was used as a control to measure 3 times with a spectrophotometer controlled at 25 ° C.
The change in absorbance at 40 nm was recorded for 3 to 4 minutes, and the change in absorbance per minute was calculated from the initial linear portion (ΔODt
est). In the blind test, 0.05 ml of K-phosphate buffer solution was added instead of the enzyme solution, and the same operation as above was performed to determine the change in absorbance per minute (ΔODblank). 1 U (unit) of malate dehydrogenase is the amount of enzyme that oxidizes 1 micromol of NADH per minute under the above-mentioned conditions.

Comparative Example 1 A solution having the composition of Example 2 was prepared as a GOT measuring reagent. However, a commercially available Thermus-derived enzyme was used as the malate dehydrogenase. The results of Example 2 are as shown in FIG. 2 and the results of Comparative Example 2 are as shown in FIG. 3, and the Thermus-derived enzyme was stored for 7 days at any temperature for 50 to 6
The enzyme derived from the genus Bacillus has a residual activity of 80 to 100%, while the residual activity of 0% is exhibited, showing the stability of the malate dehydrogenase derived from the genus Bacillus in the GOT measurement reagent. It was

[Brief description of drawings]

FIG. 1 is a graph showing GOT dilution linearity of a GOT measurement reagent using a malate dehydrogenase derived from Bacillus.

FIG. 2 is a graph showing the storage stability of malate dehydrogenase in a GOT measurement reagent using a malate dehydrogenase derived from Bacillus.

FIG. 3 is a graph showing the storage stability of malate dehydrogenase in a GOT reagent using a malate dehydrogenase derived from Thermus as a comparative example.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Shigenori Aisui 10-24 Toyomachi, Tsuruga City, Fukui Prefecture Toyobo Co., Ltd. Tsuruga Bio Research Institute

Claims (2)

[Claims] 1. Lactate dehydrogenase and malate dehydrogenase derived from Bacillus in the presence of NADH in the oxaloacetate produced by allowing L-aspartic acid and α-ketoglutarate to act on glutamic acid / oxaloacetate transaminase (GOT) in a sample. G, characterized by reacting with and measuring the NAD ⁺ produced
OT measurement method. 2. A reagent for measuring GOT, which contains NADH, L-aspartic acid, α-ketoglutarate, lactate dehydrogenase and malate dehydrogenase derived from Bacillus.