JPS6319159B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel method for measuring nucleosides using a novel nucleoside oxidase. Conventionally, methods for quantifying nucleosides, measuring the enzyme activity of enzyme reaction systems that produce nucleosides, and measuring the enzyme activity of enzyme reaction systems that use nucleosides as substrates have been extremely complicated and lacking in accuracy. Ta. The present inventors discovered that Pseudomonas isolated from the soil of an onion field in Ono City, Hyogo Prefecture.
Bacterial strain B-0781 belonging to the genus contains within its bacterial body,
At least the following general formula [] (However, in the formula, Base indicates a nucleobase residue)
has substrate specificity for the nucleoside represented by, and at least reaction formula [] and/or reaction formula [] We have discovered a new enzyme that consumes oxygen and produces hydrogen peroxide in the reaction that oxidizes the sugar moiety using at least nucleoside as a substrate.
55-110762 = Japanese Patent Application Laid-Open No. 57-58883), it was named nucleoside oxidase due to its enzymatic action and substrate specificity, and this nucleoside oxidase is a nucleoside oxidase present in the test solution. Acts on the 5'-hydroxymethyl group of nucleosides, and by quantifying the amount of oxygen consumed or hydrogen peroxide produced in the reaction, it is possible to accurately and easily measure nucleosides and utilize nucleosides. We have discovered that it is possible to measure enzyme activity in a system that uses First, the nucleoside oxidase used in the present invention has the following substrate specificity,
Any material having enzymatic action may be used. First, we will discuss the substrate specificity, enzyme action, and activity measurement method of this enzyme. (1) Substrate specificity Various compounds (nucleosides, nucleobases, sugars, nucleotides) shown in Table 1 below were used as substrates (substrate concentration was 1 mM), and their relative activities were measured.
The results are shown in Table 1. This enzyme acts on the sugar moiety of nucleosides, but not on nucleobases or sugars alone, and also acts on the sugar moieties of nucleosides such as ribose, deoxyribose, and arabinose. However, the nucleobase portion of nucleosides acts on various purine bases and pyrimidine bases. It also does not act on nucleotides. From the above, it is recognized that this enzyme has substrate specificity at least for the nucleoside represented by the general formula [].

【table】

【table】 ※※







(2) Enzyme action (a) Identification of reaction products: 10ml of 0.2M dimethylglutanate-sodium hydroxide buffer (PH6.0), 500mg of adenosine, 180U of nucleoside oxidase, catalase.
15,000U. and 90ml of distilled water were aerated and stirred to adjust the pH (as the reaction progressed,
It is recognized that the pH decreases and acidic substances are produced. ). After the reaction was carried out at 37°C for 90 minutes, hydrochloric acid was added to adjust the pH to 2.0, and the resulting precipitate was collected by centrifugation and further washed with an aqueous hydrochloric acid solution (PH3). After washing, a 1N aqueous sodium hydroxide solution was added to this to adjust the pH to 8.5, and the mixture was dissolved.
It was again precipitated with hydrochloric acid to a pH of 3, and the crystals were collected. The elemental analysis value of this crystal is C=42.62%,
H = 3.93%, N = 24.9%, O = 28.55%, its molecular weight is 281, and its molecular formula is C ₁₀ H ₁₁ N ₅ O ₅
It was hot. In addition, the infrared absorption spectrum and ultraviolet absorption spectrum of this crystal were consistent with adenosine-5'-carboxylic acid, a synthetic product. From the results, the final product of this enzymatic reaction was identified as adenosine-5'-carboxylic acid. (b) Thin layer chromatogram of the time course of reaction products: 10 ml of 0.2M dimethylglutaric acid-sodium hydroxide buffer (PH6.0), 5 ml of 10mM adenosine,
60 ml of reaction solution consisting of 20 U. of nucleoside oxidase, 1000 U. of catalase, and 45 ml of distilled water was added to 37
React at ℃, 0 minutes, 5 minutes, 10 minutes, 20 minutes after reaction.
5 ml was collected at each hour after 60 minutes and 60 minutes later.
After heat treatment at 100°C for 2 minutes, the mixture was subjected to ultraviolet filtration and used as a sample solution for thin layer chromatography. In addition, in thin layer chromatography, silica gel is used, and acetonitrile: 0.1
% ammonium chloride aqueous solution=7:3 was used. Furthermore, at that time, thin layer chromatography was performed using adenosine (indicated by "Ad") and a synthetic product adenosine-5'-carboxylic acid (indicated by "Ad-A") as standard samples. Ta. As a result, as shown in Figure 1, 5 to 10 minutes after the reaction, spots were observed where the 5'-hydroxymethyl group of the ribose of adenosine was oxidized to an aldehyde group, and furthermore, the reaction rapidly It was recognized that the final product was oxidized to a carboxyl group and showed a spot at the same position as the synthetic product adenosine-5'-carboxylic acid. (c) Oxygen consumption and hydrogen peroxide generation in the reaction system: 0.2M dimethylglutaric acid-sodium hydroxide buffer (PH6.0) 0.4ml, 0.2% N, N-diethyl-
m-toluidine 0.1ml, 0.3% 4-aminoantipyrine 0.1ml, peroxidase (50U./ml) 0.1
ml, 0.05 ml of 10 mM adenosine, and 0.25 ml of distilled water, add 3 U. of nucleoside oxidase, and measure the amount of oxygen consumed (measured with an oxygen electrode) and the excess produced. Amount of hydrogen oxide (wavelength based on the colorimetric method described in the activity measurement method)
(measured by absorbance at 545 nm). As a result, the oxygen consumption is lower than 0.05 μmol of adenosine.
The amount of hydrogen peroxide generated was 0.097 μmol, and the amount of hydrogen peroxide generated was 0.101 μmol. From this, it was recognized that 2 molecules of oxygen were consumed from 1 mole of adenosine, and 2 molecules of hydrogen peroxide were generated. (iv) Ammonia component: No ammonia component was detected in this reaction system. From the above, this enzyme converts adenosine into adenosine-5′-aldehyde through adenosine-5′-aldehyde.
It has the effect of catalyzing reactions that produce carboxylic acids. The reaction formula is as follows. As a result of the above, since this enzyme acts on various nucleosides shown in Table 1, it has substrate specificity for nucleosides represented by the general formula [], and it is clear that this enzyme has at least the reaction formula [ ] and/or reaction formula [ ], and is recognized as a novel enzyme exhibiting enzymatic action and substrate specificity that have never been reported before. In addition, the reaction formula [] and reaction formula [] for the enzymatic action of this enzyme are the following general formula [] (In the formula, Base indicates a nucleobase). (3) Activity measurement method: 0.2M dimethylglutaric acid-sodium hydroxide buffer (PH6.0) 0.2ml, 0.2% N,N-diethyl-
m-toluidine 0.05ml, 0.3% 4-aminoantipyrine 0.05ml, peroxidase (50U./ml)
0.05ml, 10mM adenosine 0.05ml and distilled water
Transfer 0.5 ml of the reaction solution consisting of 0.10 ml to a small test tube,
After prewarming at 37°C for 3 minutes, 20μ of enzyme solution is added to start the reaction. After carrying out the reaction for exactly 10 minutes at 37°C, the reaction is stopped by adding 0.5 ml of 4M urea solution (containing 0.2% cetylpyridinium chloride). Next, after adding 2.0 ml of distilled water to this, the absorbance at a wavelength of 545 nm is measured within 5 minutes using a cell with a layer length of 1.0 cm (AS). In addition, as a blind test, absorbance is measured by performing the same procedure below, using 20μ of distilled water instead of the enzyme solution. ( _AB ). The enzyme activity is determined from the absorbance difference (A _S - A _B ) between the absorbance using this enzyme solution (A _S ) and the blind absorbance (A _B ). Note that the activity is measured within the range of A _S value of 0.15 or less, and if the value is higher than that, dilute the enzyme solution and perform the same reaction. One unit (1U.) of enzyme activity is 1μ per minute when the reaction is carried out at 37℃ using adenosine as a substrate.
The amount of enzyme that generates moles of hydrogen peroxide is calculated using the following formula. Enzyme activity (U./ml) = 1.25 x (A _S - A _B ) x dilution factor Furthermore, although not limited to the novel nucleoside oxidase of the present invention, the Km value, isoelectric point, and molecular weight are as follows. , optimal PH, PH stability,
We will discuss the optimum temperature, thermal stability, the influence of metal ions, etc., and the influence of surfactants.・Km value 4.5×10 ^-5 M (relative to adenosine) ・Isoelectric point around PH4.7 (by electrophoresis using carrier amphorite) ・Molecular weight approximately 240,000 Cephalose CL-6B): Manufactured by Pharmacia (Although a few small molecules of about 240,000 are also observed, it is unknown whether they are subunits of this enzyme or not.) - Optimum pH As shown in Figure 2, 〇-〇 are glycine-hydrochloric acid buffers. solution (40mM), ●-● is dimethyl glutaric acid-sodium hydroxide buffer (40mM), △
-△ indicates the buffer used, phosphate buffer (40mM), and its oxygen consumption was determined using an oxygen electrode. As a result, although differences in activity were observed depending on the buffer used, the optimum pH was found to be 5 to 6 (in glycine-hydrochloric acid buffer). -Optimal temperature As shown in Figure 3, the reaction was carried out under various temperature conditions, and the activity was determined based on the activity measurement method. As a result, the optimum temperature for this enzyme was found to be 45-55°C.・Thermal stability As shown in Figure 4, phosphate buffer (PH6.0,
After treatment with 40mM) for 10 minutes, residual activity was determined based on the activity measurement method. As a result, in terms of its thermostability, this enzyme was stable at 60°C for up to 10 minutes.・PH stability As shown in Figure 5, in the figure, 〇-〇 is Britton-Robinson buffer (150mM), ●-● is dimethylglutaric acid-sodium hydroxide buffer (150mM) , △-△ indicates the buffer used: phosphate buffer (150mM), ▲-▲ indicates the buffer used: Tris-HCl buffer (150mM).
After treatment at ℃ for 60 minutes, each was diluted 40 times and its
The residual activity was determined based on the activity measurement method using 20μ. As a result, in terms of pH stability, this enzyme was stable at pH 8 to 9. - Effects of metal ions, etc. The effects of various metal ions, etc. on the activity of this enzyme were as shown in Table 2.

【table】

[Table] - Effects of surfactants Table 3 shows the effects of various surfactants on the activity of this enzyme.

[Table] The characteristics of the culture of the B-0781 strain on various media based on macroscopic and microscopic observations are as follows. A. Macroscopic observation The results of culturing at 30°C for 18 to 24 hours are as follows. Ordinary agar slant medium Grows in a linear pattern and is pale yellowish-gray to grayish-white in color. No soluble pigments are produced. Ordinary agar plate culture medium: Forms circular, hill-like settlements with smooth surroundings.
It is semi-gloss and has a pale yellowish-gray to grayish-white color. No soluble pigments are produced. Liquid medium becomes uniformly cloudy and then precipitates. Forms a thin bacterial film, but is easily broken by gentle shaking. Gelatin multilayer medium Grows along the barbed lines. Does not hydrolyze gelatin. BCP milk medium Becomes weakly alkaline. Do not peptonize. Anaerobic growth Does not grow. B. Microscopic observation Straight or slightly curved rods, singly, in pairs, or in short chains, motile with polar hairs. The size is
0.5 x 1.0-1.5μ, no spores are formed. No polymorphism. C Physiological and biochemical characteristics Gram staining - acid-fast staining - OF test 0 Catalase production + oxidase production + urease production SSR medium - Christensen medium + gelatin hydrolysis - starch hydrolysis - casein hydrolysis - esculin Hydrolysis − Hydrolysis of cellulose − Hydrolysis of arginine + Accumulation of poly-β-hydroxybutyrate − Production of indole − Production of H ₂ S + Production of acetoin − Reduction of nitrate − Denitrification reaction − Utilization of citrate + GC content of DNA 59% Production of acid from sugar

【table】

[Table] Growth PH range: PH4.5-9.0 〓〓 Growth temperature range: 10-37℃ Utilization of ammonia nitrogen + 〓〓 Utilization of nitrate nitrogen + The above properties of the B-0781 strain of this bacterium were Bergey's Manual of Determinative Bacteriology
Identification was performed in comparison with Determinative Bacteriology) 8th edition, 1974 and Medical Bacteria Identification Guide, 1974 (translated by Toshikazu Sakazaki). This strain is a Gram-negative bacillus that is positive for catalase and oxidase production, oxidatively decomposes glucose, and moves with polar hairs, and has the characteristics of growing well on PH7.0 ordinary agar (slant) medium. It was recognized that it corresponds to the genus. As a result of more detailed comparison, this strain was found to be Pseudomonas putida.
It was recognized that there was a good match between the results (Putida).

【table】

[Table] Based on the above, this strain was designated as Pseudomonas Putida B-0781.
(National Institute of Microbial Technology, Agency of Industrial Science and Technology, Microbial Accession Number Notification "Microorganism Accession Number, FERM-P No. 5664, FERM-P No. 5664)".
The above-mentioned enzyme is one example of the nucleoside oxidase related to the present invention, and judging from the enzymatic action and substrate specificity, the nucleoside oxidase is
Any enzyme recognized as an oxidase can be used. In addition, the test liquid that is the subject of the present invention has the following general formula [] (However, in the formula, R ₁ , R ₂ , and R ₃ represent a hydrogen atom or a hydroxyl group, and Base has the same meaning as above.)
Any sample may be used as long as it contains a nucleoside compound having at least a 5'-hydroxymethyl group represented by: Examples of such samples include the following. For example, adenosine, guanosine, thymidine,
Cytidine, inosine, xanthosine, uridine,
It is produced by reacting a reagent sample as a nucleoside such as arabinosylcytosine, arabinosyladenosine, 2'-deoxyadenosine, or RNA, DNA, or their oligonucleotides with a corresponding hydrolase, such as ribonuclease or deoxyribonuclease. Samples for measuring ribonuclease or deoxyribonuclease activity that release nucleosides by acting on nucleotides such as AMP with nucleotidase or alkaline phosphatase, samples for measuring nucleotidase activity that release nucleosides by acting on nucleotides such as AMP with nucleotidase Samples, samples for measuring enzyme activities that release nucleosides, such as the production of adenosine by S-adenosyl-L-homocysteine hydrolase, and the production of guanosine and deoxyguanosine by guanosine phosphorylase, or their enzyme substrate samples, or samples containing nucleosides. Hydrolysis of nucleosides by nucleosidase as a substrate, hydrolysis of inosine by inosinase which uses inosine as a substrate, hydrolysis of uridine by uridine nucleosidase which uses uridine as a substrate, decomposition of uridine by uridine phosphorylase which uses uridine as a substrate, Samples for measuring the enzyme activity of enzymes that degrade nucleosides using nucleosides as substrates, such as the degradation of thymidine by thymidine phosphorylase, which uses thymidine as a substrate, phosphorylation of the 5'-hydroxy group of adenosine by adenosine kinase, which uses adenosine as a substrate, and uridine. Phosphorylation of the 5′-hydroxyl group of uridine by uridine kinase, which uses thymidine as a substrate, and phosphorylation of thymidine by thymidine kinase, which uses thymidine as a substrate.
Samples for measuring enzyme activity of enzymes that phosphorylate 5'-hydroxyl groups using nucleosides as substrates, etc.
Various samples are mentioned. Next, these test solutions are selected according to the purpose, and nucleoside oxidase is allowed to act on them. Generally, the reaction can be carried out at 37°C, and the reaction time can be set to whatever time the reaction occurs. However, it is not limited in any way. The reaction is based on the reaction formula [] and/or reaction formula [] or reaction formula [] of the enzymatic action of nucleoside oxidase,
It is sufficient to quantify the amount of enzyme consumed or the amount of hydrogen peroxide produced in the reaction based on the general reaction formula []. In addition, when quantifying oxygen, it is preferable to use an electrical means such as an oxygen electrode, and when quantifying hydrogen peroxide, it is preferable to use an electric means such as a hydrogen peroxide electrode, phenol, etc. 4-aminoantipyrine, peroxidase reagent, phenol, 4-aminophenazone,
Peroxidase reagent, N,N'-diethyl-
This may be carried out using color reagents for quantifying hydrogen peroxide such as m-toluidine, 4-aminoantipyrine, peroxidase reagents, and other fluorescent reagents for quantifying hydrogen peroxide. By performing the reaction in this way, the nucleoside content in the test liquid is calculated assuming that 2 moles of oxygen are consumed and 2 moles of hydrogen peroxide are produced for 1 mole of nucleoside. Just force it. In addition, for measurement, for example, in the case of a nucleoside reagent sample, the sample is dissolved in a buffer solution, a certain amount of it is taken out, and this is added to a solution containing 1 to 20 U. of nucleoside oxidase.
℃, and the amount of oxygen consumed by the reaction is measured using an oxygen electrode, or the amount of hydrogen peroxide produced is measured using a hydrogen peroxide electrode, or instead of the above nucleoside oxidase-containing solution. ,
After adding to a solution containing N,N-diethyl-m-toluidine, 4-aminoantipyrine, peroxidase, and nucleoside oxidase and reacting at 37°C for a certain period of time, the color development was determined by
Absorbance may be measured at 545 nm. In addition, in the case of a sample for measuring enzyme activity, it is sufficient to allow the enzyme reaction to occur for a certain period of time, and then quantify the amount of nucleoside consumed or produced by the reaction using the above-mentioned method. For example, in measuring adenosine kinase activity, first add a certain amount of ATP and adenosine to an adenosine kinase-containing solution and allow it to react for a certain period of time.
The amount of adenosine consumed in the reaction may be quantified using the above-mentioned method to determine adenosine kinase activity. In the measurement of nucleotidase activity, ATP is preferably used as a substrate to determine the reaction product. This can be done by treating the nucleoside adenosine produced as a nucleoside with nucleoside oxidase and measuring the amount of oxygen consumed or hydrogen peroxide produced.
Furthermore, in measuring RNA content or ribonuclease activity, RNA is reacted with ribonuclease, converted into nucleotides, and then converted into nucleosides by the action of nucleotidase or alkaline phosphatase, and then quantified by the action of nucleoside oxidase. , also
What is necessary is to measure RNA content or ribonuclease activity. As described above, the present invention uses this novel nucleoside oxidase to quantify various nucleosides, measure the activity of enzymes using nucleosides as substrates, measure the activity of enzymes that produce nucleosides, and quantify their substrates. This is an extremely useful measurement method for various measurements such as. Next, examples and reference examples of the present invention will be described, but the present invention is not limited to these in any way. Example 1 0.2M dimethylglutarate-sodium hydroxide buffer (PH6.0) 0.4ml 0.2% N,N-diethyl-m-toluidine 0.1ml 0.3% 4-aminoantipyrine 0.1ml Peroxidase (50U./ml) 0.1 0.95 ml of a reaction solution consisting of 10 U of nucleoside oxidase and 0.25 ml of distilled water was prewarmed to 37°C, and to this was added 0.05 ml of test solution containing adenosine at various concentrations.
After adding 2.0 ml of distilled water, the absorbance was measured at a wavelength of 545 nm. As a result, as shown in FIG. 6, the adenosine content could be easily and well quantified. Example 2 Using 0.95 ml of the same reaction solution as in Example 1, at 37°C
0.05 ml of a test solution containing adenosine at various concentrations was added thereto, and the mixture was allowed to react at 37° C. for 20 minutes, after which the amount of oxygen consumed was measured using an oxygen electrode (manufactured by Ishikawa Seisakusho). As a result, as shown in FIG. 7, extremely good quantitative values were obtained. Examples 3 to 9 Instead of the adenodine-containing test solution of Example 1,
The following procedure was carried out in the same manner as in Example 1 or Example 2 using test solutions containing various concentrations of guanosine, thymidine, inosine, uridine, 2'-deoxyadenosine, arabinosyladenine, and bredeinine.
Figure 8 (Guanosine measurement results), Figure 9 (Thymidine measurement results), Figure 10 (Inosine measurement results),
Figure 11 (uridine measurement results), Figure 12 (2'-
As shown in Figure 13 (deoxyadenosine measurement results), Figure 13 (arabinosyladenine measurement results), and Figure 14 (bourdeinine measurement results (oxygen electrode method)),
Good results were obtained. Example 10 0.2M dimethylglutaric acid-sodium hydroxide buffer (PH6.0) 0.4ml 0.2% N,N-diethyl-m-toluidine 0.1ml 0.3% 4-aminoantipyrine 0.1ml Peroxidase (50U/ml) 0.1ml A reaction solution was prepared containing 5 U of nucleoside oxidase , 0.25 ml of distilled water, and 0.95 ml in total. In addition, adenosine kinase-containing solution extracted and collected from the enzyme in advance (J.Biol.Chem. (1951) 193
0.1ml (adjusted according to the method described in 481-495),
0.2M dimethylglutaric acid-sodium hydroxide buffer containing 5mM ATP and 5mM adenosine (PH6.0)
(Containing 10mM _MgCl2 ) at 37°C, 0.02ml of the reaction solution was collected at 0, 5, and 10 minutes after the reaction.
After heat treatment at 70°C, add this to the above reaction solution,
Incubate for 20 minutes at
Absorbance was measured at 545 nm. As a result, as shown in FIG. 15, it was possible to measure extremely simply and accurately. Example 11 0.2M dimethylglutarate-sodium hydroxide buffer (PH6.0) 0.4ml 0.2% N,N-diethyl-m-toluidine 0.1ml 0.3% 4-aminoantipyrine 0.1ml Peroxidase (50U./ml) 0.1 ml 20mM AMP 0.05ml Nucleoside oxidase 10U. Distilled water 0.2ml Total 0.95ml 0.95ml of the reaction solution was preheated to 37℃, and 0.05ml of the nucleotidase-containing solution (manufactured by Sigma) was added to this. After reacting at 37°C for 20 minutes, the color development was measured by absorbance at a wavelength of 545 nm. The results were as shown in FIG. 16, and the nucleotidase activity could be easily and well quantified. Example 12 0.95 ml of a reaction solution containing RNA and nucleotidase was treated with 0.05 ml of a solution containing ribonuclease (manufactured by Sigma) at 37°C for 30 minutes.
0.05 ml was collected, heated at 80°C, and added to 0.95 ml of the reaction solution having the same composition as in Example 1.
React at 37℃ for 20 minutes, and observe the color development at a wavelength of 545nm.
Absorbance was measured at As shown in FIG. 17, ribonuclease was successfully measured. Reference example 1 Peptone 1.0%, casein 1.0%, oleic acid 1.0
%, yeast extract powder not included 0.5%, KCl 0.2%,
Into an Erlenmeyer flask containing 100 _ml of a medium (PH7.0) consisting of 0.1% K 2 HPO 4 and 0.05% MgSO ₄ 7H ₂ O (sterilized at 120°C for ₂₀ minutes), Pseudomonas putida.
B-0781 strain was inoculated and cultured at 30°C for 1 day to obtain an inoculum. Next, this inoculum was transplanted into a 30-sized jar fermenter containing 20 medium (containing 0.1% Desform BC-51Y) having the same composition as above, and incubated at 30°C, 42 hours, 350 rpm, and aeration rate 20.
Culture was carried out with aeration under conditions of sterile air / min. After culturing, the culture was centrifuged at 5000 rpm for 10 minutes to collect the bacteria, and the bacteria were washed with water from step 7 and further
The cells were collected by centrifugation at 5000 rpm for 10 minutes. Next, the bacterial cells were dispersed in 20mM phosphate buffer (PH7.0) containing 10mM MEDTA in step 5, and lysozyme was added to the solution to give a lysozyme concentration of 0.5mg/ml.
The bacterial cells were destroyed by stirring at 37°C for 5 hours. Thereafter, this was centrifuged at 5000 rpm for 10 minutes to remove insoluble matter, and the supernatant 4.2 was obtained (2.86 U./
ml). Then, add cold acetone (-20℃) to this supernatant.
Add 2.5 and centrifuge at 5000 rpm for 10 minutes.
The supernatant was obtained (11150U.). Further, 2.5 g of cold acetone was added to this supernatant, centrifuged at 5000 rpm for 10 minutes, the supernatant was removed, and the precipitate was collected. The obtained precipitate was dissolved in 20mM phosphate buffer (PH7.0) 1.0, centrifuged at 5000 rpm for 15 minutes to remove insoluble matter, and the supernatant (9700U.)
was heat-treated at 50°C for 20 minutes and centrifuged at 15,000 rpm for 10 minutes to remove heat-denatured proteins and obtain a supernatant (9,400 U.). Next, ammonium sulfate (319 g) was added to this supernatant to give a saturation of 0.42, and then the mixture was heated at 15,000 rpm for 10
Centrifuge for 1 minute to remove the precipitate, and then centrifuge for 140 min.
g of ammonium sulfate was added and the precipitate was collected. Next, this precipitate was dissolved in 50 ml of 20 mM phosphate buffer (PH7.5), centrifuged at 15,000 rpm for 10 minutes to remove insoluble materials, and the supernatant (7,800 U.) was obtained. The solution was dialyzed overnight with cellophane tubes. Furthermore, after the dialysis,
Charge a column (2.6 x 60 cm) of DEAE-cellulose (manufactured by Cellbar) equilibrated with 20mM Tris-HCl buffer (PH7.5), and
20mM Tris-HCl buffer containing 0.5MKCl (PH7.5)
Elute with a KCI linear concentration gradient of 0.15~
Collect the elution fraction at 0.25MKCI concentration (5760U.),
This was further concentrated using Amicon PM-10. After that, this concentrated liquid was charged to a column (2.6 x 90 cm) of Cephacryl S-200 (manufactured by Pharmacia), and the active fraction (280 nm absorption fraction and enzyme active fraction was collected) was combined. and lyophilized to obtain 51.0 mg (3675 U.) of purified nucleoside oxidase powder.

[Brief explanation of the drawing]

Figure 1 is a thin layer chromatogram of the time course of the reaction product using nucleoside oxidase, in which Ad represents adenosine as a standard sample, and Ad-A represents adenosine, a synthetic product as a standard sample. 5'-carboxylic acid, Figure 2 shows the optimum pH curve for nucleoside oxidase, Figure 3 shows the optimum temperature curve for nucleoside oxidase, and Figure 4 shows the optimum temperature curve for nucleoside oxidase. The thermal stability curve of the fifth
The figure shows the PH stability curve of nucleoside oxidase, Figure 6 is the measurement result of adenosine, Figure 7
The figure shows the measurement results of adenosine using an oxygen electrode.
The figure shows guanosine measurement results, Figure 9 shows thymidine measurement results, Figure 10 shows inosine measurement results, Figure 11 shows uridine measurement results, Figure 12 shows 2'-deoxyadenosine measurement results, and Figure 13 shows arabinosyl adenine. Measurement results, Figure 14 shows Bredeinin measurement results,
Figure 15 shows the adenosine kinase measurement results,
FIG. 16 shows the results of nucleotidase activity measurement, and FIG. 17 shows the results of ribonuclease activity measurement.

Claims (1)

[Claims] 1. A nucleoside represented by at least the following general formula [] (However, in the formula, Base indicates a nucleobase residue)
A novel method for measuring nucleosides, which comprises applying nucleoside oxidase to a test solution containing . 2. The method for measuring nucleosides according to claim 1, wherein the nucleoside oxidase is a nucleoside oxidase having at least the following substrate specificity and physicochemical properties of enzymatic action. - Substrate specificity: It has substrate specificity for at least the nucleoside represented by the following general formula []. (However, in the formula, Base indicates a nucleobase residue) Enzyme action: At least the reaction formula [ ] and /
or has the effect of catalyzing the reaction represented by the reaction formula [] 3. The method for measuring nucleosides according to claim 1, wherein the test liquid is a nucleoside-containing liquid or a liquid that liberates nucleosides. 4. The method for measuring nucleosides according to claim 3, wherein the liquid that releases nucleosides is a liquid containing at least nucleotidase and nucleosides released by nucleotides. 5. The nucleoside according to claim 3, wherein the liquid that releases the nucleoside is a liquid containing nucleoside released by at least ribonuclease or deoxyribonuclease and nucleotidase or alkaline phosphatase. Measurement method.