JPH03180200A - High-sensitivity determination - Google Patents

Abstract

PURPOSE:To accomplish the title determination of putrescine and cadaverine by acting specific enzymes on putrescine and/or cadaverine and by determining the resulting product. CONSTITUTION:Putrescine and/or cadaverine is (are) subjected to putrescine transaminase (pref. putrescine: pyruvic acid transaminase), an amino group donor (e.g. L-alanine, glycine) and a polyamine oxidase (e.g. putrescine oxidase of microbial origin) (normally at a pH6-9 at 25-45 deg.C for 10min-5hr) to carry out an amplificative reaction by enzymatic cycling technique, and the resulting reaction products such as hydrogen peroxide, ammonia and alpha-keto acid are determined, thus quantifying the putrescine, cadaverine, etc.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a novel method for quantifying putrescine and/or cadaverine with extremely high sensitivity, and is also applicable to the examination of the state of each organ in a living body. It is effective and can be widely used in the medical field such as clinical testing.

[Prior Art] Conventionally, putrescine and cadaverine have been quantified by a method using a high-performance liquid chromatography device, an enzymatic colorimetric method, or a method using chemiluminescence.

[Problem to be solved by the invention] In recent years, there has been active development of methods for accurately measuring trace amounts of samples, which can be called a proposition of analytical chemistry.
It has also been desired to develop a method for quantifying trace amounts of putrescine and cadaverine, which are medically important metabolic components. Various high-sensitivity measurement methods have been developed, such as fluorescence measurement and luminescence measurement, but when the sample is body fluid, errors are likely to occur due to re-enching, which increases the blinded value due to substances contained in the body fluid. In true liquid chromatography and enzymatic colorimetric assay, the limit of quantification is approximately 1 μH at most. Furthermore, although the chemiluminescence method allows measurement in the nM region, it has the drawback of requiring special equipment for measurement.

On the other hand, the principle of the enzyme cycling method itself is a method that has been known for a long time (Biochemistry Experiment Course 5, Enzyme Research Methods, Volume 1, 121-135. Nippon Kagaku Complete Edition, Tokyo Kagaku Doujin), which was used for trace analysis of putrescine and cadaverine. It was not known as a technology applied to

[Means for Solving the Problems] As a result of intensive research to solve the above-mentioned conventional drawbacks, the present inventors have discovered that enzyme cycling can be achieved by adding putrescine transaminase together with a substrate to a conventional enzymatic quantitative determination system. The present invention was completed based on the discovery that hydrogen peroxide, ammonia, and α-keto acid, which are the actual measurement targets, can be amplified and generated.

That is, the present invention involves treating putrescine and/or cadaverine with putrescine transaminase, an amino group donor, and a boria ξ oxidase, performing an amplification reaction by an enzyme cycling method, and measuring the resulting cycling reaction product. This is a highly sensitive method for measuring putrescine and/or cadaverine.

Putrescine transaminase used in the present invention is an ω-
An enzyme that transfers an amino group to aminoalkyl aldehyde and regenerates putrescine and cadaverine, and is necessary for establishing the enzyme cycling system. As such enzymes, there are several conventionally known putrescine transaminases as shown below.

That is, diamine:α-ketoglutarate transaminase derived from Escherichia coli B described in Journal of Biological Chemistry Vol. 239, p. 783 (1964) and Agricultural Biological Chemistry Vol. 43, p. 1043 ( ω- derived from Pseudomonas bacterium F-126 described in (1979)
Aminoll: pyruvate transaminase or γ-anobutyric acid: α-ketoglutarate transaminase derived from Pseudomonas aeruginosa as described in Journal of Bacteriology, Vol. 128, p. 722 (1978).

These putrescine transaminases can also be used, but the Km value and pH stability of the enzyme may vary.

It is more advantageous to use putrescine:pyruvate transaminase described in Japanese Patent Application No. 1-45416, which is excellent in terms of thermostability and the like.

Putrescine:pyruvate transaminase will be described in detail below.

■ Action As shown in the following formula, a transamination reaction that acts on putrescine, which is an amino group donor, and pyruvic acid, which is an amino group acceptor, to produce 4-aminobutanal and L-alanine, and its reverse reaction. catalyze.

Nl2(CH2)4NH2+CH3COC00I(□N
H2(CH2)3CHO+ CH2Cl(Nl2)G
OQH■Substrate specificity (1) Amino group donors Putrescine, cadaverine, supermine; acts on gin and spermine, but has no effect on L-lysine and L-ornithine.

(2) Amino group receptor For pyruvic acid and glyoxylic acid and has no effect on α-ketoglutaric acid.

The strength of the action on these amino group donors and amino group acceptors is shown in Table 1 as relative activity values, with the action on putrescine and pyruvic acid set as 100, respectively.

Table 1 Substrate specificity Substrate relative activity (%) Amino group donor Putrescine Cadaverine Spermidine Spermine L-Lysine L-Ornithine Amino group acceptor Pyruvate Glyoxylate α-Ketoglutarate 00 23.8 30.7 11.9 00 72.7 ■Optimal pHpH9.5-10.5 (shown in Figure 1)■
pH stability pn 4.5 when stored at 30°C for 1 hour
It has residual activity of 90% or more at ~13.0.
(As shown in Figure 2) Other physical and chemical properties of putrescine:pyruvate transaminase are as follows.

1. Optimum temperature: 55-60°C for reaction at pH 10, 5, and 30 minutes. (shown in Figure 3) 2. Temperature stability P) I 7.5. Under the treatment conditions for 1 hour,
It has a residual activity of more than 90% at temperatures up to 70°C. Moreover, it is completely inactivated by treatment at 80°C for 1 hour.

(As shown in Figure 4) 3. Molecular weight 192,000±s, oo.

(Biosil TSK-250; measured by gel filtration method manufactured by Tosoh Corporation) 4. Molecular weight of subunit 48,000±5,00
0 (measured by SO3-polyacrylamide gel electrophoresis) 5. Number of subunits: 4 8. Km value: Using putrescine and pyruvate as substrates,
The Km value for putrescine determined under conditions of pH 10, 5, and 30°C is 0.21 M, and the Km value for pyruvic acid is 1.0 mA.

7. Coenzyme Pyridoxal Phosphoric Acid 8, Absorption Spectrum Has maximum absorption at 280 nm and 416 nm. (shown in Figure 5) 9. Inhibitor Various reagents and metal ion concentrations IIIM
Table 2 shows the effects on enzymes. It is strongly inhibited by enzyme inhibitors containing pyridoxal phosphate as a coenzyme, such as hydroxylamine, phenylhydrazine, and D-cycloserine. It is also strongly inhibited by parachloromercribenzoic acid, silver ions, and mercury ions.

Table 2 Effect of reagents and metal ions Relative activity of reagents and metal ions (%) Putrescine Table 3 shows the results of comparing the physical and chemical properties of pyruvate transaminase and conventionally known transaminases that can act on putrescine. .

The conventional transaminases in Table 3 are those described in the Journal of Biological Chemistry (J,
Biol, Che+s, ), vol. 239, p. 783 (1964).
Diamine from scherichiacolt B):
α-ketoglutarate transadenase, Agricultural Biological Chemistry (Agric,
Biol, Che+*, ), vol. 43, p. 1043 (
Pseudomonas (1979)
ω-amino acid: pyruvate transaminase from F-128, a bacterium belonging to the genus P. onas), and the Journal of
Bacteriology (J, Bacteriol,), 1
Pseudomonas aeru described in Vol. 28, p. 722 (1976).
It is a γ-aminobutyric acid:α-ketoglutarate transaminase derived from S. ginosa.

As shown in Table 3, putrescine:pyruvate transaminase is an enzyme that differs in substrate specificity from the conventionally known putrescine transaminase.

In addition, although it was obtained from a microorganism belonging to the genus Streptomyces, which is an actinomycete that grows at room temperature,
It has high heat resistance, with more than 90% residual activity even after one hour of heat treatment.

It was also found that it has an excellent property of being stable over a wide pH range of pn 4.5 to 13.0. The excellent heat resistance and pn stability of putrescine/pyruvate transaminase as described above are very useful properties when using this enzyme in industrial or clinical testing fields.

The method for measuring the enzyme activity of putrescine; pyruvate transaminase and the method for displaying the enzyme activity are as follows.

2mM putrescine, 2mM pyruvate and 0.02
2.0 ml of 0.1 M phosphate buffer (pH 7.5) containing % 0-A major nobenzaldehyde and 0.2 ml of analyte containing putrescine:pyruvate transaminase.
After mixing and reacting at 30°C for 30 minutes to 1 hour, the absorbance at 435 nu is immediately measured. From the increase in absorbance per minute (A), use the following conversion formula (1)
Subject 1. Calculate the enzyme activity value of putrescine:pyruvate transaminase per Oml.

The enzyme activity value is calculated by calculating the amount of enzyme that produces 1 μmole of 1-pyrroline per minute as 1 unit (μmole/win).
).

+ A 2.2 Enzyme activity value −-2:f ”0:2- (1
) putrescine: pyruvate transa[nase] The specific activity value of a completely purified enzyme is approximately 4.5 units/m
Indicates g-tan height. Furthermore, a single protein band is observed in polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate.

The amino group donor in the present invention may be any substrate for putrescine transaminase necessary for regenerating putrescine or cadaverine from ω-aminoalkyl aldehyde, depending on the action of the enzyme. Specifically, L-alanine, glycine,
Examples include L-glutamic acid and L-aspartic acid. Moreover, ω-aminoalkylaldehyde is 4-aminobutanal and 5-aminopentanal, and in an aqueous solution, 1-pyrroline and 2,3,4,5. - in equilibrium with tetrahydropyridine.

In the present invention, the boria ξ oxidase is not particularly limited as long as it catalyzes the reaction of oxidizing putrescine or cadaverine to produce ω-aminoalkyl aldehyde, hydrogen peroxide, and ammonia; enzymes may be used without particular restriction. For example, putrescine oxidase originating from microorganisms such as Rokotsucus, Nocardia, Aspergillus, and Pseudomonas;
Examples include putrescine oxidase derived from plants such as germinated soybeans, and diamine oxidase derived from animals such as pig kidney.

In addition, cycling reaction products refer to compounds that can be amplified and produced by enzymatic cycling reactions, and refer to hydrogen peroxide, ammonia, and α-keto acids. Examples of α-keto acids include glyoxylic acid, pyruvic acid, oxaloacetic acid, and α-ketoglutaric acid generated from amino group donors.

An example of the principle of the reaction in the present invention is as follows.

In addition, according to the present invention, compounds that produce putrescine and cadaverine, or 4-aminobutanal and 5-aminopentanal can be converted to putrescine and cadaverine, so compounds that produce these ω-aminoalkyl aldehydes can also be converted to putrescine and cadaverine. Be able to measure with high sensitivity.

A specific example of such a case is when puttingrescine oxidase derived from Micrococcus roseus or Pseudomonas fluorescens is applied to Spell-based gin to produce 4-aminobutanal, and then an enzyme cycling reaction is performed. For example, putrescine is produced by allowing Penicillium chrysogenum or rat liver-derived boria ξone oxidase to act on the main gin or spermine, and then enzymatic cycling is performed.

Any cycling product amplified by the enzyme cycling method can be quantified by conventionally known methods. For example, ammonia and α-ketoglutarate can be determined by an enzymatic method using L-glutamate dehydrogenase and NADH, pyruvate can be determined by an L-lactate dehydrogenase method, and oxaloacetate can be determined by an oxaloacetate decarboxylase method. Quantification of hydrogen peroxide can be easily performed, for example, by the conventionally known 4-aminoantipyrine-peroxidase method. Among the cycling reaction products, quantifying hydrogen peroxide is advantageous in that it can be measured with high sensitivity.

Furthermore, a more sensitive measurement method can be achieved by measuring amplified hydrogen peroxide using a chemiluminescence method.

For the above cycling reaction, the reaction solution volume is usually 0.
．． It is carried out in the range of 1 to 5 ml. The minimum quantifiable concentration of putrescine or cadaverine, which is the polyamine to be measured, is about 1 to 10 nM.

The concentration of putrescine transaminase used is usually in the range of 0.5 to 50 units per ml of reaction solution.

Amino group donors are usually used in a concentration range of 5 to 50 mW.

The concentration of polyamine oxidase used is usually 1 ml of reaction solution.
It ranges from 0.5 to 50 units per unit.

Typically, these enzymes and reagents are present simultaneously in the same solution to form an enzyme cycling reaction.

The above reaction is carried out in a pH range and temperature range in which both putrescine transaminase and polyamine oxidase can act, and usually the pH is 8-92 and the reaction temperature is 25°C-45°C.

The reaction time can be arbitrarily set within the range of enough time to sufficiently amplify and generate the cycling reaction product until it can be detected, but it depends on other reaction conditions and is usually in the range of 10 minutes to 5 hours.

The enzyme cycling reaction is stopped by adding a stopping reagent at the end of a predetermined period of time, and 1,8-cyaminooctane, pyruvic acid, etc. are used as the stopping reagent.

[Effect] The highly sensitive measurement method for putrescine and/or cadaverine of the present invention can detect extremely small amounts of putrescine and/or cadaverine with simple operations.
It also provides a method that can accurately quantify cadaverine, and is expected to make a significant contribution to the medical field such as clinical testing.For example, it enables application to polyamine analysis in serum. .

Next, the present invention will be specifically explained with reference to Examples, but the present invention is not limited thereto.

[Example] Production example 1 0.5% glucose, 0.4% polypeptone, 0.5%
Fish meat extract, 0.3% acetylputrescine, 0.2%
Medium consisting of salt and 0.02% antifoam (pH 7.5)
Pour ijl into a 52-inch Erlenmeyer flask and heat at 120℃ for 20 minutes.
After autoclaving for a minute, Streptomyces averanius R-20 [Feikoken Bokuyori No. 5443] was seeded in this medium at 28°C. After culturing at 28° C. for 24 hours, the culture solution was added to a sterilized jar fermenter containing 150 L of a medium having the same composition as above for main culture. Culture conditions were 28℃;
After culturing for 18 hours at a stirring speed of 300 rpm and aeration of 1004 IL/win, the culture solution was centrifuged to collect bacterial cells.

Approximately 2.7 kg (wet bacterial weight) of the obtained bacterial cells was reduced to 40%.
10mM phosphate buffer containing ethanol (pH 7.5)
The cells were suspended for 12 minutes, and the suspension was continuously passed through a Dynomil cell crusher to disrupt the bacterial cells. The disrupted fluid was centrifuged using a continuous centrifuge to obtain supernatant synovial fluid.

The total activity of putrescine:pyruvate transaminase in this synovial fluid is 22,000 units, and the specific activity is 0.
It was 072ヱnift/B-tan 8°.

This supernatant synovial fluid was preliminarily soaked in 10mM phosphate buffer (pH 7,
5) was added to 41° DEAE-cellulose (Whatman ml) and stirred for 1 hour.
, 5) Washed with 15 Jl. Then, 10 mM phosphate buffer (pH 7,5) containing 0.5 M saline 5.
The enzyme component was eluted with f. [a enzyme activity = 15,
400:I nif), specific activity = o.

56 UNIFFT/B-TANK COVER] After desalting this enzyme solution by ultrafiltration, it was incubated at 65°C for 3
Heat treatment was performed for 0 minutes, and the resulting precipitate was removed by centrifugation. Ca enzyme activity = 12,200 units, specific activity = 2. lunift/B-tan^°ri] The enzyme solution thus obtained was preliminarily dissolved in 10mM phosphate buffer (pH'y, s).
The mixture was passed through a column of 1·i (7) DEAE-cellulose that had been equilibrated with 1·i (7) for adsorption. Column with similar buffer 2. After washing with I2, putrescine:pyruvate transaminase was eluted with a linear saline gradient. [a Enzyme activity = 8,820 units, specific activity = 3.
After desalting this eluate by ultrafiltration, ammonium sulfate was added to a concentration of 20%, and then 10 mM phosphate buffer (pH 7.5) containing 20% ammonium sulfate in advance was added. The enzyme was adsorbed through a column of 0 and 11 Butyl Toyopearl 850M (manufactured by Tosoh Corporation) that had been equilibrated with . After washing the column with the same phosphate buffer, putrescine z pyruvate transanase was eluted with a reverse linear gradient of ammonium sulfate. [Total enzyme activity = 7.43
0 units, specific activity = 4.4 units/B-Tambaco After concentrating the obtained enzyme solution by ultrafiltration, 1.
The active fraction was collected by gel filtration through a column filled with Sephacryl S-400 (manufactured by Pharmacia) in 7). [Total enzyme activity = 7,380 units, specific activity = 4
．． When the purity of the enzyme thus obtained was examined by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate, only one band was observed in both cases. , was confirmed to be pure putrescine:pyruvate transaminase.

In addition, the molecular weight of one enzyme was measured by gel filtration using Biosil TSK-250 (Tosoh Corporation II).
It was estimated to be about 192,000. Furthermore, when the molecular weight of the subunit was measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it was found to be approximately 48.0
Estimated to be 00. The molecular weight and the molecular weight of the subunits indicate that putrescine:pyruvate transaminase is an oligomeric enzyme composed of four subunits.

Next, the optimal PI (, pH stability,
The optimum temperature, temperature stability, and absorption spectrum were investigated.

[Optimal pH] 0.1M acetate buffer (pH 3, 2, 4, 4, 5, 7) containing 2mM putrescine, 2d pyruvate or 0.1
M phosphate buffer (pH 5, 0, 8, 0, 7, 0, 7, 5
) or 0.1M) lis(hydroxymethyl)aminomethane-hydrochloric acid buffer (pH 7, 0, 8, 2, 9, 2) or 0.1M carbonic acid 11fr solution (pH 9, 0, 9, 4, 1)
0, 8, 11, 7) or 0.1 M tetrahedron buffer (p
) (10, 5, 11, 4, 12, 1, 13, 0) 1.
0.2 ml of enzyme preparation (0,002 units) in 8 ml
were added and mixed, and the reaction was carried out at 30"C for 30 minutes. To 1.0+1 of this reaction solution, 0.21 of a 20% trichloroacetic acid aqueous solution was added and left at 0°C for 20 minutes. Then, , 0.02% 0-aminobenzaldehyde
．． After adding 1.01 ml of 5M phosphate buffer (pH 7.5) and leaving it at room temperature for 30 minutes, the absorbance at 435 nm was measured, and the enzyme activity value for each was calculated. After the above operations, calculate the relative activity with the highest enzyme activity value as 100%,
Figure 1 was obtained by graphing. From Figure 111, it can be seen that the optimum pH of this enzyme is in the range of 9.5 to 10.5.

[pH-stable 0mM vinegar W1 buffer (pH 3, 5, 4, 5, 5, 5,
5,9) or 50mM phosphate buffer (PI(5,6,
6,2,6,7,7,5°8.3) or 50mM tris(hydroxymethyl)aminomethane-hydrochloric acid buffer (
pH 7, 8, 8, 1, 8, 9) or 50 °C M borate buffer (pH 8, 2, 9, 3, 9, 9) or 50 mW carbonate buffer (pH 9, 4, 10, 6, 11, 7) ) or 5
0mM tetraborate buffer (pH 10, 8, 12, 7, 1
3,2,13,7) 0.95 Ugly 1 and enzyme preparation 0.05
m1 (50 units) and left at 30°C for 1 hour, then 0.02ml of each solution was added to 20mM phosphate buffer (
pH 7,5) 1.98 ml were mixed. Add 0.2ml of this enzyme solution to 2.0ml of 0.1M phosphate buffer (pH 7.5) containing 2mM putrescine, 2+aM pyruvate and 0.02% 0-aminobenzaldehyde, and react at 30°C for 1 hour. 435 n immediately after
The absorbance at m was measured, and each enzyme activity value was calculated.

After the above operations, the relative activity was calculated with the highest enzyme activity value as 100% and graphed to obtain Figure 2.

As is clear from Figure 2, one enzyme has a pH of 4.5 to 13.
．． It has a residual activity of 90% or more in the range of 0.

[Optimal temperature: 0 containing 25mM putrescine and 25mM pyruvate]
, IN carbonate buffer (pH 10,5) 1.8 ml and 0
, mix 2 ml of enzyme preparation (0,013 units), 30. 39, 44, 50. 56°80, 88.
The reaction was carried out for 30 minutes at each temperature of 79°C. 0.2 ml of 20% trichloroacetic acid solution was added to 1 ml of each reaction solution, and the mixture was left at 0° C. for 20 minutes. Then 0.02%
After adding 1.0 ml of 0.5 M phosphate buffer (pH 7.5) containing 0-aminobenzaldehyde and leaving it at room temperature for 30 minutes, the absorbance at 435 nu was measured, and the enzyme activity value for each was calculated. After the above operations,
Calculate the relative activity with the highest enzyme activity value as 100%,
93 figures were obtained by graphing. From FIG. 3, it can be seen that the optimum temperature for this enzyme is 55 to 60°C.

[Temperature-stable enzyme preparation (0,034 units/ml) diluted with 25mM phosphate buffer (pH 7.5) in 4. 30.
40. 50. 60. The samples were left at temperatures of 70°C, 75°C, and 80°C for 1 hour. 0.2ml of this enzyme solution to 2ml
M putrescine, 2mM pyruvate and 0.02% 0
- Add to 2.0 ml of 0,1 phosphate buffer (pH 7,5) containing aminobenzaldehyde and
Immediately after reacting for a period of time, the absorbance at 435 cm was measured, and the enzyme activity value for each was calculated.

After the above operations, the relative activity was calculated with the highest enzyme activity value as 100%, and graphed as shown in FIG. 4.

As is clear from FIG. 4, this enzyme has a residual activity of 90% or more at temperatures up to 70°C.

[Absorption spectrum] Enzyme preparation prepared by diluting purified enzyme with 20+aM phosphate buffer (pH 7,5) - (a) (180 μg/ml
), the absorption spectrum in the ultraviolet region was measured, and Figure 5-(a) was obtained. Enzyme preparation prepared in the same manner (
b) (910 μg/ml) was used to measure the absorption spectrum in the visible region to obtain Fig. 5-(b). Fifth
The figure shows that this enzyme has absorption maxima at 280 nm and 418 nm.

Example 1 To 2.0 ml of putrescine aqueous solutions of various concentrations, 10
0 mM) Lis-HCl buffer (pH 8,0),
1.0mM 2.4-dichlorophenol, 0.6mM
4-71 noantipyrine, putrescine oxidase from Micrococcus flavidus (5 units), putrescine from Streptomyces averanius R-20; pyruvate transaminase (62 units), peroxidase (1 unit) and 15 mM L-alanine Add 2.0 ml of cycling solution consisting of 3
The reaction was carried out at 7°C for 30 minutes. 20 d 1,8-diaminooctane solution (100% Tris-HCl buffer,
After stopping the reaction by adding pi 8.0) 0.111, the amplified hydrogen peroxide was quantified by measuring the absorbance at 510 rv. As a blind test, 2.0 ml of water was used instead of the putrescine aqueous solution.

The results are shown in FIG. 6(A), and an extremely good straight line was obtained, indicating that the cycling reaction was performed accurately. Moreover, FIG. 6 shows that this method is very sensitive, and the sensitivity was about 100 times that of the case where the cycling reaction was not performed.

Example 2 The same procedure as in Example 1 was performed except that 2.0 ml of cadaverine aqueous solutions of various concentrations were used instead of 2.0 ml of putrescine aqueous solutions of various concentrations. The results obtained are shown in Figure 6. As shown in (B). The calibration curve was a good straight line, and the measurement sensitivity was about 25 times that of the case where no cycling reaction was performed.

Comparative Example 1 Putrescine: The same operation as in Example 1 and Example 2 was performed except that pyruvate transaminase was not added, and the results obtained were as shown in FIGS. 6(C) and (D). was below the limit of quantification.

[Brief explanation of drawings]

Figure 1 shows the pH activity curve of putrescine: pyruvate transaminase (Ow acid buffer, Δ phosphate buffer, Lotris-hydrochloric acid buffer, carbonate buffer, and tetraborate buffer); The figure shows the same < pH stability (OII'
Figure 3 shows the temperature activity curve, Figure 4 shows the temperature activity curve. In FIG. 5, (a) shows the absorption spectrum in the ultraviolet region, and (b) shows the absorption spectrum in the visible region. FIG. 6(A) shows a calibration curve for quantifying putrescine according to the present invention, and FIG. 6(B) shows a calibration curve for quantifying cadaverine. FIG. 6(C) shows the results of quantifying putrescine without performing a cycling reaction, and FIG. 6(D) shows the results of quantifying cadaverine. Patent Applicant Tokuyama Soda Co., Ltd.Tsukuen 1st page H 5th closed page &('C) 50 00 50 00 50 r Original (nm) Figure 6 ICO200300400 Concentration (nM) 00 7. Contents of amendment Procedure amendment Document (Method) 1. Indication of the case Patent Application No. 1-318758 2゜Name of the invention High sensitivity measurement method 3゜Relationship with the case

Claims (1)

[Claims] Putrescine, characterized in that putrescine and/or cadaverine are reacted with putrescine transaminase, an amino group donor, and a polyamine oxidase to perform an amplification reaction by an enzyme cycling method, and the resulting cycling reaction product is measured. and/or a sensitive assay for cadaverine.