JPS6311848A - Handy measurement of compound related to adenosine-3-phosphoric acid - Google Patents

Abstract

PURPOSE:To expand the application scope of a measuring method, by enabling discrimination and assay of AdR (adenosine) and AMP (adenosine- monophosphate) utilizing a specified enzyme. CONSTITUTION:Before a compound action of NP (nucleoside phosphorylase) and XO (xanthine oxidase) is caused upon a liquid to be inspected, AcP (qacid phosphatase), NT (nucleotidase) or ADA (adenosine deaminase) is made to act thereupon or after the action of NT thereupon, ADA is made to act thereupon and IMP (inosinic acid), AdR and AMP are measured separately to determine at least one of the compounds. Here, coarse AP (alkaline phosphatase) is added to the liquid being inspected beforehand to cause a preliminary reaction and ATP + ADP (adenosine-dephosphoric acid) + AMP + AdR + IMP + NxR (inosine) + Hx (hypoxanthine), AMP, AdR, IMP, HxR and Hx are measured by an electrochemical signal of decrease of dissolved oxygen or generation of hydrogenperoxide from a mixture of compounds related to ATP (adenosine-3- phosphoric acid).

Description

[Detailed description of the invention] I' The present invention can be used in various fields such as aquatic nests, food industry, analytical equipment industry, and analytical reagent industry.

Traditionally, chromatographic methods and enzymatic methods have been used to analyze the composition of ATP (adenosine triphosphate)-related compounds, that is, ATP decomposed products, but these procedures require tens of minutes to several hours. , and required an expensive liquid chromatography device and ultraviolet spectrophotometer, but the inventors of the tree have developed a method to quickly and easily measure dissolved oxygen (DO) to analyze the composition of ATP decomposed products in just a few minutes. We have found a method for determining the freshness of fish and meat using a device (Japanese Patent Application Laid-open No. 59-232097 and Japanese Patent Application Laid-Open No. 60-4-H5).

In JP-A-59-232097, xanthine oxidase (XO) and nucleoside phosphorylase (N
P) + xanthine oxidase (XO), alkaline phosphatase (AP) + nucleoside phosphorylase (NP) + xanthine oxidase (XO) using the reduced amount of dissolved oxygen (DO) under the action of each enzyme, hypoxanthine (H11), inosine (HX R)
, and the amount of inosinic acid (IMP) were determined.

In JP-A-60-471395, by using crude alkaline phosphatase (crude AP) instead of highly purified alkaline phosphatase (AP), crude AP+
Due to the combined effect of NP+XO, the scope of application of the above application is expanded and ATP+ADP book A I'vl P+ I M
P+H! R+H! This made it possible to measure

ATP is a key substance in the energy metabolism of animals.
e), the 411 constant of ATP-related compounds is important. As mentioned above, the present inventors have already measured ATP-related compounds by decreasing Do under the action of a specific enzyme, but in the present invention, the types of enzymes used are further expanded. This is an attempt to further develop the developed technology, and in particular, the purpose is to enable the separate quantification of AMP and Ad R, and to expand the scope of use of the all standard method developed by the present inventors.

The present invention is an electrochemical method for determining the amount of dissolved oxygen decreased or the amount of hydrogen peroxide produced under the combined action of the enzymes nucleoside phosphorylase (NP) and xanthine oxidase (XO) when quantifying ATP-related compounds. In the method of determining all by the target signal, before subjecting the test liquid to the combined action of NP and xo,
(i) act with acid phosphatase (Ac P) or nucleotidase (NT), or (ii) act with adenosine deaminase (ADA), but (iii) act with adenosine deaminase (ADA).
After acting with nucleotidase (NT), ADA
By acting on IMP and Ad R, respectively.
, AMP, and thus at least one of these compounds; and crude alkaline phosphatase (11
By adding AP) and pre-reacting, ATP+ADP+AMP+Ad is obtained from the mixture of ATP-related compounds.
R+I MF+Hz R+Hx (hereinafter referred to as total ATP-related compounds), AMP, Ad R, IMP, HX
R.

The present invention provides a method for determining HX by ΔIII based on an electrochemical signal of the amount of dissolved oxygen decreased or the amount of hydrogen peroxide produced.

In the measurement of ATP-related compounds, the present invention sequentially enzymatically changes each component based on the enzymatic reaction shown in the flow sheet (1) below, and finally converts hyboxanthin (Hz) to xanthine using xanthine oxidase (XO). In the process of being oxidized to uric acid (UA) through (X),
The main idea is to use an electrochemical sensor to detect two molecules of 02 absorbed or two molecules of H2O2 produced per molecule of HX.

The term crude AP (crude alkaline phosphatase) in Flow Sheet h(1) was originally published by the present inventors in the earlier application (Japanese Patent Laid-Open Publication No.
-47895), ATP, ADP, AMP
This is an AP enzyme solution that also acts on
It is known that dR is also decomposed. Although some APs are of high purity, they only convert IMF to HxR and do not effectively act on ATP, ADP, and AMP.

As the sensor used in the present invention, a known sensor such as a polarographic type, a two-cell galvanic oxygen sensor, or a polarographic hydrogen peroxide sensor can be used. The output signals of these sensors are All! Since the current corresponds to twice the fixed molar concentration, it is possible to quantify the component to be measured without using a standard solution of the component to be measured, using pure water with a known concentration of oxygen or hydrogen peroxide as a reference. Furthermore, there is an advantage that even if there is an ultraviolet absorbing substance, color, or tinge in the sample, there will be no problem in all cases.

(The device used in the invention is small, lightweight, and easy to handle, so it can be used at production sites other than laboratories, on farms, and outdoors.

FIG. 1 is a system diagram of a reaction tank and equipment used in the present invention. That is, 1 in the figure is a reaction tank, and a small one having a volume of about 1 to 21 is advantageous in terms of saving enzymes and reagents. Reference numeral 2 denotes a sealing plug for the reaction tank, and a thin hole 3 having a diameter of about 1 mm, for example, is provided in the center for liquid injection. The liquid entering this small hole has a water seal effect, that is, a Kusterer stirrer, and 6 is a temperature control jacket for circulating constant temperature water 7 from the outside.The shape of the reaction tank is not particularly limited, but It is essential to have a structure that allows constant mixing and stirring of the reaction solution, which is convenient for controlling the reaction temperature and injection of reagents, and which prevents oxygen from the outside world from dissolving into the reaction solution.

As the sensor 8, any one of those described above can be used. 9 is an amplifier. Reference numeral 10 indicates an arbitrary mV recorder manufactured by Ichisaka, which is capable of recording at a speed of approximately 100 mV at a full scale of 10 mV. Reference numeral 11 is a computer that automatically calculates the measurement results, but this is not necessary.

Next, the enzyme used in the present invention will be explained.

Table 1 shows the enzymes used in water precipitation and their reaction conditions. The sample solution is preliminarily reacted with the three enzymes, alkaline phosphatase (crude AP), l'd phosphatase (Ac P), and nucleotidase (N T ) in a separate container. In addition, the occurrence of 02 due to the contamination of catalase, which tends to occur in enzyme preparations, is a major hindrance to the implementation of water precipitation, so to prevent this, sodium azide (NaN3) is added to the phosphate buffer.
It is desirable to add about M.

/' 7, / /' /-m--, -- In the following examples, the pure products of the ATP compounds shown in Table 2 are mixed, and various ATPI''A-related compounds are mixed. The liquid was prepared from A and used.

Note 2: Purity was calculated without considering water of crystallization. (The loft of this reagent is Cl0H1IN4 N a20.lP ・7.5 Hz
0) 1- Prepare an IQ mM aqueous solution of the named reagent alone as a stock solution. First, mix each stock solution appropriately, and further dilute with 0.1M phosphate buffer to obtain the specified concentration of each component. A test solution having a concentration of was prepared by rA.

Example (a) Quantification of inosinic acid (IMP) The operating procedure will be described for the case where an oxygen sensor is used, but the same applies to the case where a hydrogen peroxide sensor is used.

Test solution: A test solution containing three components, IMF, HK R, and Hz, each at a concentration of 0.2 mM, was prepared by passing the sample through L.

Preliminary reaction conditions: In order to change the IMF in the test solution to Hz R in advance, the enzyme AcP or NT was added to a portion of the test solution for a preliminary reaction. This test liquid is designated as 52. The conditions for the preliminary reaction are shown in Table 3.

Table 3 Preliminary Reaction Condition Setting 111 Operation: A 2000 μ volume reaction tank (Figure 1 (1)) was filled with air-saturated P, B, and buffer solutions at 37°C, sealed tightly (2), and the tube was sealed. Inject test liquid S+ with a microsyringe through hole (3).
(Do not pre-react) Inject 50 ILj. And the instructions on the DO recorder became stable.
P If) IA synthetic enzyme solution 25gj (MO=20μ,
NP=5μ). An enzymatic reaction occurs immediately,
As shown in Figure 2, DO derived from the oxidation of Hz +Hx R
decrease dTo+)! xR is recorded on recorder 1-.

After confirming that the DO decrease has stopped, inject 2100μ of the mixed solution S2 (this amount corresponds to 5150μ) pre-reacted as described in L, this time Hz + HX R + I
Mpt: derived DO decrease di(we)lxR+l1
IP is recorded.

dIMP: dH! ◆H! R-IMP'
H! +H! RThe second stage and the first stage glue as shown in this formula.

The difference between the decrease values corresponds to the Do decrease width corresponding to the IMF concentration. Of course, instead of using the enzyme xO and NP in the form of a mixed solution, it is also possible to first add xo and record the corresponding DO decrease, and then add NP and record the DO decrease at that time. In this case, dI(!+I(IR=d)It
10d 1 (tR). In this method for quantifying IMF, the enzyme Ac P or N used in the preliminary reaction
When T is injected into the reaction tank as S2, I in S1
It takes advantage of the fact that the MF component does not undergo chemical changes. That is, A
c Since the action of P is inhibited by phosphate ions in the P, B, and l buffer solutions, S2 is present in the P, B, and buffer solutions in one reaction tank.
Even if NT is injected, the IMF component in S1 does not change, and when NT is used, even if alkaline S2 solution is injected, the pH of the reaction tank remains almost the same based on P, B, and the slow infusion solution. This is because NT does not affect the IMF component in S1 because it remains unchanged and remains near neutrality.

Next, we will explain how to calculate the concentration. A 0.5M Na2SO3 solution 10044 to which a trace amount of cobalt chloride was added was injected into a tightly closed reaction tank filled with air-saturated water at 37°C.
A decreasing curve from DO saturation to Do zero is obtained, and the decreasing width d
Measure o. This length is 02 saturation 10 at 37℃
．． Since it corresponds to 214 g mat/aj, the linear (2
) The concentration of IMF is determined by the formula.

However, in formula (2), C: concentration of quantitative component (u ll1o l/m1) d:
Do reduction width for quantitative components (cm) do: Do reduction width for air-saturated water (CII) Go'2: Oxygen concentration in air-saturated water (pmol/ml) 0.2 at 37°C
14 (gmol/m1) 2: Number of oxygen units V Volume of two reaction vessels (IIJ) vs: Volume of test liquid (Sl) (μ) Thus, the determined IMP, Hz R, Hz The concentration of each component is It was quantitative and showed a value close to the initial 0.20M.

Figures 6 (1) and (2) show the IMF concentration and oxygen consumption (this consumption is the Do reduction value when using the standard solution) using a standard solution with a known IMF concentration using Ac P and NT, respectively. According to the calibration curve, which is calculated from the relationship between the
It has been quantitatively shown that 0□2 mol is consumed per 1 mol of MF, and there is significance in the method of the present invention in which the degree of IMPc is determined by an electrochemical signal based on 02 consumption.

In addition, in order to avoid measurement errors, the dissolved oxygen (DO) remaining at the end of one reaction (measurement) must be at least 1
It is necessary that 0 to 20% remain. For this purpose, the amount of the test liquid to be injected into the reaction tank is sufficiently adjusted in advance.

(b) Fill an adenosine (Ad R) fixing reaction tank with P, B, sufficiently saturated with air at 37°C, and after sealing, H! , Hz R and Ad R are respectively 0.
．． Inject 50μ of test solution containing 2mM, then enzyme xO+
Inject 25gj of NP. As shown in Figure 3, the first D
Since the decrease in O is recorded on the recorder, after confirming that the reaction has stopped, when the enzyme ADA5μ is further injected, the decrease in DO (dAdR) is recorded again. Thus obtained Ad R,H! The RlHz concentration each showed a value close to the initial 0.2mM. Figure 7 shows Ad R
This is a calibration curve of the enzyme consumption amount determined by the above method for the standard solution of AdR, and it can be seen that 2 moles of 02 are absorbed per 1 mole of AdR.

Therefore, practically, Ad R can be immediately quantified using equation (2) without particularly preparing such a calibration curve.

(c) G, B, 180ILj, NT20gj, in AMP quantitative containers.
H! Take 200 ILj of test solution containing 0.2mM of each of HxR, AMP, IMF, and Ad R, and pre-react at 37°C for about 5 minutes.After filling the reaction tank with P and B saturated with air at 37°C, Seal tightly and add 100μ of the mixed solution S pre-reacted as above (this amount is equivalent to 50g of the original test solution).
j) and then XO+NP25μ, Hx+HxR+IMP as mentioned in (a)
A DO reduction derived from the above is obtained. When ADA5μ is injected after confirming the stoppage of DO decrease with a recorder, AMP
+Do decrease derived from AdR AMP and AdR are obtained (Figure 4 (1)), this is because NT converts IMP into HXR.
This is because it has the property of not only converting AMP to Ad R, but also converting AMP to Ad R. Next, after newly filling the reaction tank with P and B saturated with air at 37°C, the v: is stoppered, and the original test solution is 50μ
and repeating the same operation as described in (b) to obtain dAdR (Figure 4 (2)) -dAMp
= dAMp + AaR - Do decrease corresponding to the amount of AMP by claaR' [d AMP is obtained, but at this time, the two bonds shown in Fig. 4 (1) and (2).

A decreasing curve can be used. Thus obtained d
The AMP concentration is calculated by substituting AMP into equation (2).

The thus determined concentrations of AMP and other components were close to the initial value of 0.2 mM, respectively. −[−
The method described above can also be carried out by sequentially injecting xO and NP instead of the XO+NP mixture and recording the DO reduction width corresponding to the HX and HXR contents.

FIG. 8 is a line showing the relationship between AMP concentration and oxygen consumption obtained using a standard solution.

(d) H! in a mixture of ATP-related compounds! %HX
Methods for determination of RX, l/IMF, Ad R, AMP and total ATP related compounds.

J of HX, Hz R, 1ATP related compounds described in JP-A-80-471395 previously filed by the present inventors.
11 When the above (a), (b) and (c) are combined with the conventional method, HX, HX RlIM can be obtained from a mixture of ATP-related compounds.
The fifth-order components of F, Ad R, and AMP can be quantified in a short time.

(d-1) In another container A, G, B, 180gj, NT20tLj
, HX , HX R, prepared as described in the notes to Table 2
0.2mM of IMP, AdR, AMP, ADP, ATP
Take 200μ of the test solution containing the sample and pre-react it at 37°C for about 5 minutes (this will be referred to as test solution S2). Also, in a separate container B, take G, 8.18Qμ, crude AP2Qμ, and 200μ of the same test solution as above, and pre-react at 37°C for about 5 minutes (this will be the test solution S3). After filling the tube with air-saturated P and B at 37°C, seal the tube tightly and inject 50μ of test solution S according to Fig. 5 (1).

When the recorder indication is stable, inject X02Qμ and H
Read the DO reduction value di corresponding to the amount of X, then NP
Inject 5gj and read dl corresponding to the amount of HxR0
Next, inject 52,100μ of the pre-reacted liquid in another container A (this amount is equivalent to the content of S, 50μ) and add Hx.
+Hx R+Record d3 corresponding to the amount of IMF0
Next, inject 5μ of ADA and record d, which corresponds to the amount of AMP + 2AdR (2AdR indicates the sum of the contents in Sl and S2).
Inject 3100μ (this amount corresponds to S, 50μ or 52100μ) into the reaction vessel and record d5, which corresponds to the total ATPrA conjugate. As previously mentioned, by performing preliminary reactions in separate containers A and B on the alkaline pH side and performing reactions in the reaction vessels near neutrality, it is possible to use each reaction depending on the difference in optimum pH.

[-Since it is not possible to directly quantify AMP and AdR from the results described above, the following experiment is continued. The same conditions as in the above experiment, but the injection order was changed to 4g, XO,
For NP, ADA, S2, and Sl, a decreasing curve as shown in FIG. 5 (2) is obtained.

At this time, each decrease width is as follows: dl corresponds to the amount of HX,
dl is the amount of Hz R, d3' is the amount of Ad R, d4
' is HX + HX R + IMF + Ad R + AMP, and d corresponds to the amount of total ATP-related compounds. Therefore, dHx=d+, dHxR= dl, p in dI
= d3 (dl + dl)
, d AdR= d:+' , d AM
P = +L -2d3' = dn' (d3+
d3'). By substituting these values into equation (2), the concentration of each component is determined. Also, by substituting d5, a, -d, ; into equation (2), the concentrations of total ATP-related compounds and ADP+ATP can be determined, respectively. The concentration of each component determined in this way is quantitative, and the initial concentration of 0.2mM
It showed a value close to .

(d-2) HxO, 1mM HXR, IMF, AdR, AMP, ADP,
ATP 0.2mM (total ATP-related compounds 1
．． The same measurements as in (d-1) were performed twice using 3mM). The determined concentrations of each component are as shown below, and were approximately quantitative.

NoI O, 130, 190, 150, 130, 25
1,29No2 0.14 0.20 0.29 0.
14 0.28 1.41 (unit: mM) (d-3) ) (!, HX R, IMP, AMP, ADP, ATP
Figure 5 (1) in (d-1) using a test solution containing 0.2mM of each (1.2mM of total ATP 155 compound (excluding Ad R))
A11l constant operation was performed four times according to the procedure. The concentration 11 of each component determined is as shown below, and was approximately standard.

(not including) Not O, 170, 170, 120, 221, 15
No2 0.14 0.18 0.27 0.22
1.27No3 0.13 0.18 0.28 Q
,22 1.28No4 0.13 0.17 0.
48 0.27 1.27 (unit: mM) (d-4) Hllo, 11! IM HXR, IMF 0.1511 each IMAMP, ADP
, ATP 4 times in the same manner as in (d-3) using a test solution containing 0.2 mM each of total ATP-related compounds (but not including AdR) and 1.0 mM.
Two operations were performed. The determined concentrations of each component are as follows, and were approximately constant HL.

Not including total ATP-related compounds) Not O, 130, 170, 08-1, 08 No2
0.12 0.15 0.14 0.18 0.9
9No3 0.11 0. +4 0.18 0.22
0.98No4 0.12 0.17 0.0?
0.39 0.98 (unit: mM) (d-5) HX O, LmM HE R, Ad R80, 15a+M AMP', ADP, ATP each 0.2a+M total AT
Figure 5 (2) in (d-1) using a test solution containing 1.0mM of P-related compound (but not IMF)
The measurement operation was performed four times according to the following. The determined concentrations of each component are as shown below, and were approximately constant.

■Does not include liver) Nol O, 130, 160, 1? 0.32
1.113No2 0.15 0.20 0.12 0
．． 30 1.18No3 0.14 0.19 0.
15 0.28 1.05No4 0. +3 0.1
9 0.1B 0.24 1.19 (unit: mM) In the 11111 constants of Examples (a) to (d), each enzyme reaction ends in around 1 minute, so in order to obtain the Do reduction curve, (a) to In case (c), it takes only about 5 minutes, and (d
), it is about 10 minutes. Note that in the present invention, it is important that each enzyme agent is free from contamination with other enzymes in order to utilize the specificity of enzymes to perform many complex actions in succession.

As is clear from the above explanation, the method of the present invention can quickly analyze the composition of ATP-related compounds in about 10 minutes, whereas conventional chromatography and fermentation methods require several hours. This method is a further improvement on the method previously invented by the present inventors for analyzing the composition of ATP-related compounds using electrochemical signals to determine the amount of Do reduction or H2O2 produced. That is, it has become possible to quantify AdR and AMP by using specific enzymes. Therefore, the method of the present invention allows detailed analysis of the composition of ATPI 38 compounds, which is useful not only for measuring the freshness of seafood and meat, but also for a wide range of fields such as biochemistry, pharmacy, agriculture, medicine, and biotechnology. The amount of enzymes used in the present invention, which is an analytical technique, is small and is extremely economical.

Based on the recorded Do reduction value and H2O2 production amount, it is easy to calculate the concentration of each component not only manually but also automatically using a computer.

Compared to conventional techniques, the measurement method of the present invention can be easily implemented not only in laboratories but also in production and distribution sites, and is extremely desirable from the viewpoints of promoting the food industry, improving food hygiene, and protecting consumers. It is something.

[Brief explanation of drawings]

FIG. 1 is a longitudinal sectional view of a reaction tank and a system diagram of an apparatus suitable for carrying out the method of the present invention. Figures 2 to 5 are the constant product of IMF; constant product of Ad R:
, : , Determination of A M p ;) ; H in a mixture of A T P related compounds! ,H! RlIMP, Ad R
and quantification of AMP and total ATP-related compounds; Showing a decreasing curve. Figures 6 to 8 show the tests for IMF, Ad R, and AMP, respectively.
, is a line. FIG. 6(1) and FIG. 6(2) show the cases where Ac P and NT were used in the preliminary reaction, respectively. Patent applicant: Oriental Yeast f-Gyo Co., Ltd. Oriental Electric Co., Ltd. Agent: Tadashi Wakabayashi (
(valve) Figure 2 Figure 4 Figure 5 Fosse - ← E participating boats - F! ：砒－

Claims (1)

[Claims] 1. Nucleoside phosphorylase (NP) and xanthine oxidase (XO) are combined to act on adenosine triphosphate (ATP)-related compounds to reduce the amount of dissolved oxygen or the amount of hydrogen peroxide produced. In the method of measurement using chemical signals, before the test liquid is subjected to the combined action of NP and XO, (i) acid phosphatase (AcP) or nucleotidase (NT) is applied, or (ii) adenosine deaminase is (ADA) or (ii
i) After acting with nucleotidase (NT), A
By acting with DA, inosinic acid (
1. A method for measuring adenosine triphosphate-related compounds, which comprises measuring adenosine monophosphate (IMP), adenosine (AdR), and adenosine monophosphate (AMP), and thus measuring at least one of these compounds. 2. In the method described in claim 1, crude alkaline phosphatase (crude AP) is allowed to act on the test liquid before subjecting it to all operations (i), (ii), and (iii). adenosine triphosphate (ATP) + adenosine diphosphate (ADP) + adenosine monophosphate (A
MP) + adenosine (AdR) + inosinic acid (IMP)
+ inosine (HxR) + hypoxanthine (Hx) (however, there is no problem even if one of each component is missing), AMP, AdR, IMP, adenosine triphosphate-related by measuring each of HxR and Hx How to measure compounds.