JPS623800A - Determination of ammonia or atp - Google Patents

Abstract

PURPOSE:To enable the quantitative determination of ammonia or ATP in high sensitivity, by reacting a specimen liquid with a glutamine synthetase in the presence of L-glutamic acid and one of the ammonia and ATP other than the one to be determined and measuring the compound produced by the kinase reaction. CONSTITUTION:A specimen liquid is made to react with a glutamine synthetase in the presence of L-glutamic acid and one of the ammonia and ATP other than the one to be determined (ATP in the case of determining ammonia and ammonia in the case of determining ATP). A kinase is added to the produced ADP and a phosphorus compound for kinase substrate, and the produced kinase reaction product is determined. The reaction completes within about 3min by the use of a glutamine synthetase in contrast with the reaction with glutamic acid dehydrogenase which lasts longer than 6-7min.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a method for quantifying ammonia or ATP using an enzymatic method.

[Conventional technology]

Conventionally, a chemical measurement method called the indophenol method has been mainly used to quantify ammonia. Furthermore, as an enzymatic measurement method, a method using 1-glutamic acid dehydrogenase (for example, see Japanese Patent Publication No. 57-21995) is known. The problem is that a large amount of glutamic acid dehydrogenase is required for one measurement. Furthermore, as other enzymatic measurement methods, a method using carbamate kinase (see JP-A-59-213399) or a method using carbamoyl phosphate synthase (see JP-A-60-47698) is known.

It has been reported that the carbamate kinase used in the former method is generally unstable and is inactivated by treatments such as dialysis. Therefore, it is necessary to purify or preserve the enzyme in the presence of an SH group protecting agent such as reduced glutathione or 2-mercaptoethanol. However, these SH
When quantifying ammonia in a test solution using a carbamate kinase preparation containing a group-protecting agent and using a system that includes an oxidase-heroxidase-hydrogen donor chromogen reaction system, the presence of an SH group-protecting agent is due to peroxidase. Since the color development system of the hydrogen donor is inhibited, accurate measurement values cannot be obtained and a negative error is given. Since the carbamoyl phosphate synthase used in the latter method can only be obtained from animal organs, there is a problem in supplying the enzyme in large quantities and at low cost.

In addition, as a method for quantifying ATP, there is a conventional method in which darcochloride is applied and the generated NADPH is measured at 340 nm [Methods Op Enzymatic Analysis,
Vol. 7, p. 346 (1985)), or a method is known in which luciferase is allowed to act on ATE in the presence of luciferin and Mg2+, and ATP is quantified from the generated light intensity at 562 nm [Vol. 7, p. 346 (1985)]. Page 357 (1
985)).

However, in the former method, more NADPH than one ATE molecule
Since only one molecule is produced, the sensitivity is low, and the latter method has problems in that luciferin and luciferase are expensive.

[Problems that the invention attempts to solve]

The purpose of the present invention is to: 1. In view of the above-mentioned current situation, 1. ammonia or A.
The object of the present invention is to provide a highly sensitive, simple, and inexpensive method for quantifying TP.

[Means for solving problems]

To summarize the present invention, the present invention provides ammonia or ATP.
In quantifying ammonia or ATP in a test solution, glutamine synthetase is allowed to act on the test solution in the presence of L-glutamic acid and another substance that is not the component to be quantified, The method is characterized in that a kinase is made to act on the generated ADP and a phosphorus compound for a kinase substrate, and the generated kinase reaction product is quantified. The other substance that is not the component to be quantified is ATP in the case of ammonia quantification, and ammonia in the case of ATP quantification.

The present invention is a quantitative method that utilizes a cycling reaction, and is highly sensitive.1 Colorimetric determination is possible. In particular, the glutamine synthetase of Micrococcus spp. and Brevibacterium spp.
% and is extremely stable, making it a new ammonia or AT product in the field of clinical testing.
This provides a method for quantifying P.

The test liquid used in the present invention includes ammonia or A
It is sufficient that the test liquid contains either one of T]', and examples thereof include a test liquid that contains ammonia or ATP in advance, and a test liquid that contains ammonia or ATP produced by an enzyme reaction. Examples of reactions that produce ammonia by enzymatic reactions include those listed below, and it is possible to measure their enzyme activity and determine the amount of substrates or products.

1.7 Sparaginase (EC! 3.5.1.1) L-asparagine + HIO-+ J, -asparagine + N
H32, aspartase (ICO4,3,1,1)L-
Aspartic acid → fumaric acid 1NH.

3. Adenine deaminase (Mg3.5.4.2) Adenine 10HzO → Hyboxanthin 10NH34, Adenosine deaminase (to3.5.4.4) Adenosine 10H
xO→Inosine 1NHs5, adenosine monophosphate deaminase (EC3/5/4/6) AMP + Hto
→ Engineering MP + ME s6, adenosine IJ acid theaminase (to3.5.4.17) adenosine phosphate + H, O → inosine phosphate + mHs7, -yminedehyde a genease (KOl, 4.99.3) 8, arginine>
Deaminase (to3.5.3.6) L-arginine →
L-citrulline NHs9, amidase (KO3,5,
1,4) Monocarboxylic acid amide + J (,0 → monocarboxylic m + yns10, ω-amidase (EC3,5,1,
3) 2-ketoglutamic acid + H, O → 2-ketoglutaric acid + NH311, urease (KO3, 5, 1, 5
) Urea 10H! O → 2 NHz + OO! 12, ureidosuccinase (B:03.5.1.7) 13, guanine deaminase (KO3,5,4,3) guanine + H,
O → xanthine + NH.

14, Guanosine deaminase (IO3,5,4,15
) Guanosine + H, O → Xanthosine + NH3 Nicotinamide 1 H, O → Nicotine m + NHsL-Histidine →
Urocanic acid 1NH.

In addition, there are the following examples of reactions that generate ATP by enzymatic reactions.

It is possible to measure enzyme activity and quantify substrates or products.

L-Tyrosine+ATP-)-tRNA2.7 Cetyl-OoA Synthetase (EO6,2,1,1)AMP-1
-PPi+acetyl-0oA ATP+acetic acid+OoA
SHAMP + birophosphoric acid + AMP 2 ATP + deamide-NAD + NH.

These are merely examples and do not limit the scope of the present invention in any way.

Next, although the glutamine synthetase used in the present invention is present in the brains and livers of various higher animals, bean seeds, Escherichia coli, and other microorganisms, Micrococcus spp. and Brevibacterium spp. Enzymes obtained from glutamine synthetase-producing bacteria selected from the genus
First, the properties of the glutamine synthetase used in the present invention will be shown.

Enzyme-chemical and physicochemical properties of glutamine synthetase (1) Action: This enzyme catalyzes the reaction of synthesizing glutamine from L-glutamic acid and ammonia using the chemical energy of ATP, as shown in the following formula.

L-glutamic acid + ammonia + ATP → L-glutamine + ADP + inorganic phosphate (2) Substrate specificity As a niamin group receptor, this enzyme shows extremely high specificity for L-glutamic acid. Approximately 30% activity was also observed when hydroxylamine was used instead of ammonium chloride.

(J optimal pH and pH stability: The optimal pH of this enzyme is 7.0 to 8.0. Also, when this enzyme was treated with each pkl at 50'C for 10 minutes, the pH was 6.0 to 8.0. C that is stable in the range of ~9.0
[4) Optimal temperature and thermostability: The optimal temperature of this enzyme is around 50°C, and when treated at each temperature for 10 minutes at pa 7.0, the temperature of 50°C
It is stable up to C.

(ω Molecule Company: The molecular weight of this enzyme was determined by the sedimentation equilibrium method, and the partial specific volume was determined to be 0475.
Assuming that, it is approximately 500,000.

Moreover, since the 5DS-polyacrylamide gel electrophoresis method shows that the amount is approximately 60,000 to 65,000, this enzyme is an octamer consisting of eight identical subunits.

(6) Inhibition: After examining the damage caused by various metabolites, there are only a few medical records for amino acids such as glycine, L-tryptophan, and D-threonine, but for nucleotides,
Inhibition by nucleosides is large; adenosine, A
M.P.

Its activity is strongly impaired by ADP and the like.

('r) Effect of metal ions: Enzyme reactions require Mg 1+ as metal ions, and SM
34% activity even with n"0 (8) Km value: When the Km value for each substrate was determined, it was 7.9 x 10-" for L-glutamic acid and 5.9 for M% ammonium chloride. 0 x 10-'M and 1.2 x 10-'M for ATP.

(9) Enzyme activity measurement method: Enzyme activity was measured as follows.

soomM sodium glutamate solution 0mi
x250mM ammonium chloride solution 0.1ffiA! ,
75mM ATP solution 0.1ml s 300mM
Mg01g solution 0.1ml, 1M imidazole-hydrochloric acid solution (pH 7,0) Q, lml, water 0.4rn
l and appropriately diluted enzyme solution Q, l mi , reaction liquid fl 1. The reaction was carried out at 37°C for 10 minutes in Orat, and the produced inorganic phosphoric acid was measured using the method of Fuisuka-Sabarou, and the produced glutamine was separated using paper chromatography and measured using the ninhydrin color method. One unit of the determined ◇glutamine synthesis activity was expressed as the amount of enzyme that produces 1 μM of inorganic phosphoric acid or glutamine per minute in the above reaction system.

The method for producing glutamine synthetase is described in JP-A-57-33594.

[For production]

Quantification method of ammonia and ATP The present inventors have conducted extensive studies on a method for quantifying ammonia or ATP in a test solution. ・The quantification of ammonia or ATP is highly sensitive, simple, and inexpensive by using glutamine synthetase. I found the law. In the presence of L-glutamic acid and ATP, ammonia in the test solution produces L-glutamine, ADP, and inorganic phosphate by the action of glutamine synthetase, and ATP in the test solution produces L-daltamic acid and ammonia. 1 by the action of this enzyme in the presence of
Produces L-glutamine, ADP and % inorganic phosphorus.

When a kinase is allowed to act on ADP produced by the enzymatic reaction in the presence of a phosphorus compound for the kinase substrate, a kinase reaction product and ATP are produced, and the cycling reaction progresses.
TP is amplified. An example of this reaction is ADP and phosphoenolpyruvate by pyruvate kinase.
An example is a reaction that produces TP and pyruvic acid. A known method can be used to quantify the produced pyruvic acid. For example, by reacting pyruvate with NADH and lactate dehydrogenase, it is determined by the decrease in absorbance of NADH at 340 nm, or by reacting pyruvate oxidase and measuring the consumed oxygen with an oxygen electrode, or by reacting with pyruvate oxidase and measuring the consumed oxygen with an oxygen electrode, or by reacting with pyruvate oxidase and measuring the consumed oxygen with an oxygen electrode, or by reacting with pyruvate oxidase and measuring the consumed oxygen with an oxygen electrode, or by reacting with pyruvate oxidase and measuring the consumed oxygen with an oxygen electrode, or by reacting with pyruvate oxidase and measuring the consumed oxygen with an oxygen electrode. can be quantified at 500 nm using a color reaction using a peroxidase system, for example, the 4-aminoantipyrine-phenol-peroxidase method.

The buffer used in the reaction is not particularly limited, and phosphate buffer, imidazole buffer, Tris buffer, glycylglycine buffer, etc. are suitable.Buffers with pH 6 to 9S and preferably pH 7 are used. Glutamine synthetase is usually used in an amount of 0.2 to 2 Q units, preferably 1 to 5 units. Kinases such as hahirubin increase kinase are used in amounts of 1 to 1 Q0 units, preferably 5 or more units. Also, L-glutamic acid concentration is 1 to 5 QmM, preferably 5 to 1 QmMs
Phosphorus compounds 1 for ATP and kinase substrates, such as phosphoenolpyruvate and NADH, may be used in an amount at least equal to or greater than the molar amount of ammonia or ATP in the test solution. The reaction temperature is 20 to 400C and the reaction time is 1 to 20 minutes.

〔Example〕

The present invention will be explained below with reference to examples, but the present invention is not limited to the scope of the following examples.

Example 1 Quantification of ammonia (pyruvate kinase-lactate dehydrogenase reaction system) 1 M imidazole-hydrochloric acid buffer (pH, 7.0)
0.3ml 150mmL-sodium glutamate
Q, l mi3 Q mM ATE
Q, 1mt3QmM
Phosphoenolpyruvate 011d
7.5mM NADHQ, 1ml 1.5M MgO1x
0.1 engineering! 2.7M KOI
0.1m 130 units 7mb
Glutamine synthetase (synthetic activity) 0.11
rLl water
To 1.3 ml of the above mixed solution 0.1 ml of 1.2, 3.4 and 5 mM ammonium chloride solutions were added, respectively, and reacted at 37° C. for 5 minutes. As shown in FIG. 1, a good linear relationship was obtained between the amount of ammonium chloride added and the amount of decrease in absorbance at 340 nm.

Example 2 Reaction thread) 1 M imidazole-hydrochloric acid Q bacterial solution (pH 7.0)
0.3m/150mM Sodium L-glutamate Q, l m113 Q mM A
TP 0.1m
130 mM Phosphoenolpyruvate 0.112
4.6mM 4-7 Minoantivirine
Q, lm1420mM phenol
0.1 rILtl, 5 M
MgO1z Q,
1mA2.7M KOI
O,1mA6mM Thiamine pyrophosphate 0.1 mAlmM XFA
D 0.1rrl
A30 monowmt glutamine synthetase (synthetic activity),
Q, 1mj water
1.4 Mixed solution 3. Onle; 1, 2, 3.4 and 5mM ammonium chloride solution Q
, 1 ml were added, and reacted at 37°C for 5 minutes. @2 As shown in the figure, a good linear relationship was obtained between the amount of ammonium chloride added and the increase in absorbance at 500 nm. Example 3 Quantification of ATP 1 M imidazole-hydrochloric acid buffer (pH 7,0)
0.3ml115QmML-Sodium Glutamate
0.1 ml 50 mM ammonium chloride Q, l mi13 Q m
M Phosphoenolpyruvate Q, l
mi7.5mM NADH041m1j 1.5M Mg011
0.1m12.7M KOI
o, xmt30 single V'mt
Glutamine synthetase (synthetic activity) o, 1
mi water 1
．． 3ml of the above mixed solution 3. Q 0.25 in ml, 0
．． 50.0.75 and l, QmM ATP solution 0.1
Add each elm.

and reacted at 37°C, and NADlK after 3 and 5 minutes.
The difference in decrease was measured. As shown in FIG. 3, a good linear relationship was obtained between the amount of ATP added and the amount of decrease in NADH.

Reference Example: Comparison of reaction rates when quantifying ammonia using glutamine synthetase and glutamate dehydrogenase. When quantifying, we conducted a comparative test to determine which method reaches the end point more quickly. When using glutamine synthetase, the mixed solution composition is the same as in Example 1 except for using 45 units/rnl (synthetic activity) of glutamine-mediated enzyme solution Q, lmi, and when using glutamate dehydrogenase. The mixed solution composition is 300mM To'J-HCl buffer (pH 8,0) 1.0mls 225mM 2-
'r) Gluta A/Wisteria solution 0.1 mε, 5 mM
NADPH solution 01, 300 mM ammonium chloride solution Q, l mt·45 units/me glutamic acid dehydrogenase solution Q, l mA and water 1.5 mi 0
Both of the above mixed solutions 3. 0.5°10 and 2. to Qrnb. 50 μl of Q mM ammonium chloride solution was added to each, reacted at 37° C., and the absorbance at <340 nm was measured over time based on the decrease in NADH. As shown in Figure 4, when glutamine synthetase is used (A), the reaction is completed in 3 minutes at any ammonia concentration, whereas when glutamate dehydrogenase is used (B), the reaction is completed within 3 minutes. After ~7 minutes had elapsed, the reaction had not been completed at any ammonia concentration.

〔Effect of the invention〕

As explained in detail above, according to the quantitative method of the present invention, ammonia or ATP can be quantitatively determined with high sensitivity and in a convenient manner. Furthermore, the glutamine synthetase used in the present invention can be prepared at a lower cost than microorganisms, and has excellent effects as a clinical test sample.

[Brief explanation of drawings]

FIG. 1 is a graph showing the relationship between the amount of ammonia and the absorbance difference at 340 nm for the first test line in Example 1 of Method No. 1 of the present invention. Figure 2 shows Example 2 of the quantitative method of the present invention.
2 is a graph showing the calibration curve in terms of the relationship between the amount of ammonia and the difference in absorbance at 500 nm. Figure 3 shows the calibration curve in Example 3 of the quantitative method of the present invention with ATP residue and 340 nm.
It is a graph shown in relation to the absorbance difference. Figure 4 shows the reaction time (minutes) and the reaction rate at 340 nm for quantifying ammonia using the glutamine synthetase (A) used in the present invention and the conventional glutamate dehydrogenase (B). This is a graph shown in relation to the absorbance difference.

Claims (1)

[Claims] 1. In quantifying ammonia or ATP in a test solution, glutamine and a synthetic enzyme are allowed to act on the test solution in the presence of another substance other than the component to be quantified and L-glutamic acid. A method for quantifying ammonia or ATP, which comprises causing a kinase to act on the generated ADP and a phosphorus compound for a kinase substrate, and quantifying the generated kinase reaction product. 2. The method for quantifying ammonia or ATP according to claim 1, wherein a glutamine synthetase, a kinase, and a phosphorus compound for substrate are present simultaneously in the test solution.