JPH0773514B2 - Ammonia determination method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial application] The present invention relates to a method for quantifying ammonia using an enzyme.

[Conventional technology]

Conventionally, a chemical measurement method called an indophenol method has been mainly used as a method for quantifying ammonia. As an enzymatic assay method, a method using glutamate dehydrogenase (see, for example, Japanese Patent Publication No. 57-21995) is known.
This method has a problem that a large amount of glutamate dehydrogenase is required for measurement because the Km value of the glutamate dehydrogenase used for ammonia is large. As another enzymatic assay method, a method using carbamate kinase (JP-A-59-213399) or a method using carbamoyl phosphate synthase (JP-A-60-4769).
No. 8) is known. It is reported that the carbamate kinase used in the former method is generally unstable and is inactivated by treatment such as dialysis. Therefore, it is necessary to purify or store the enzyme in the presence of SH group protecting agents such as reduced glutathione and 2-mercaptoethanol. However, when using these carbamate kinase preparations containing SH group protecting agents to quantify mammonia in the test solution in a system containing oxidase-peroxidase-hydrogen donor chromogen, the presence of SH group protecting agents Since it interferes with the coloring system, an accurate measurement value cannot be obtained and a negative error is given. The latter method has a problem in that the carbamoylphosphate synthase used can only be obtained from animal organs, and that the enzyme can be supplied in large quantities and at low cost.

[Problems to be solved by the invention]

In view of the above situation, an object of the present invention is to provide a rapid and accurate method for quantifying ammonia using an enzyme that can be easily supplied.

[Means for solving problems]

When the present invention is outlined, the present invention relates to a method for quantifying ammonia, in which alcohol dehydrogenase, which requires an ammonium salt for activity expression in quantifying ammonia in a test solution, its substrate, electron acceptor and When the test solution is made to react, the activity of the alcohol dehydrogenase is expressed according to the amount of ammonia in the test solution, and therefore the ammonia is quantified by measuring the activity.

As a result of intensive studies on the method for quantifying ammonia, the present inventors have found that there is a proportional relationship between the reaction rate of an alcohol dehydrogenase, which requires an ammonium salt for activity expression, and the ammonium salt concentration, and completed the present invention.

The test liquid to be used in the present invention may be any liquid as long as it contains ammonia, and there are biological samples such as serum and urine containing ammonia in advance and foods, and there is a liquid containing ammonia produced by an enzymatic reaction. . Examples of reactions that produce ammonia by an enzymatic reaction include the following, but it is possible to measure their enzymatic activities and quantify their substrates or products.

1. asparaginase (EC3.5.1.1) L- aspartic + H ₂ O → L- aspartic acid + NH ₃ 2. aspartase (EC4.3.1.1) L- aspartic acid → fumaric acid + NH ₃ 3. adenine deaminase (EC3. 5.4.2) Adenine + H ₂ O → hypoxanthine + NH ₃ 4 adenosine deaminase (EC3.5.4.4) Adenosine + H ₂ O → inosine + NH ₃ 5. Adenosine monophosphate deaminase (EC 3.5.4.6) AMP + H ₂ O → IMP + NH ₃ 6. Adenosine phosphate deaminase (EC3.5.4.17) Adenosine phosphate + H ₂ O → Inosine phosphate + NH ₃ 7. Amine oxidase (EC1.4.3.4) (EC1.4.3.6) Amine + H ₂ O + O ₂ → Aldehyde + NH ₃ + H ₂ O ₂ 8. Amine dehydrogenase (EC 1.4.99.3) Amine + Oxidized phenazine methosulfate + H ₂ O ₂ → Aldehyde + Reduced phenazine methosulfate + NH
₃ 9. arginine deiminase (EC3.5.3.6) L- arginine → L-citrulline + NH ₃ 10. amidase (EC3.5.1.4) monocarboxylic acid amide + H ₂ O → monocarboxylic acid + NH ₃ 11.ω- amidase (EC3.5.1.3) 2-ketoglutamic acid + H ₂ O → 2-ketoglutaric acid + N
H ₃ 12. Urease (EC3.5.1.5) Urea + H ₂ O → 2NH ₃ + CO ₂ 13. Ureidosuccinase (EC3.5.1.7) N-carbamoyl-L-aspartic acid + H ₂ O → L-aspartic acid + CO ₂ + NH ₃ 14. Guanine deaminase (EC3.5.4.3) Guanine + H ₂ O → xanthine + NH ₃ 15. Guanosine deaminase (EC3.5.4.15) Guanosine + H ₂ O → xanthosine + NH ₃ 16. Glutaminase (EC3.5.1. 2) L-glutamine + H ₂ O → L-glutamic acid + NH ₃ 17. Creatinine deiminase (EC3.5.4.21) Creatinine + H ₂ O → N-methylhydantoin + NH ₃ 18. Cytidine deaminase (EC3.5.4.5) Cytidine + H ₂ O → uridine + NH ₃ 19. Cytosine deaminase (EC3.5.4.1) Cytosine + H ₂ O → uracil + NH ₃ 20. Citrulinase (EC3.5.1.20) L-citrulline + 2H ₂ O → L-ornithine + CO ₂ + NH ₃ 21 .Transglutaminase (EC2.3.2.1 3) N ² -R-glutaminyl peptide + RNH ₂ → NH ₃ + N ² -R-N ⁵ -R-glutaminyl peptide 22. Threonine dehydratase (EC4.2.1.16) L-threonine + H ₂ O → 2-ketobutyric acid ＋ NH ₃ ＋ H ₂ O 23. Nicotinamide amidase (EC3.5.1.19) Nicotinamide + H ₂ O → Nicotinic acid + NH ₃ 24. Nicotinamide nucleotide amidase (EC3.5.1.4
2) Nicotinamide mononucleotide + H ₂ O → nicotinic acid-D-ribomucreotide + NH ₃ 25.L-histidine ammonia lyase (EC4.3.1.3) L-histidine → urocanic acid + NH ₃ 26. Phenylalanine ammonia lyase (EC4.3.1.3) EC4.3.1.5) L-phenylalanine → trans-cinnamic acid + NH ₃ or more, these are examples and do not limit the scope of the present invention.

Next, the alcohol dehydrogenase used in the present invention catalyzes the reaction of primary alcohol + electron acceptor → aldehyde + reduced electron acceptor and is classified as enzyme number 1.1.99.8. Is an enzyme that requires. This enzyme is generally methane and methanol-assimilating bacteria,
For example, Pseudomonas AM-1 (NCIB913
3), Syudomonas SB M-27, or Methylococcus capsulatu
Purified samples are easily obtained from s).

This enzyme activity is usually obtained by reacting phenazine methosulfate (PMS) as an electron acceptor in the presence of an ammonium salt using a primary alcohol such as methanol, ethanol or n-propanol, usually methanol as a substrate, It can be measured by quantifying the product formaldehyde or reduced PMS by a known method. The amount of formaldehyde can be quantified, for example, by allowing aldehyde oxidase to act on formaldehyde and measuring the amount of oxygen consumed or the amount of hydrogen peroxide produced. on the other hand,
The amount of reduced PMS can be quantified by the decrease in the absorbance of DCPIP when conjugated with the dye 2,6-dichlorophenol indophenol (DCPIP). When a tetrazolium salt is used as the dye, it can be quantified from the increase in the absorbance of the formazan dye produced.

This enzyme activity is indicated by 150 mM Tris-HCl buffer (pH 9.
0) 1.0 ml, 500 mM ammonium chloride 0.1 ml, 200 mM methanol 0.1 ml, 10 mM PMS 0.1 ml, 2 mM DCPIP 0.1 ml, distilled water 1.5 ml and appropriately diluted enzyme solution 0.1 ml, total reaction volume 3.0 ml , 1 μmol DC per minute at 37 ℃
The amount of enzyme that reduces PIP was defined as 1 unit.

Although it was known that an ammonium salt was required for the elementary activity, the present invention was completed by finding that there is a proportional relationship between the ammonium salt concentration and the enzymatic reaction rate in the presence of a low concentration of ammonium salt. .

The buffer solution used in the present invention is not particularly limited, and a phosphate buffer solution, an imidazole buffer solution, a Tris buffer solution, a glycylglycine buffer solution, and the like are suitable, and a pH 7 to 10, preferably pH 8 to 9 buffer solution. Is used. The alcohol dehydrogenase is usually used in an amount of 0.01 to 1.0 unit, preferably 0.05 to 0.5 unit. As the electron acceptor, in addition to phenazine methosulfate, phenazine ethosulfate, Meldola blue, etc. are suitable, and usually used at a concentration of 0.1 to 1 mM. When using 2,6-dichlorophenol indophenol as a dye, 0.01-1 mM, nitroblue tetrazolium, 3- (4,5-dimethyl-2-thiazolyl)
-2,3-Diphenyltetrazolium bromide, 3-
(P-Indophenyl) -2- (p-nitrophenyl)
When a tetrazolium salt such as -5-phenyltetrazolium chloride or tetranitroblue tetrazolium is used, a concentration of 0.1 to 1 mM is suitable.

According to the present invention, the amount of ammonia can be accurately determined within 1 minute after the start of the reaction, which can be said to be an effective measurement method for high-speed trace measurement.

〔Example〕

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

Example 1 (phenazine methosulfate, 2,6-dichlorophenol indophenol system) 0.3M glycylglycine-NaOH buffer 0.5 ml solution (pH 9.0) 0.2M methanol 0.1 ml 10 mM phenazine methosulfate 0.1 ml 2 mM 2,6-dichlorophenol 0.1 ml indophenol 0.5 unit / ml alcohol dehydrogenase 0.1 ml H ₂ O 2.1 ml To the above mixed solution 3.0 ml, 0.1 ml of 2,4,6,8 and 10 mM ammonium chloride solution was added. Add and react at 37 ℃ to start reaction 1
The change in absorbance between 5 seconds and 45 seconds was measured. As shown in FIG. 1, a good linear relationship was obtained between the amount of ammonium chloride added and the amount of change in the absorbance at 600 nm.

Example 2 (phenazine methosulfate, nitroblue tetrazolium system) 0.3M Tris-hydrochloric acid buffer solution (pH 8.0) 0.5 ml 0.2M methanol 0.1 ml 10 mM phenazine methosulfate 0.1 ml 2 mM nitroblue tetrazolium 0.3 ml 6 units / ml Alcohol dehydrogenase 0.1 ml H ₂ O 1.9 ml To each 3.0 ml of the above mixed solution, 0.1 ml of 2,4,6,8 and 10 mM ammonium chloride solution was added, and the mixture was reacted at 37 ° C for 15 seconds. The absorbance change was measured after 45 seconds. As shown in Fig. 2, the amount of ammonium chloride added and 560n
A good linear relationship was obtained for the change in absorbance at m.

[Reference example]

An example of the production of the alcohol dehydrogenase used in the present invention is shown.

Methanol-assimilating bacterium C. domonas AM-1 (Ps
eudomonos AM-1) [NCIB9133] with 0.5% methanol,
Peptone 0.1%, yeast extract 0.2%, (NH ₄ ) SO ₄ 0.26
%, KH ₂ PO ₄ 0.24%, K ₂ HPO ₄ 0.56%, MgSO ₄ -7H ₂ O 0.0
A 5% crude medium (pH 7.0) was inoculated, and flask culture was carried out at 30 ° C. for 32 hours to obtain a seed culture solution. Put the same composition medium 20 in 30 jar fermenter, sterilize, and then 600 ml of the seed culture
Inoculate and incubate at 30 ° C for 28 hours at an air flow rate of 10 per minute and 250 per minute.
When cultivated at a stirring speed of rotation, 0.1
Units of alcohol dehydrogenase were produced. Since this enzyme is defective in cells, solid-liquid separation is first performed by continuous centrifugation.
After suspending the obtained bacterial cells in 50 mM phosphate buffer (pH 7.0),
The supernatant obtained by ultrasonic treatment and centrifugation was used as a crude enzyme solution. The enzyme solution was adjusted to pH 4.5 with 1N hydrochloric acid, the resulting precipitate was removed by centrifugation, and then dialyzed against 20 mM Tris-hydrochloric acid buffer solution (pH 8.0). Next, the dialyzed enzyme solution is passed through a DEAE-Sepharose CL-6B column (φ2.5 cm × 10 cm) (manufactured by Pharmacia) equilibrated with 20 mM Tris-hydrochloric acid buffer solution (pH 8.0) in advance and the non-adsorbed section is subjected to the collodion bag. After concentrating with Sef Acrylic S
It was passed through a −300 column (φ2.5 × 100 cm) (Falmatia). As a result, the active fraction was eluted as a single peak. This active fraction is dialyzed against 10 mM phosphate buffer (pH 7.0) and then lyophilized to give a standard activity of 1.6 units / mg, 475 mg of the standard product.
Got The total absorptivity was 38%, and the specific activity increased 7.6 times. The purified sample showed a single band by polyacrylamide gel electrophoresis.

〔The invention's effect〕

As described in detail above, according to the quantification method of the present invention, ammonia can be quantified with high sensitivity and specificity.
Further, the alcohol dehydrogenase used in the present invention can be prepared at a lower cost than microorganisms and has an excellent effect as a clinical test reagent.

[Brief description of drawings]

FIG. 1 shows the calibration curve in Example 1 with the amount of ammonia.
2 is a graph showing the relationship with the amount of decrease in the absorbance of 2,6-dichlorophenol indophenol at 600 nm. FIG. 2 is a graph showing the relationship between the amount of ammonia and the amount of increase in the absorbance of the formazan dye at 560 nm in the calibration curve in Example 2.

Claims (1)

[Claims] 1. When quantifying ammonia in a test solution, an alcohol dehydrogenase, which requires an ammonium salt for activity expression, is allowed to react with a test solution in the presence of a substrate and an electron acceptor. A method for quantifying ammonia, characterized in that the amount of ammonia is measured from the reaction rate of.