JPS63236963A - Determination of superoxide anion - Google Patents

Abstract

PURPOSE:To make effective determination of anion by acting a reducing agent or the reducing agent and the adjuvant thereof to a sample contg. superoxide anion to convert the anion to hydrogen peroxide and measuring the same. CONSTITUTION:A metal salt, metal complex, protein contg. metal, etc., are utilized as the reducing agent to be used for the determination of the anion and the salts of manganese, chromium, vanadium, titanium, tin, etc., are used as the metal salt. Porphyrin iron such as hemin, ferrocene, chlorophyll, etc., are used as the metal complex. and a chelate agent, cyclic hydrocarbon and compd. such as protein which assists donating reaction of O2<-> with electrons are used as the reduction adjuvant. The anion is converted to the hydrogen peroxide in such a manner and chromogen is oxidized and condensed by using H2O2 in the presence of peroxidase so that light is emitted. The light absorbancy thereof is measured by a coloration method. The chromogen is similarly made to emit light by P-hydroxyphenylacetic acid and homovanillic acid and the fluorescence is measured.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for quantifying superoxide anion (02-).

(Prior Art) It is known that superoxide anion (02-) is generated by various reactions in living organisms.

For example, the production of lipid peroxide is 0! -, and the production of 0□- is attracting attention in relation to oxygen toxicity. Measuring O2- is considered important in understanding the generation of active oxygen in living bodies and its mechanism of action. Methods for measuring 0□- include measuring the amount of reduction of 1,000-tochrome C; measuring chemiluminescence due to the oxidation of luminol in the absence of an oxidation catalyst; and There is a known method for measuring the complex ■.

The generation of superoxide anions is known in vitro, especially in oxidase reactions.

For example, it is known that in the oxidation process of hypoxanthine-xanthine-uric acid by xanthine oxidase (XOD) shown by the reaction formula below, 0□- is produced in addition to nzoz.

Hyboxanthin + Ot -> Xanthine +11.0
The oxidase that generates □+Ot-(1)0□- is a one-electron reductase enzyme, and the oxidase that generates H2O2 is a two-electron reductase enzyme, but the above XOD actually has both properties. There are many H, as mentioned above.
Both 0□ and 02- are generated. Oxidases that exhibit the above reactivity include milk-derived xanthine oxidase and aldehyde oxidase.

Examples include flavin enzymes that do not form complexes with sulfite, such as diamine oxidase. In addition to these, there are oxidases that generate H2O2 and 0□- due to their reaction characteristics with oxygen.

By the way, the oxidase reaction mentioned above is a biological component.

For example, for the measurement of guanase, etc., it is used in clinical tests in conjunction with the 11□02 measurement method described below. for example,
(1), (I(to
A commonly used method (Trinder method) is to allow □ to act on a chromogen in the presence of peroxidase, oxidize the chromogen, develop a color, and measure its absorbance. but.

In such a measurement method, as described above, 820□
In addition to 0□- is generated. Since it is difficult to measure 11tO□ and 0□- separately, 1 usually 0□- is measured as 11□
A method of changing it to 0□ is adopted. 0□-HtOt
In order to change to 1f20□, ■ leave the reaction system alone and change 02- to 1f20□ by natural disproportionation reaction; and ■
Superoxide anion dismutase (5
A method is known in which the disproportionation rate of 0□--II□0□ is increased by allowing 0□--II□0□ to coexist. However, the method (2) has the disadvantage that the presence of catalase in one reaction system causes the generated 11□0□ to decompose. In the method (2) using SOD, the reaction occurs quickly, so decomposition by catalase is unlikely to occur. but.

In this method, even if the amount of SOD is increased, the color recovery rate in the Trinder method is 10% of the theoretical amount.
It was found through experiments by the inventors that it does not reach 0%.

The reason for this is that 02- generated with H20□ production combines with peroxidase to form a complex ■.

This complex■ is 0□ due to its catalase action.
- is decomposed before being disproportioned to H2O2.

Its speed is faster than the disproportionation reaction of SOD, among others.

This is thought to be due to the fact that the catalase action of complex (1) is extremely promoted when a chromogen as a hydrogen donor is present. Once generated, the dye may be reduced and faded by 0t-, but this will be oxidized again and recovered over time. The reason why the above-mentioned recovery rate does not reach the theoretical amount is that peroxidase combines with O2- to form complex ■ and exhibits catalase action.
This is different from direct fading due to −.

On the other hand, NADII and NA generated by dehydrogenase
As a method for measuring DPH, a method has been adopted in which HzO2 is generated using an oxidase reaction or an electron carrier and then measured. For example, when NADH is reacted with oxygen using phenadium methyl sulfate (PMS) or microorganism-derived diaphorase, the resulting H20□
There are known methods to measure In this system as well, a method is adopted in which the chromogen is oxidized and colored with 11□02 in the presence of peroxidase, and the degree of color development is measured by visible spectrophotometry.

In this method, the above-mentioned NADH and NADP
The sensitivity is higher than that of directly measuring 11 with an absorbance meter, and it is possible to avoid contamination (coloring) of the measuring instrument due to the formazan coloring method. However, even in this type of reaction, since PMS or diaphorase is basically a one-electron reduction type, 0□- is generated, and the above-mentioned XOD
As with other types of oxidase reactions, accurate measurements cannot be obtained. Similarly to the case of XOD, the addition of SOD does not provide a sufficient effect.

As described above, at present, no method has been developed for quantifying superoxide anions, particularly for effectively quantifying 0□-, which is generated simultaneously with H20□ produced by an oxidase reaction or an electron carrier such as PMS.

(Problems to be Solved by the Invention) The present invention solves the above conventional problems, and its purpose is to provide an effective method for quantifying superoxide anions. Another object of the present invention is to provide a method for accurately quantifying superoxide anion, which is generated together with H,0□, by an oxidase reaction or the like, as H,0□.

It is about providing.

(Means and effects for solving the problem) The inventors have developed the Oz''→11202
We investigated ways to change this effectively and without interference from other factors such as catalase.

First, in the reaction system of fl) and (2) described in the prior art section, milk-derived xanthine oxidase is used as ×OD, and SOD is added in a reaction system in which the generated 11□02 is measured by the trisider method, The effect was investigated by varying its concentration. However, even when SOD was added at a final concentration as high as 2000/-, the color recovery rate (calculated from the molecular extinction coefficient) was 80% of the theoretical value. And at this time.

The amount of uric acid produced was the same regardless of the amount of SOD added. This is because the generated 0□- combines with the peroxidase present in the system to form a complex ■, and this complex ■ has a catalase action. This is thought to be because the reaction rate is much faster than the disproportionation reaction rate of SOD. Especially when the chromogen present in the system acts as a hydrogen donor, the complex ■
The catalase action of is promoted.

Based on the above considerations, the inventors conducted experiments using various oxidizing agents, reducing agents, disproportionating agents, and enzymes related to these agents, and found that 02- forms a complex ■ and undergoes decomposition by the action of catalase. We investigated a method that can be quickly converted to l1zOt without any problems. As a result, they discovered that metal salts and the like can extremely quickly reduce 02- and convert it into 1ItO2, and have arrived at the present invention.

Namely, in the method for quantifying superoxide anions of the present invention, a reducing agent or a reducing agent and its auxiliary agent are applied to a sample containing superoxide anions to convert the superoxide anions into hydrogen peroxide, and the superoxide anions are converted into hydrogen peroxide. It is characterized by measuring hydrogen.

The reducing agent used in the method of the invention is a metal salt.

Metal complexes, proteins containing metals, etc. are used. The term "metal salt" as used herein refers to a metal salt in which the oxidation number of the metal forming the salt can be easily changed by reaction and can donate electrons in a reaction solution. Such metal salts include manganese, chromium, and vanadium.

Examples include salts such as titanium and tin. Metal complexes are also electron-donating metal-containing organic compounds, including porphyrin irons such as hemin, ferrocene, and chlorophyll. Examples of metal-containing proteins include hemoglobin, hemocyanin, and cytochrome. The reduction aid is a compound that can assist the reaction in which the reducing agent donates electrons to 02-, and includes, for example, chelating agents, cyclic carbohydrates, and proteins. Chelating agents include ethylenediaminetetraacetic acid (EDT^), iminodiacetic acid (to ID), and ethylenediaminediacetic acid (EDDA).
).

Examples include triethylenetetraminehexaacetic acid (TTH^).

Cyclic carbohydrates include cyclodextrin, crown glucan, and crown ether.

Examples of proteins include bovine serum albumin (BSA) and various globulins.

To quantify superoxide anions according to the present invention, the above-mentioned reducing agent and, if necessary, the above-mentioned reduction aid are added to the system containing 02-. The reducing agent.

Usually 10 μM to 1 μM in 2 reaction solutions. It is added at a rate of I. The concentration of the reducing aid is usually 0.1 to 100 mM.

0 included in the reaction system! - is quickly reduced by the above-mentioned reducing agent and converted to HtO□, so this 11□Ot is quantified by a conventional method. For the measurement of H,0, a method using a peroxidase reaction is adopted.

For example, a method in which a chromogen is oxidized and condensed with H,0□ in the presence of peroxidase to form a color, and its absorbance is measured (color method); Examples include a method of measuring the fluorescence produced by oxidative condensation of leucodichlorofluorescein, etc. (fluorescence method); a method of measuring the luminescence produced by oxidizing luminol or isoluminol with HJ□ in the presence of peroxidase (luminescence method). It will be done.

The method of the present invention is suitably used to measure the group X (e.g., hyboxanthin, diamine, uric acid, etc.) and enzyme activity (e.g., xanthine oxidase, etc.) in the reaction system of oxidase that produces 02-. In addition, the method of the present invention has 0
Combining the reaction system of oxidase that produces 2- with other enzymatic reactions is also suitably used for measuring substrates (eg, guanine, inorganic phosphorus, etc.) and enzyme activities (eg, guanase). Furthermore, the method of the present invention can also be used to measure NADH and MADP11 produced by dehydrogenase.

For example, the enzymatic reaction (
When the above-mentioned reducing agent and, if necessary, a reduction aid are added to the system of formulas (1) and (2)), 02- quickly converts into H, Of
is converted to When this H2O2 is allowed to act on 4-aminoantipyrine and phenols under an oxidation catalyst having peroxidase activity.

The 4-aminoantipyrine and phenol undergo oxidative condensation to develop color. Hypoxanthine is quantified by measuring the degree of color development. In addition to such measurement by the Trinder method, known H,0□ quantitative methods such as absorption method and fluorescence method 9 luminescence method may be employed.

The oxidases that generate OX- include xanthine oxidase, leukocyte NADPH oxidase, aldehyde oxidase, diamine oxidase, NAD
These include H diaphorase, old yellow enzyme, sarcosine oxidase, and uricase. In addition to such oxidase, 0□- is non-enzymatically produced by NADH, NADP.
It is also produced by oxidizing ll with, for example, PMS.

Examples of the catalysts having peroxidase activity include various peroxidases such as horseradish peroxidase, lactoperoxidase, myeloperoxidase, peroxidase derived from microorganisms, and microperoxidase; non-catalytic enzymes such as hemin and hematin; and proteins such as methemoglobin.

Next, It 20 generated by xanthine oxidase
The present invention will be described in detail using a method of measuring 2 and 02- as H, O□ by the Trinder method.

The conventional oxidase reaction without adding a reducing agent is 9
As shown in the prior art section, the following equations (1) and (2)
).

XOD Hypoxanthine +0. −〉Xanthine+H,0□+(h
-(1) For example, as a reducing agent in this reaction system, Mn'' (M
nC1z, etc.) is present at around 0.5M, the following 9-order reaction occurs.

As shown here, all oxidase reactions in the presence of a reducing agent such as Mn'' are one-electron reduction type reactions, and Ot- is formed instead of 11□ islands (Formula (1)
-a and formula (2) -a). A total of 4 molecules of Oz- are formed in this way, which are converted to H, O, (formula (3)) 4 molecules of 110. is generated. (
The reactions of 1) -a, (2) -a and (3) proceed extremely rapidly, and the presence of this M n t + does not have a negative effect on the reaction 1. In the above system, uric acid is It does not undergo decomposition and does not become a hydrogen donor in the reduction reaction (3) of rat□.

The equilibrium of the above reaction system is tilted toward uric acid production (p146
．． 3) uric acid is produced in a larger amount than in the absence of 4-aminoantipyrine (4AA) and N in the presence of peroxidase, as shown in formula (4). -ethyl-N-(
It acts on 2-hydroxy-3-sulfopropyl)-m-1-luidine (TOOS) to form a quinoneimine dye.

2ToOz+4 -7minoantibiline +TOOSOn the other hand, in conventional reaction systems (1) and (2), free 02- binds to peroxidase,
Because catalase activity is expressed, sufficient peroxidase reaction does not proceed.

(Experimental Example) A comparison between the present invention and a conventional example regarding the above reaction system will be explained using an experimental example.

First, 20mM MES buffer (pH 6,3
), hyboxanthin (final concentration 10.6 μM), X0, 4A^, and TOO5 were dissolved and allowed to react for a sufficient amount of time. Equations (1) and (2) are completed and 0
□- was converted to 11202 by a natural disproportionation reaction.

Next, peroxidase was added to this reaction system, and the produced quinoneimine dye was quantified by measuring absorption at 550 nm. The result is shown as trace A in FIG.

This amount is the molecular extinction coefficient 1 determined by the uric acid-uricase system.
6.4mM-'cm-', in good agreement with uric acid (290n
It can be seen that two molecules of H,0□ are generated per one molecule (measured at +*).

Next, the above buffer 7-medium XOD, 4AA, TO
The reaction is started by dissolving O5 and peroxidase and adding hyboxanthin.

The quinone imine produced in the same manner was measured. The results are shown as trace B in Figure 1. In this system,
0! generated in equations (1) and (2)! - binds to peroxidase and exhibits catalase action, so H
tO□ decomposes, resulting in a lower amount of quinoneimine dye produced.

Next, in the system of Trace B, SOD was added at a ratio of 2000/M1 per reaction and a similar reaction was carried out.

This result is shown as trace C in FIG.

In this system, the amount of quinoneimine dye is higher than in the trace B system because the disproportionation of 0□-'-HtO□ is promoted, but in the trace A system where the natural disproportionation reaction has been completed. has not yet reached the level of

Next, in the system of Trace B, 1 g of MnC was added at a ratio of 0.5M and a similar reaction was carried out. The result is shown as trace D in FIG. In this system, the above (1
)-a, (2)-a, (3) and (4) proceed rapidly and in succession, four molecules of nzo□, that is, twice as many IhO□ (quinone imine dye) as in the trace A system, are produced. generate. When we measured the amount of uric acid produced in this system, we found that
It was confirmed that the amount produced was about 1.18 times the amount produced in the system of traces A-C. Thus, in this system, the reaction leans towards the uric acid production system. The amount of quinone imine produced in Trace D is approximately 2.36 times that of Trace A.
Through this reaction, the theoretically valid quinone imine dye u+zo
It can be seen that z) is recovered.

Next, we investigated the concentration of the reducing agent using MnC1z. In the system shown in trace D of FIG. 1 above, the MnC1□ concentration was varied from 0 to 600 mM.

The absorbance of the quinone imine dye was measured. The result is the second
This is shown as trace A in the figure. Furthermore, add 1 EDTA to the same system.
A similar reaction was carried out in a system in which the solution was added at a concentration of mM. The results are shown as trace B in FIG. From trace A in Figure 2, if 0.5M MnC1g exists, 0
It can be seen that all 2- is reduced to lI20□. Furthermore, when a reduction aid (EDTA) is present, the frequency decreases to 0□-→Hz
The reaction of O□ is promoted. It can be seen that when EDTA is present at the above concentration, trace B is saturated at 0.4 M of MnC1, and a sufficient conversion reaction to H,02 takes place.

(Example) The invention will be explained below with reference to examples.

On the tip [0! - Quantification] 10mM TOO30, 3rd lomM 4-AA
0.15d100U/-peroxidase
20mM ME containing 0.03+d2M MnClt
S buffer y - (pl(6,3)0.75+d 20mM MBS buffer (pH 6,3)
At 1.75 d, 0□ was dissolved in crown ether to form IM, and 0.02 - of the solution was added to the above reaction composition.

02- generated from KOt was converted to 11□02 and measured as the color development of the quinone imine dye due to oxidation.

As a result, 20 μH of KO2 was determined. On the other hand, in the system not containing 1 g of MnC, no color development occurred, so quantification was not possible.

Backyard 1 [Quantification of Hyboxanthin] 10mM TOO30,3wd lonM 4-AA 0
．． 15++d1000U/-peroxidase
0.03d150/-xanthine oxidase
20mM containing 0.02rn12M MnC1z
MES buffer y - (pH 6,3) 0.75mffi 20mM MES buffer (pH 6,3)
1.7:3d Hyboxanthin solution (0
~20 μM) 0.02- was added and the absorbance of one reaction system was measured. When the hypoxanthine concentration increases, Ht produced
O! The amount of quinone imine dye increases and therefore the absorbance increases. The results are shown in FIG. Thus, a linear relationship between hypoxanthine concentration and absorbance was obtained up to a hypoxanthine concentration of 20 μh. When hyboxanthin was not added (blank), the absorbance was extremely low and showed a constant value.

Practical scale [Quantification of guanase] 200μN guanine 200μM MBTI + 1mM ESPAS O, 2U/d ~100/7! Add 0.021 n1 of guanase sample containing a proportion of .

Absorbance was measured for 10 minutes (by rate method), and the results showed a linear relationship between guanase concentration and absorbance up to IOU/6. This is approximately 2.5 times more sensitive than the system measured by adding superoxide anion dismutase at a rate of 200 υ/cell instead of MnCl2.

(Effects of the Invention) According to the method of the present invention, superoxide anions can be measured accurately and in a short time.

This method can be suitably used to measure substrates and enzyme activities using an oxidase reaction system that generates 0□- and H20□.

4. Figure 1 is a curve showing the absorbance when HtOt produced in the oxidation reaction of hypoxanthine in the presence of xanthine oxidase is converted into a quinone imine dye and measured by the conventional method and the method of the present invention; Figure 2 is a graph showing the relationship between the amount of reducing agent used in the method of the present invention and H, 0□, produced by the oxidase reaction; and Figure 3 is a graph showing the amount of hyboxanthin determined by the method of the present invention.

It is a graph showing the relationship between the hypoxanthine and absorbance.

that's all

Claims (1)

[Claims] 1. Adding a reducing agent to the sample containing superoxide anions,
Alternatively, a method for quantifying superoxide anion, which comprises converting the superoxide anion into hydrogen peroxide through the action of a reducing agent and its auxiliary agent, and measuring the hydrogen peroxide.