SU1636772A1 - Method for determination of hydrogen peroxide in biological objects - Google Patents

Abstract

This invention relates to methods for biochemical analysis and can be used to determine hydrogen peroxide in biological samples and generating systems. The purpose of the invention is to increase the sensitivity of the method while it is possible to carry out the method in the presence of heme-containing pigments. Buffer solution, peroxidase, and a mixture of gva cola and p-aminodiethylaniline at a concentration of each component of 0.05-0.5 mM are added to the test sample. The sample is incubated until the increase in optical density is stopped and spectrophotometrized against a control sample not containing hydrogen peroxide. The content of hydrogen peroxide is determined by the value of optical density. The method allows to increase the sensitivity by 2.5 times. 2 tab. with 5S (L

Description

The invention relates to methods for biochemical analysis and can be used to determine hydrogen peroxide in biological samples.

The purpose of the invention is to increase the sensitivity of the method while it is possible to carry out the method in the presence of heme-containing pigments.

The method is carried out as follows.

Buffer solution and peroxidase are added to the test sample and, as a chromogen, a mixture of gba cola and p-aminodiethylaniline with a concentration of each component of 0.05-0.5 M. The sample is incubated until the increase in

optical density and spectrophotometry against a control sample that does not contain hydrogen peroxide. The content of hydrogen peroxide is determined by the value of optical density.

Example 1. Analysis of hydrogen peroxide in solutions.

400 µl of 0.5 M phosphate buffer, pH 7.0-7.5, 400 µl of 2 mM gua-cola and 400 µl of 2 mM n-aminodiethylaniline sulphate are added to 10-2000 µl of the sample being analyzed, containing 10-500 nmol H20g. (or p-aminodimethylaniline dihydrochloride). The sample volume was adjusted to 3.8 ml with water. The reaction is started by adding 200 ml of a 0.1% solution of horseradish peroxidase and after 5 minutes (it is possible through

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5-30 min.) The optical density of the solutions is measured at 625 nm on a spectrophotometer or at 670 nm on a KFK-2MP photoelectric colorimeter against a control containing water instead of hydrogen peroxide. The content in the initial sample is determined by a calibration chart using the following formula, mM:, J

r -, s -,

where p is the dilution of the original sample

before introduction into the incubation mixJ

V is the volume of the introduced sample, ml;

D is the optical density of the solution versus the control that does not contain FLO:

k is the coefficient for, found from the calibration test (it is usually 0.35 under the described conditions for a KFK-2MP photoelectric colorimeter). Example 1. The original sample, obsessed, diluted 100 times. 0.1 ml was taken for analysis. The optical density of the photometric solution was 0.210 (PEC KFK-2MP, 670 nm, 1 cm). The calibration coefficient (i.e. the number of micromoles H, 0 g in 4 ml of the sample corresponding to optical density 1) for these conditions is equal to 0.35.

Therefore, the initial concentration is C 73.5 mM.

Example 2. Analysis of hydrogen peroxide generated in the oxidase reaction (method for determining the activity of oxidases).

A. Determination of alcohol oxidase activity. 400 μl of 0.5 M phosphate buffer, pH 7.0-7.5, 400 μl of 2 mM gua cola, 400 μl of 2 mM n-aminodiethylaniline sulphate (or dichlorohydrate) are added to the analyzed sample (5-100 μg of protein). -aminodimethylaniline), 400 µl of 100 mM methanol, the sample volume was made up to 3.8 ml with water. The reaction is started by adding 200 µl of horseradish peroxidase to 0.1% (Reanal RZ 0.6) and after 30 s an optical density increment is measured for 1 min at 670 nm of the KFK-2MP photoelectric hydrometer in the Active mode. The specific activity of the enzyme (generated hydrogen peroxide for 1 min per 1 mg of protein) is calculated by the formula, μmol:

Q

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and O 5

40

0 5

45

A; & R / min. k- n

where db / min is the optical increment

density for 1 min; p - dilution of the solution

enzyme before adding reagents; V is the volume of the enzyme solution introduced into the sample, ml

Sy - baseline protein concentration, mg / ml; k is the coefficient for, found from calibration experiments, (B of the described conditions, it is equal to 0.35).

The original sample of the cell-free extract of methylotrophic yeast containing alcohol oxidase was diluted 100 times. The protein concentration in the initial extract is 5.5 mg / ml. 0.2 ml was taken for analysis. The increment of optical density for 1 min was 0.125 (FEK KFK-2MP-, 670 nm; 1 cm). The calibration factor under these conditions is 0.35. Consequently, the specific alcohol oxidase activity in the initial extract is A 3.98 µmol / min mg of protein.

B. Determination of glucose oxidase activity. The analysis and calculation was carried out in the same way as in Example 2A with the only difference that 100 mM glucose was used instead of methanol, and 0.5 M citrate-NaOH pH 6.0 was used as a buffer.

The boundary values of the chromogen concentration (HgOj concentration, 50 µM) of the proposed method are summarized in Table 1.

The sensitivity of the proposed method has increased 2.5 times.

Table 2 gives a comparison of the sensitivity of the determination of hydrogen peroxide by the proposed method (the chromogen is a mixture of gua-cola and p-aminodiethylaniline) and the known method (the chromogen is p-phenylenediamine). Conditions: 50 mM phosphate buffer, pH 7.0-, chromogen concentration 0.25 mM horseradish peroxidase (RZ 0.6) 0.05 mg / ml. Spectrophotometer SF-26, 1 cm.

It is possible to determine the hydrogen peroxide in the presence of hem-containing pigments, in particular blood hemoglobin.

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The effect of blood hemolysate on the determination of hydrogen peroxide according to the proposed method (the chromogen is a mixture of gua cola and n-aminodiethylaniline) and 1 known (the chromogen is p-phenylenediamine) is shown in table 3. Conditions are similar to the table ...

and n-aminodiethylaniline at a concentration of each component of 0.05-0.5 mM.

Table 1

As follows from Table 3, when analyzing in samples containing blood (hemolysate), using a known method, the optical density of the blank (control) is dramatically increased to 0.510 when diluted 100 times, which drastically impairs the convenience, accuracy and sensitivity of the analysis. In the case of the use of the proposed chromogenic system (gwa col + + n-aminodiethylaniline), the optical density of the control in the presence of blood is 7.5 times lower than that known, which is due to the non-overlapping absorption spectra of the reaction product and hemoglobin.

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0.01

0.025

0.05

0.1

0.25

0.375

0.5

0.75

1.0

2.5

five

25

Claims (1)

SUMMARY OF THE INVENTION A method for determining hydrogen peroxide in biological objects by adding a buffered solution ·, peroxidase and chromogen to the test sample, incubating the sample until the increase in optical density and spectrophotometry ceases, which involves increasing the sensitivity of the method while the possibility of implementing the method in the presence of heme-containing pigments, a guaiacol mixture is used as a chromogen .θ Table 1 The concentration of guaiacol and p-aminodiethylaniline, mm Optical control density (without Hg0 ₂ ) The increase in optical density, ^P Ng.O ₂ 0.01 0.005 0,090 0,025 0.010 0.447 0.05 0.015 0.517 0.1 0,028 0.550 0.25 0.104 0.560 0.375 0.161 0.550 0.5 0.272 0,500 0.75 0.915 0.412 1,0 1,197 0.327 2,5 2,102 0,096 5 2,33 0,064 table 2 The concentration of Hg0 ₂ , μm (guaiacol + + p-aminodiethylaniline) ^D 52O (η — Phenylenediamine) 12.5 0.140 0,055 25 0.288 0.115 fifty 0.556 0.222 75 0.820 0.322 100 1,080 0.417 Table 3 i Series Try HjOj concentration, μm Guaiacol + p-aminodiethylanilia 2-phenylenediamine ^D fi50 ° 52o Φ IgG. ” The control 0 0.01 3 - 0,025 - Without 1 25 0,301 0.288 0.440 0.115 laziness hemo 2 fifty 0.569 0.556 0.247 0.222 lysate The control 0 0,034 - 0.258 - With hemol 1 25 0.268 0.234 0.350 0,092 then (times - 2 fifty 0.437 0.403 0.413 0.155 conducting - 200) With hemol The control 0 0,068 • '· * 0.510 - then (times 1 25 0.250 0.182 0.571 0,061 management 2 fifty 0.386 0.318 0.641 0.131 100) D _K )