JPH0640838B2 - Enzyme activity or substrate assay - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Object of the Invention] The present invention relates to a method for measuring enzyme activity or substrate. More specifically, ornithine-keto acid amino transferase (EC 2.6.1.13, hereinafter referred to as “OKAT”), α-ketoglutarate (hereinafter referred to as “α-KG”), pyrroline 5
-Ornithine carbamoyl tranferase by using a reagent composition containing carboxylate dehydrogenase (EC 1.5.1.12, hereinafter referred to as "P5CDH") and oxidized pyridine nucleotide coenzyme (hereinafter referred to as "NAD (P)") as essential components (EC 2.1.3.3, below "O
"CT"), ornithine or citrulline.

2. Description of the Related Art Conventionally, the measurement of OCT or citrulline has been performed by the method described below. Reichard et al. Used the reaction of OCT to produce carbamoylarsenic acid and ornithine from citrulline in the presence of arsenate, using a radiolabeled substrate or ammonia generated by nonenzymatic decomposition of carbamylarsenic acid. The method of measurement is reported (J.Lab.Clin.Me
d., 52, 709-717, 1958). Brown et al. And Hamanaka et al. Reported a method for colorimetrically determining citrulline produced by the action of OCT on ornithine with diacetyl monooxime (J.Lab.Clin.Med., 54, 617-620). ， 1959
Year; Japanese Examined Sho 56-36919).

Regarding the measurement of ornithine, Matsuzawa et al.
KG, reduced pyridine nucleotide coenzyme, pyrroline 5
-A method for activating carboxylate reductase and OKAT to measure reduction of reduced pyridine nucleotide coenzyme has been reported (Anal. Biochem., 106, 1-6).
Page, 1980; Japanese Examined Patent Publication 59-15639).

Each of the above-mentioned prior arts has a safety problem due to the use of radioactive elements, takes a long measuring time, requires deproteinization operation, the reagent used is unstable, and is interfered by urea coexisting in a serum sample, The procedure is complicated and dangerous because it involves a heating reaction under strong acidity.A method for measuring the reduction of reduced pyridine nucleotide coenzyme requires a blank reaction, and the measurement range is narrow, and the measurement accuracy is low. There are drawbacks such as.

Problems to be Solved by the Invention An object of the present invention is to provide a method for measuring OCT, ornithine or citrulline by the total enzyme method. Another object of the present invention is to solve the above problems in the prior art and to provide a highly sensitive measurement method.

[Structure of Invention]

Means for Solving Problems According to the present invention, there is provided a method for measuring an enzyme activity or a substrate by using a reagent composition containing OKAT, α-KG, P5CDH, and NAD (P) as essential components. It More specifically, first, OKAT,
Ornithine is measured by allowing a reagent composition consisting of α-KG, P5CDH and NAD (P) to act and measuring the resulting product. Secondly, OCT is measured by further adding citrulline and phosphoric acid to the above essential reagent composition. Third, citrulline is measured by further adding OCT and phosphoric acid to the above essential reagent composition.

The invention is based on the use of a combination of reactions catalyzed by the enzymes mentioned above. The steps of each reaction are represented as follows.

Reaction (A): OCT is caused to act on citrulline in the presence of phosphoric acid to convert it to ornithine and carbamoylphosphate.

Reaction (B): OKAT is allowed to act on ornithine produced in the above reaction (A) or contained in the sample in the presence of α-KG to convert it to pyrroline 5-carboxylic acid and glutamic acid.

Reaction (C): P5CDH is allowed to act on the pyrroline 5-carboxylic acid produced in the above reaction (B) in the presence of NAD (P), and glutamic acid and a reduced pyridine nucleotide coenzyme (hereinafter referred to as “NAD (P) H”) Convert.

The above reaction formula is shown below.

The present invention utilizes an OCT-catalyzed reaction equilibrium in which citrulline is produced from ornithine, and the reverse reaction thereof is utilized. However, by using the reagent composition disclosed in the present invention, the reaction It was totally unexpected that the equilibrium moved in the opposite direction.

In the present invention, the target enzyme activity or substrate can be measured by quantifying the product produced in any of the above reactions (A) to (C). The product to be measured is preferably NAD (P) H or glutamic acid. NA
D (P) H and glutamic acid can be quantified by known methods. NAD (P) H is quantified by measuring its own absorption or fluorescence in the ultraviolet region, inducing a formazan dye by allowing a redox indicator such as a tetrazolium compound to act in the presence of diaphorase, and measuring its absorption in the visible region. , Or by measuring the absorption in the visible or visible region of resorufin produced by reacting resazurin in the presence of a catalyst. Glutamic acid is measured by a method of measuring NAD (P) H produced by causing glutamate dehydrogenase (hereinafter referred to as “GLUDH”) to act in the presence of NAD (P), or by causing glutamate oxidase (hereinafter referred to as “GLUOX”) to act. It is performed by measuring the amount of hydrogen peroxide formed.

The reaction of GLUDH is represented by the following formula.

In the present invention, N produced by allowing GLUDH to act on the glutamic acid produced in the above reactions (A) to (C)
When measuring AD (P) H, NAD (P) H derived from glutamic acid and NAD (P) ⁺ produced in reactions (B) and (C) respectively and N produced in reaction C were measured.
Since 3 equivalents of AD (P) H, NAD (P) H, is to be measured, the unexpected effect of significantly improving the sensitivity was obtained. In addition, GLUOX is added to the produced glutamic acid.
When glutamic acid is quantified by measuring the hydrogen peroxide produced by reacting with, the sensitivity of the glutamic acid produced in the reactions (B) and (C) is 2 equivalent. The effect of remarkably improving was obtained.

In the present invention, the reaction conditions can be varied widely. For example, the temperature is 25-50 ° C, preferably 30-45
The temperature and pH are preferably 6.5 to 8.5.

As the test sample, whole blood, serum, plasma, urine, tissue extract and the like are used. In the case of mass screening, it is preferable to use so-called blood filter paper in which blood is soaked in filter paper and dried, and then hemoglobin is immobilized.

In the present invention, OCT, OKAT, P5CDH, GL
The enzyme activity of UDH and GLUOX was 1 at 37 ° C.
The amount of enzyme that changes 1 μmol of substrate per minute was defined as 1 unit.

As described above, OKAT, α-KG, P5C of the present invention
The present invention provides a method for assaying enzyme activity or substrate by using a reagent composition containing DH and NAD (P) as essential components. The present invention includes all methods for measuring enzyme activity or substrate that can be linked to the reaction system disclosed by the present invention, including OCT, ornithine, and citrulline.

The present invention will be described in more detail with reference to the following examples.

Example 1 Measurement of OCT activity 0.2M phosphate buffer (pH 7.5) 0.685 ml, 0.12M NA
DP ⁺ 0.05 ml, 25 mM α-KG 0.005 ml, 165 U / ml O
KAT 0.05 ml, 190 U / ml P5CDH 0.02 ml, 0.2 M
0.5 ml of citrulline, 1.0 ml of water, and 0.2 ml of serum sample were mixed and incubated at 37 ° C for 20 minutes. The increase in absorption of the reaction solution at 340 nm was measured, and OC in the sample was measured.
The T activity was calculated.

For reference, the same serum sample was measured with a commercially available reagent composition for measuring OCT activity (trade name: OCT-TEST wako, manufactured by Wako Pure Chemical Industries, Ltd.).

The correlation between the value of the method of the present invention and the value measured by the known commercially available reagent composition was 0.989, which was extremely well in agreement (shown in FIG. 1).

Example 2 Ornithine measurement 0.2 M phosphate buffer (pH 7.5) containing 0.05% NADP ⁺
2.0 ml, 200 mM α-KG 0.1 ml, 90 U / ml OKAT
0.05 ml, 30 U / ml P5CDH 0.05 ml water 0.1 ml, and
0.2 ml of a sample containing a known concentration of ornithine of 0.25 to 2 mM
Were mixed and incubated at 37 ° C for 15 minutes. When the decrease in absorption of the reaction solution at 340 nm was measured and the relationship between the ornithine concentration and the absorbance was plotted, good linearity was obtained as shown in FIG.

Example 3 Measurement of ornithine NADP ⁺ 100 nmol, α-KG 50 nmol, OK in 0.1 ml.
AT 0.5 units and P5CDH 0.5 units were added
0.2 M containing 0.1 mM dithiothreitol, 0.2 mM ethylenediaminetetraacetic acid and 0.01% sodium azide
Mixing PIPES-potassium hydroxide buffer (pH 7.5) and immobilized filter paper (diameter 3 mm) containing known concentration of ornithine,
Incubated at 37 ° C for 20 minutes. Water in the reaction solution
2.5 ml was added and the fluorescence intensity was measured. When the relationship between ornithine concentration and fluorescence intensity was plotted, good linearity was obtained as shown in FIG.

Example 4 Measurement of citrulline NADP ⁺ 100 nmol, α-KG 50 nmol, O in 0.1 ml.
KAT 0.5 units, OCT 30 units, and P5CDH
0.1 mM dithiothreitol supplemented with 0.5 units, 0.2
0.2 M PIPES-potassium hydroxide buffer (pH 7.5) containing mM ethylenediaminetetraacetic acid and 0.01% sodium azide, and a test sample solution containing citrulline of known concentration.
01 ml was mixed and incubated at 37 ° C for 20 minutes. 2.5 ml of water was added to the reaction solution, and the fluorescence intensity was measured. When the relationship between the citrulline concentration and the fluorescence intensity was plotted, good linearity was obtained as shown in FIG.

Example 5 Measurement of OCT activity 0.2M phosphate buffer (pH 7.5) 0.685 ml, 0.12M NA
DP ⁺ 0.05 ml, 25 mM α-KG 0.005 ml, 160 U / ml P
5CDH 0.02ml, 165 U / ml OKAT 0.05ml, water 0.99
ml and serum sample 0.2 ml were mixed and incubated at 37 ° C. for 10 minutes. 0.2 M citrulline 0.5 in the reaction solution
After adding ml and incubating at 37 ° C. for 20 minutes, the increase in absorbance at 340 nm of the filtered reaction solution was 0.016, and the OCT activity in the sample was calculated to be 1.6 U /.

In the above reaction, 90 U / ml GL was added to the reagent composition.
When the same operation was performed except that 0.1 ml of UDH was added and 0.89 ml of water was used, the absorbance was about 3 times the above.

Example 6 Measurement of OCT activity 0.1 mg of 4-aminoantipyrine or 0.5 mg of TOOS [N-ethyl-N- (2-hydroxy-3-sulfopropyl-m-toluidine)] and 5 units of peroxidase and 14.3 units of ascorbate oxidase. , Sodium azide 0.34 mg
And 0.2 M phosphate buffer containing 17.5 mg citrulline (pH
7.5) 0.685 ml, 0.12 M NADP ⁺ 0.05 ml, 25 mM α
-KG 0.005 ml, 165 U / ml OKAT 0.05 ml, 17 U /
ml GLUOX 0.02 ml, water 0.99 ml, and serum sample 0.2
The mls were mixed and incubated at 37 ° C for 10 minutes.
TOOS 0.5 mg or 4-aminoantipyrine in the reaction solution
Add 0.5 ml of a solution containing 0.1 mg, and add 5 at 37 ° C.
After incubation for minutes, the increase in absorbance at 555 nm was 0.017.

In the above reaction, 160 U / ml P was added to the reagent composition.
When the same operation was performed except that 5CDH (0.02 ml) was added and water was changed to 0.97 ml, the absorbance was about twice the above.

Example 7 Measurement of OCT activity 0.2 M phosphate buffer (pH 7.5) containing 0.8 mg of NTB (nitrotetrazolium blue) and 5 units of diaphorase.
0.685 ml, 0.12 M NADP ⁺ 0.05 ml, 25 mM α-KG
0.005 ml, 160 U / ml P5CDH 0.02 ml, 165 U / ml
OKAT 0.05 ml, 90 U / ml GLUDH 0.1 ml, water 0.89 m
l, and 0.2 ml of the same serum sample as in Example 5 were mixed and
Incubated at 0 ° C for 10 minutes. 0.2M in the reaction solution
When 0.5 ml of citrulline was added and incubated for 20 minutes at 37, the increase in absorbance at 560 nm was 0.0.
It was 90.

〔The invention's effect〕

The present invention relates to OKAT, α-KG, P5CDH, and NA.
The present invention provides a method for measuring enzyme activity or substrate by using a reagent composition containing D (P) as an essential component. According to the present invention, highly sensitive measurement of enzyme activity or substrate has become possible.

[Brief description of drawings]

FIG. 1 is a diagram showing the correlation when the OCT activity of a serum sample is measured by the method of the present invention and a known method. FIG. 2 is a diagram showing the relationship between the ornithine concentration in a sample and the absorbance at 340 nm in the measurement of ornithine by the method of the present invention. FIG. 3 is a diagram showing the relationship between the ornithine concentration in the sample and the fluorescence intensity in the measurement of ornithine by the method of the present invention. FIG. 4 is a diagram showing the relationship between the concentration of citrulline in a sample and the fluorescence intensity in the measurement of citrulline by the method of the present invention.

Claims (9)

[Claims] 1. A test sample is treated with a reagent composition containing ornithine-keto acid aminotransferase, α-ketoglutarate, pyroline 5-carboxylate dehydrogenase and oxidized pyridine nucleotide coenzyme, and the resulting product is obtained. A method for measuring an enzyme activity or a substrate in a test sample, which comprises measuring. 2. The assay method according to claim 1, wherein the reagent composition further contains citrulline and phosphoric acid, and the enzyme activity to be assayed is ornithine carbamoyl transferase. 3. The measuring method according to claim 1, wherein the substrate to be measured is ornithine. 4. The measuring method according to claim 1, wherein the reagent composition further contains ornithine carbamoyl transferase and phosphoric acid, and the substrate to be measured is citrulline. 5. The method according to claim 1, wherein the product is a reduced pyridine nucleotide coenzyme. 6. The measuring method according to claim 5, wherein the reduced pyridine nucleotide coenzyme is measured by absorption in the ultraviolet, fluorescence intensity or absorption of the produced dye in the presence of a redox indicator. 7. The measuring method according to any one of claims 1 to 4, wherein the product is glutamic acid. 8. The measuring method according to claim 7, wherein the produced glutamate is allowed to act on glutamate dehydrogenase and an oxidized pyridine nucleotide coenzyme to measure the produced reduced pyridine nucleotide coenzyme. 9. The measuring method according to claim 7, wherein glutamate oxidase is allowed to act on the produced glutamate to measure the produced hydrogen peroxide.