JPH0677519B2 - Method for quantifying substrate or enzyme activity using oxidase reaction - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a method for quantifying a substrate or an enzyme using an oxidase reaction which produces hydrogen peroxide and superoxide anion, and more specifically, a by-product with hydrogen peroxide. The present invention relates to a method for accurately quantifying substrate or enzyme activity by converting superoxide anion to hydrogen peroxide, and measuring both hydrogen peroxide generated by the oxidase reaction and superoxide anion as hydrogen peroxide. .

Of (prior art) oxidase reaction, for example, the following (1) enzyme type oxidase reaction to produce hydrogen peroxide (H ₂ O ₂₎ as an electron acceptor and, as shown in equation, the H ₂
By measuring O ₂ , the substrate and enzyme activity utilizing the reaction have been measured. H ₂ O ₂ is, for example, as shown in the formula (2), in the presence of peroxidase (POD), a chromogen [for example, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine (TOOS). ); And 4-aminoantipyrine], and the chromogenic substance is oxidized and condensed to form a color, which is measured by an oxidative coloring method (Trinder method).

Such a measuring method is widely used especially in the field of clinical chemistry. However, as a result of the inventors' detailed examination of this method, for example, the amount of formaldehyde or glycine produced in the sarcosine oxidase reaction in the above formula (1) and the amount of H ₂ O ₂ (the H ₂ O ₂ 2) It is converted into a dye by the Trinder method and calculated from the molecular extinction coefficient of the reaction solution), which does not match stoichiometrically, that is, the amount of H ₂ O ₂ is smaller than that of formaldehyde or glycine. I understood it. It is considered that this is because superoxide anion (O ₂ ⁻ ) is produced in part in addition to H ₂ O ₂ in the oxidase reaction of the above formula (1).

It is generally known that among oxidases, oxidases containing flavin but not forming a complex with sulfite are one-electron reducing oxidases, which form O ₂ ⁻ . A typical example is milk-derived xanthine oxidase, and this aldehyde oxidase and diamine oxidase also produce O ₂ ⁻ . However,
Many other oxidases have also been overlooked to produce O ₂ ⁻ in a small amount in addition to H ₂ O ₂ . For example, even in the reaction of the above formula (1), it is considered that the production of O ₂ ⁻ lowers the H ₂ O ₂ production rate.

The inventors believe that a more accurate measurement value of H ₂ O ₂ can be obtained by converting the generated O ₂ ⁻ into H ₂ O ₂ , and the reaction system of equations (1) and (2) can be superposed. was added dismutase (SOD), O ₂ ^- disproportionation reaction to H ₂ O ₂ from (2O ₂ ^- +2
An attempt was made to promote H ⁺ → H ₂ O ₂ + O ₂ ). However, high concentration of SOD
It has been found that the addition of is not consistent with the theoretical yield.
This is because the by-produced O ₂ ^{− in} formula (1) combines with POD to form complex III, and this complex III
O ₂ ^- Results expressing decomposition, O ₂ present in the system ^{- -} O ₂ relative is considered to decompose. In (2), since the chromogen as a hydrogen donor are present, the O ₂ ^-
The decomposition action is further promoted.

Although quantification of various substances using oxidases is widely used in the field of clinical chemistry to measure biological components, H ₂ O ₂
In the reaction system that produces, since the amount of the product that is stoichiometrically consistent with the amount of the substance to be measured is not obtained as described above, the apparent molecular extinction coefficient of the substance to be measured is used as a standard substance. We obtained it and used it to calculate the quantitative value. Therefore, it is difficult to obtain an accurate quantitative value.

(Problems to be Solved by the Invention) The present invention solves the above-mentioned conventional drawbacks, and an object thereof is to produce a by-product in an oxidase reaction in which oxygen is used as an electron acceptor to generate hydrogen peroxide. superoxide anion of peroxidase with the O ₂ ^- converted to hydrogen peroxide without decomposition by the decomposition action of hydrogen peroxide was determined by color reaction or Hatsuhotaru photoreactive peroxidase system, to quantify accurately substrates or enzymatic activity To provide a profitable method.

(Means for Solving Problems) In the oxidase reaction in which an enzyme is used as an electron acceptor and hydrogen peroxide is produced as described above, the inventors of the present invention bond the by-produced superoxide anion with the peroxidase existing in the system. Form complex III, and
This complex III is O ₂ ^- to have a decomposition O ₂ ^-
The present invention has been completed as a result of further studies based on the finding that the O ₂ ⁻ decomposes before being disproportionated to H ₂ O ₂ .

The method for quantifying a substrate or enzyme activity using the oxidase reaction of the present invention produces hydrogen peroxide and superoxide anion in a substance produced by an enzyme reaction of an enzyme or a substrate to act on the substrate or enzyme to be quantified. Reacting by reacting an oxidase that reacts with the oxidase, the oxidase reaction is completed, and the generated superoxide anion is converted into hydrogen peroxide. It is characterized by being measured by a fluorescence reaction.

The substrate here means oxygen as an electron acceptor and H ₂ O ₂ and
O ₂ ^- matrix of the resulting oxidase reaction, and for example sarcosine, hypoxanthine, xanthine, aldehydes, diamines, such as uric acid, further associated with the oxidase reaction reaction (e.g., product the oxidase reaction caused by certain enzymatic reactions It also includes substrates involved in the reaction of becoming a substrate of () such as creatinine, creatine, and guanine. Examples of the enzyme to be measured in the present invention include an oxidase that produces H ₂ O ₂ and O ₂ ⁻ by using oxygen as an electron acceptor, and an enzyme involved in a reaction related to the oxidase reaction, such as guanase.

The oxidase used in the method of the present invention is an oxidase that acts on a substrate in the presence of an enzyme to generate hydrogen peroxide and superoxide anion using oxygen as an electron acceptor. Examples include the following oxidases and combinations of substrates involved in the oxidase reaction.

O ₂ In the present invention a method, as a by-product in the oxidase reaction ^-
After converting it to H ₂ O ₂ without decomposing it, hydrogen peroxide is measured by peroxidase-based color reaction or fluorescence reaction. O ₂ ^- as a method for converting a H ₂ O _2, left O ₂ The reaction ^- a method for changing the H ₂ O ₂ by the spontaneous disproportionation reaction, disproportionated agent to the reaction system or reducing agent, or There is a method of causing a reducing agent and its auxiliary agent to act. Preferably, O ₂ ^-
By promoting the conversion reaction into H ₂ O ₂ using a disproportionation agent to complete rapidly the. Examples of the disproportionation agent include superoxide dismutase (SOD), galactose oxidase, a compound capable of producing Cu ²⁺ , EDTA-Mn, and EDTA-Fe. SOD is less than 200U / ml, preferably 10-200U / ml
And each of EDTA-Mn and EDTA-Fe is contained in the reaction solution at 1 mM or less, preferably 0.1 to 1 mM. Of these disproportionation agent, for example, the addition of SOD O ₂ ^- disproportionation reaction of the ^{_{H 2 O 2 (2O 2 -}} + 2H + → H 2 O 2 +
Coloring failure peroxidase color lines derived from is avoided ^- O ₂₎ O ₂ to be promoted. By adding a disproportionation agent, the time during which O ₂ ⁻ is present in the reaction system is shortened.
It is possible to avoid the O ₂ ⁻ being oxidized or reduced by the substance in the sample. Furthermore, in order to avoid interference by other substances in the sample, a remover for the interfering substances, such as ascorbate oxidase, may be added. Ascorbate oxidase is contained in the reaction solution at a rate of 500 U / ml or less, preferably 5 to 50 U / ml. O ₂ ^- to H ₂ O ₂
After conversion reaction to, for H ₂ O ₂ is to be degraded by such catalase contained in long existing the sample in a free state, H ₂ O ₂ decomposition inhibiting agent (catalase inhibitor) Adding is also effective. As the decomposition inhibitor, an azide compound (for example, NaN ₃ ) is added in a concentration range that does not inhibit the intended reaction. The azide compound is usually contained in the reaction solution in an amount of 1.0% or less, preferably 0.01 to 1.0%.

H ₂ O ₂ peroxidase-based color reaction (Trinder method)
In a method for measuring the presence of the H ₂ O ₂ peroxidase, and measuring the absorbance of the dye was oxidized color is reacted with a chromogen. As the chromogen, a compound that undergoes oxidative coloring by H ₂ O _{2 in the} presence of peroxidase is used. Such compounds include the following combinations.

4-aminoantipyrine / aniline derivative system 4-aminoantipyrine / phenol derivative system benzothiazolinone hydrazone derivative / aniline derivative system Specific examples of the aniline derivative include N-methyl-N-hydroxymethyl-3-methylaniline, N- Ethyl-N-hydroxyethyl-3-methylaniline, N-ethyl-N-
Sulfopropyl-m-toluidine, N-ethyl-N- (2
-Hydroxy-3-sulfopropyl) -m-toluidine, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-anisidine and the like can be mentioned.

Specific examples of the phenol derivative include p-chlorophenol, p-bromophenol, 3,5-dichlorophenol sulfonic acid, 3-hydroxy-2,4,6-triiodobenzoic acid and the like.

Further, a specific example of the benzothiazolinone hydrazone derivative is 3-methyl-2-benzothiazolinohydrazone.

The use temperature of the aniline derivative and the phenol derivative is not particularly limited, but about 0.1 to 20 mM is optimal.
The concentration of 4-aminoantipyrine used is preferably about 1 to 20 mM. The concentration of the benzothiazolinone hydrazone derivative used is preferably about 0.1 to 2 mM. The method of measuring H ₂ O ₂ by a fluorescence reaction is a method of measuring the amount of fluorescence by reacting with a precursor of a fluorescent substance (a compound having a property of being oxidized to generate fluorescence). As the fluorescent substance precursor, for example, homovanillic acid is used.

In order to measure the substrate or enzyme activity by the method of the present invention, the substrate, enzyme (including oxidase) and, if necessary, the above additives are mixed in a reaction solution to carry out an enzymatic reaction (including oxidase reaction). . Enzyme reactions (including oxidase reaction) is complete, is contained in the reaction system O ₂ ^- is H ₂ O
After conversion to ₂ , peroxidase and for example the above chromogen are added. The degree of color development or fluorescence is measured by an absorptiometer or a fluorescence spectrophotometer to quantify the substrate or enzyme activity.

In the present invention, peroxidase and a chromogen or a fluorescent substance precursor are added to the reaction system after completion of the conversion reaction from O ₂ ⁻ to H ₂ O ₂ to measure H ₂ O ₂ . Therefore, both O ₂ ⁻ and H ₂ O ₂ produced by the oxidase reaction are quantitatively measured as H ₂ O ₂ . A more effective measurement can be performed by adding a disproportionation reaction promoter from O ₂ ⁻ to H ₂ O ₂ or a H ₂ O ₂ decomposition inhibitor.

(Example) Hereinafter, the present invention will be described with reference to Examples.

Example 1 [Determination of uric acid using uricase] The components shown in the above Reaction Composition 1 were mixed, a uric acid sample was added thereto, and a uricase reaction was carried out at 37 ° C. After 5 minutes, a reaction was carried out by adding peroxidase, and the absorbance of the reaction solution was measured at 550 nm. The results are shown in FIG. 1 as a graph plotted with black dots.

Next, add 300U to each component shown in reaction composition 1
The same reaction was carried out by adding 0.1 ml of a / ml superoxide dismutase solution, but no difference in absorbance was observed.

Next, the components shown in the above reaction composition 2 were mixed, 0.1 ml of a uric acid sample was added thereto and reacted for 5 minutes, and the absorbance was measured at 550 nm in the same manner as above. The results are shown in FIG. 1 as a graph plotted with white dots.

From FIG. 1, the degree of color development is higher in the method of the present invention in which peroxidase is added after completion of oxidase, as compared with the conventional method in which pereoxidase is simultaneously added.
It can be seen that in the method of the present invention, O ₂ ⁻ produced together with H ₂ O ₂ by the uricase reaction is sufficiently disproportionated to H ₂ O ₂ and is quantified without being decomposed.

Example 2 [Quantification of creatinine using sarcosine oxidase] After mixing the components shown in the above reaction composition to make a 3 ml solution, 10 μl of the creatinine sample and 10 μl of the superoxide dismutase solution (3000 U / ml) were added and reacted at 37 ° C. for 10 minutes. 10 μl of a peroxidase solution was added thereto to develop color, and the absorbance at 550 nm was measured. The results are shown in FIG. 2 as a graph plotted with black dots.

Next, the components shown in the composition of the above reaction and the peroxidase solution were mixed to prepare a 3 ml solution.
The creatinine sample was added to this and reacted for 10 minutes, and then the absorbance at 550 nm was measured. The result is shown in FIG. 2 as a graph plotted with white dots.

It can be seen from FIG. 2 that according to the method of the present invention, the degree of color development by the sarcosine oxidase reaction is higher than that of the conventional method, and more accurate quantification of creatinine can be performed.

(Effects of the Invention) According to the present invention, both hydrogen peroxide and superoxide anion generated by an oxidase reaction can be measured as hydrogen peroxide by a peroxidase-based color reaction or fluorescence reaction. Since the degree of color development or fluorescence due to the peroxidase-based color reaction is higher than that of the conventional method, the substrate or enzyme can be quantified with high accuracy.

[Brief description of drawings]

FIG. 1 is a graph showing the relationship between the uric acid concentration and the absorbance when uric acid was quantified by the present invention and the conventional method; and FIG. 2 was creatinine quantified by the present invention and the conventional method. It is a graph which respectively shows the relationship between the creatinine density | concentration and light absorbency at this time.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (56) References Fumiharu Maruo One person supervised "Enzyme handbook" Asakura Shoten (1982-12-1) P. 87-89, 202-203 Japan Society for Agricultural Chemistry, "ABC Series Enzymes-Guidelines for Biotechnology-II" Asakura Shoten (1985-3-20) P. 36-37

Claims (2)

[Claims] 1. A substrate or enzyme to be quantified is reacted with an oxidase that produces hydrogen peroxide and superoxide anion, by reacting the substance produced by the enzymatic reaction of the enzyme or the substrate with the oxidase. After the reaction is completed and the produced superoxide anion is converted to hydrogen peroxide, the hydrogen peroxide contained in the reaction system is measured by a color reaction or a fluorescence reaction of a peroxidase system. Substrate or enzyme quantification method using oxidase reaction. 2. A method of converting the produced superoxide anion into hydrogen peroxide is a method of leaving the reaction system and converting the superoxide anion into hydrogen peroxide by a natural disproportionation reaction, or a disproportionation in the reaction system. A method for quantifying a substrate or an enzyme using an oxidase reaction according to claim 1, which is a method of reacting an agent or a reducing agent or a reducing agent with an auxiliary agent thereof.