RU2525137C2 - Method of determining peroxidase activity and substrate mixture for determination of peroxiadse activity - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: group of inventions relates to biotechnology and can be applied in creation of analytic methods with application of peroxidases. Method includes preparation of substrate mixture, introduction of peroxidases in substrate mixture with the following registration of intensity of formed luminescence. Substrate mixture includes the following components, given in final concentrations: buffer solution 10-125 mM, luminol 0.05-8 mM, hydrogen peroxide 0.05-8 mM, 4-aminopyridines 0.1-10 mM, N-carboxyphenothiazine, or N-(carboxymethyl)phenothiazine, or N-(2-carboxyethyl)phenothiazine 0.1-10 mM. And pH of buffer solution constitutes 7.9-9.0.

EFFECT: invention ensures obtaining of high intensity of chemiluminescence and, accordingly, high sensitivity of peroxidase activity determination.

4 cl, 8 ex

Description

The invention relates to biotechnology and can be used to create analytical kits based on the use of enhanced chemiluminescence reaction catalyzed by peroxidases (A. Roda, M. Guardigli, Analytical chemiluminescence and bioluminescence: latest achievements and new horizons // Analyt. Bioanal. Chem. 2012 Vol.402, P.69-76; CAMarquette, LJ Blum, Chemiluminescent enzyme immunoassays: a review of bioanalytical applications // Bioanalysis. 2009, Vol.1, P.1259-1269; US patent No. 0192736. 2002). Determination of peroxidase activity is most often used in biochemical and enzyme immunoassay kits to determine a variety of biologically active substances.

In most cases, peroxidase activity is determined using the colorimetric method using chromogenic substrates. Although various peroxidase substrates are used, the most popular of these is 3.3 ', 5.5'-tetramethylbenzidine. However, in those cases where a decrease in the detection limit and an extension of the working range of the analysis is required, a chemiluminescent method for determining peroxidase activity is used.

The chemiluminescent method for determining peroxidase activity is based on the reaction of luminol oxidation with hydrogen peroxide, which proceeds in an alkaline medium. It is known that peroxidase isolated from horseradish roots (HRP) is a low activity catalyst for this reaction (Thorpe G.H.G., Kricka L.J. // Meth. Enzymol. 1986. Vol.133. Part B. P.331-353). Soya peroxidase (PS), although it is a more active catalyst in relation to luminol (ISAlpeeva, I.Yu.Sakharov // J. Agric. Food Chem. 2005. Vol.53. P.5784-5788), but this enzyme exhibits low catalytic efficiency for its practical application.

It was previously found that a wide range of compounds, including derivatives of 6-hydroxybenzothiazoles, substituted phenols, naphthols and anilines, can accelerate the peroxidase oxidation reaction of luminol (GHGThorpe, LJKricka, E.Gillespie, R.Moseley, R.Amess, N.Buggett , TPWhietehead // Anal. Biochem. 1985. Vol.145. P.96-100; TJNCarter, CJGroucutt, RAWStott, GHGThorpe, TPWhitehead // Europ. Patent 87959, 1982; GHGThorpe, LJKricka, R. Moseley, TPWhietehead // Clin. Chem. 1985. Vol.31. P.1335-1341). The combined amplifier and luminol exhibit synergism during co-oxidation, which is expressed in the non-additivity of the observed signal and signals for individual oxidation of the substrates. The enhancement of chemiluminescence can reach several hundred times and depends on the nature of the substrate-enhancer, the concentration of reagents and the reaction conditions (G.H.G. Thorpe, L.J. Kricka // Meth. Enzymol. 1986. Vol.133. Part B. P.331). The greatest enhancing effect is exerted by 3- (10'-phenothiazinyl) propane-1-sodium sulfanate (FTPS), used in conjunction with 4-aminopyridines (E. Marzocchi, S. Grilla, L.Della Ciana, L.Prodi, M. Mirasoli, A. Roda // Anal. Biochem. 2008. Vol.377. P.189-194; MMVdovenko, L. Delia Ciana, I. Yu. Sakharov // Anal. Biochem. 2009. Vol.392. P.54- 58; US Patent No. 7,855,287, 2010; US Patent No. 0053200, 2011).

In studies of the enhanced chemiluminescence reaction (J. Lind, G. Merenyi, T.E. Ericksen // J. Amer. Chem. Soc. 1983. Vol.105. P.7655; SBVlasenko, AAArefyev, ADKlimov, BBKirn, ELGorovits, APOsipov, EMGavrilova, AMYegorov // J. Biolum. Chemilum. 1989. Vol.4. P.164; PMEaston, ACSimmonds, A.Rakishev, AMEgorov, LPCandeas // J . Amer. Chem. Soc. 1996. Vol.118. P.6619) the following mechanism of the co-oxidation of luminol and an amplifier was postulated. At the first stage, a series of reactions of the enzymatic oxidation of the amplifier proceeds in accordance with the "ping-pong" mechanism:

E + H ₂ O ₂ ⇒EI

EI + SH⇒EII + S ^•

EII + SH⇒E + S ^• ,

where SH is the substrate amplifier, S ^• is the radical oxidation product of the substrate amplifier, E, EI and EII are various forms of peroxidase.

Subsequently, a series of non-enzymatic reactions proceeds, the main of which are oxidation of the enhancer of the luminol anion molecule by radicals, the formation of luminol diazoquinone during the interaction of luminol radicals with oxygen or during the disproportionation of luminol radicals, the formation of luminol peroxide during the interaction of luminol radicals with superoxide radicals or the interaction of luminol diazoquinone with anion hydrogen peroxide and the decomposition of luminol peroxide into nitrogen and 3-aminophthalate, which is electronically excited Mr. condition. The transition of 3-aminophthalate to the base state is accompanied by the emission of a quantum of light. These reactions are presented below in the form of chemical equations:

S ^• + AH ^- ⇒SH + A ^{• -}

A ^{• -} + O ₂ ⇒ A + O ₂ ^{• -}

A ^{• -} + O ₂ ^{• -} + H ⁺ ⇒AO ₂ H ^-

2A ^{• -} ⇒A + AH ^-

A + HO ₂ ^- ⇒AO ₂ H ^-

AO ₂ H ^- ⇒3-AP + N ₂ + H + hν,

where AN ^- is the anionic form of luminol in an alkaline medium. A ^{• -} is the radical product of luminol oxidation, A is luminol diazoquinone, AO ₂ H ^is luminol peroxide and 3-AP is 3-aminophthalate.

Although the use of enhanced chemiluminescence reactions allowed the development of a highly sensitive method for determining peroxidase activity, in some cases bioanalytical practice requires increasing the sensitivity of this analysis method.

The objective of the invention is to develop a highly sensitive method for determining the enzymatic activity of peroxidase.

The problem is solved in that in the method for determining the activity of peroxidases, including the preparation of a substrate mixture consisting of a buffer solution, luminol, hydrogen peroxide, 4-aminopyridines and an amplifier, the introduction of peroxidases into the substrate mixture with subsequent registration of the intensity of the resulting glow, according to the invention as an amplifier use N-carboxy derivatives of phenothiazine, while the pH of the buffer solution is 7.9-9.0.

The problem is also solved by the fact that the substrate mixture used in the method is characterized by the above composition in the following final concentrations of the components:

Buffer solution 10-125 mm

Luminol 0.05-8 mm

Hydrogen peroxide 0.05-8 mm

4-aminopyridines 0.1-10 mM

N-carboxy derivatives of phenothiazine 0.1-10 mM,

while N-carboxyphenothiazine, or N- (carboxymethyl) phenothiazine, or N- (2-carboxyethyl) phenothiazine, or N- (3-carboxypropyl) phenothiazine are used as N-carboxyphenothiazine derivatives, and 4- aminopyridines are used morpholinopyridine, or 4-dimethylaminopyridine, or 4-pyrrolidinopyridine.

Thus, a method for determining the activity of peroxidases with chemiluminescent detection involves the reaction of enzymatic oxidation of luminol with hydrogen peroxide in the presence of 4-aminopyridines, peroxidases and an amplifier, where N-carboxyphenothiazine derivatives are used as an amplifier. As 4-aminopyridines, 4-morpholinopyridine, 4-dimethylaminopyridine and 4-pyrrolidinopyridine are preferably used.

The use of N-carboxy derivatives of phenothiazine as an amplifier instead of 3- (10'-phenothiazinyl) propap-1-sulfonate, used in the prototype method for chemiluminescent determination of peroxidase activity, allowed a sharp increase in the sensitivity of the developed analysis method.

There is currently no theoretical basis that could predict the emergence of new highly effective peroxidase-dependent chemiluminescence enhancers. In this connection, the solution of the problem posed is associated with the carrying out of research and experimental work, the result of which was the discovery that N-carboxy derivatives of phenothiazine are highly effective amplifiers. Confirmation of the “non-obviousness” of the solution found is also the discovery of the fact that the other phenothiazine derivatives that we studied, namely diprazine, chloracisin, nononlazine, ethacyzine and perphenazine, did not possess the properties of a peroxidase-dependent chemiluminescence enhancer.

The inventive method for determining the activity of peroxidases includes preparing a substrate mixture, introducing peroxidase into the substrate mixture, followed by recording the intensity of the resulting glow. In this case, the substrate mixture has the following composition of components with their final concentration:

Buffer solution 10-125 mm

Luminol 0.05-8 mm

Hydrogen peroxide 0.05-8 mm

4-aminopyridines 0.1-10 mM

N-carboxy derivatives of phenothiazine 0.1-10 mM.

The best result is achieved when the pH of the buffer solution is selected from the range of 7.9-9.0, and when N-carboxyphenothiazine, or N- (carboxymethyl) phenothiazine, or N- (2-carboxyethyl) phenothiazine is used as the N-carboxyphenothiazine or N- (3-carboxypropyl) phenothiazine, and also when 4-morpholinopyridine or 4-dimethylaminopyridine or 4-pyrrolidinopyridine is used as 4-aminopyridines.

In the process of implementing the method, the following reactions occur:

a) the reaction of peroxidase with hydrogen peroxide to form Compound I;

b) the reaction of Compound I with N-carboxyphenothiazine derivatives to form a radical cation of phenothiazine derivatives;

c) reactions of the radical cation of phenothiazine derivatives with luminol with the formation of 3-aminophthalate and the formation of chemiluminescence.

The use of N-carboxy derivatives of phenothiazine as enhancers in the reaction of enzymatic oxidation of luminol with hydrogen peroxide allows the determination of the activity of plant peroxidases with high sensitivity.

The invention is illustrated by the following examples.

Example No. 1.

In 100 mM Tris buffer solution, pH 8.3, luminol, hydrogen peroxide, N- (2-carboxyethyl) phenothiazine and 4-morpholinopyridine were added at concentrations of 1, 3, 5.2, and 9.3 mM, respectively. To initiate the enzymatic reaction, horseradish peroxidase was added to the prepared substrate mixture (in the control experiment, the enzyme was not added), and then the intensity of the formed luminescence was recorded using a luminometer. The sensitivity of the method for determination of peroxidase was 525150 cu luminescence intensity / pM peroxidase (the sensitivity of the method for determining peroxidase is numerically equal to the tangent of the calibration curve in its operating range).

Example No. 2.

In 60 mM Tris buffer solution, pH 8.3, luminol, hydrogen peroxide, N- (2-carboxyethyl) phenothiazine and 4-morpholinopyridine were added at concentrations of 0.5, 3, 4.2 and 7.5 mM, respectively. To initiate the enzymatic reaction, horseradish peroxidase was added to the prepared substrate mixture (in the control experiment, the enzyme was not added), and then the intensity of the formed luminescence was recorded using a luminometer. The sensitivity of the method for determining peroxidase in the operating range was 573,000 cu luminescence intensities / pm peroxidase.

Example No. 3.

In 100 mM Tris buffer solution, pH 8.3, luminol, hydrogen peroxide, N- (carboxymethyl) phenothiazine and 4-morpholinopyridine were added at concentrations of 1, 3, 5.2, and 9.3 mM, respectively. To initiate the enzymatic reaction, horseradish peroxidase was added to the prepared substrate mixture (in the control experiment, the enzyme was not added), and then the intensity of the formed luminescence was recorded using a luminometer. The sensitivity of the method for determining peroxidase in the working range was 467100 cu luminescence intensities / pm peroxidase.

Example No. 4.

In 100 mM Tris buffer solution, pH 8.3, luminol, hydrogen peroxide, N-carboxyphenothiazine and 4-morpholinopyridine were added at concentrations of 1, 3, 5.2, and 9.3 mM, respectively. To initiate the enzymatic reaction, horseradish peroxidase was added to the prepared substrate mixture (in the control experiment, the enzyme was not added), and then the intensity of the formed luminescence was recorded using a luminometer. The sensitivity of the method for determining peroxidase in the working range was 325050 cu luminescence intensities / pm peroxidase.

Example No. 5.

In 100 mM Tris buffer solution, pH 8.3, luminol, hydrogen peroxide, N- (3-carboxypropyl) phenothiazine and 4-morpholinopyridine were added at concentrations of 1, 3, 5.2, and 9.3 mM, respectively. To initiate the enzymatic reaction, horseradish peroxidase was added to the prepared substrate mixture (in the control experiment, the enzyme was not added), and then the intensity of the formed luminescence was recorded using a luminometer. The sensitivity of the method for determining peroxidase in the working range was 455300 cu luminescence intensities / pm peroxidase.

Example No. 6.

In 100 mM Tris buffer solution, pH 8.3, luminol, hydrogen peroxide, N- (2-carboxyethyl) phenothiazine and 4-dimethylaminopyridine were added at concentrations of 1, 3, 5.2 and 9.3 mM, respectively. To initiate the enzymatic reaction, horseradish peroxidase was added to the prepared substrate mixture (in the control experiment, the enzyme was not added), and then the intensity of the formed luminescence was recorded using a luminometer. The sensitivity of the method for determining peroxidase in the working range was 499,400 cu luminescence intensities / pm peroxidase.

Example No. 7.

In 100 mM Tris buffer solution, pH 8.3, luminol, hydrogen peroxide, N- (2-carboxyethyl) phenothiazine and 4-pyrrolidinopyridine were added at concentrations of 1, 3, 5.2 and 9.3 mM, respectively. To initiate the enzymatic reaction, horseradish peroxidase was added to the prepared substrate mixture (in the control experiment, the enzyme was not added), and then the intensity of the formed luminescence was recorded using a luminometer. The sensitivity of the method for determining peroxidase in the operating range was 471800 cu luminescence intensities / pm peroxidase.

Example No. 8.

In 100 mM Tris buffer solution, pH 8.3, luminol, hydrogen peroxide, N- (2-carboxyethyl) phenothiazine and 4-morpholinopyridine were added at concentrations of 1, 3, 5.2, and 9.3 mM, respectively. To initiate the enzymatic reaction, soy peroxidase was added to the prepared substrate mixture (in the control experiment, the enzyme was not added), and then the intensity of the luminescence formed was recorded using a luminometer. The sensitivity of the method for determining peroxidase in the working range was 475900 cu luminescence intensities / pm peroxidase.

Example No. 9 (comparative).

In 50 mM Tris buffer solution, pH 8.3, luminol, hydrogen peroxide, sodium salt of 3- (10'-phenothiazinyl) propane-1-sulfonate and 4-morpholinopyridine were added at concentrations of 0.75, 0.5, 1, and 1 mM, respectively. A comparative experiment was carried out according to the method described in (E. Marzocchi, S. Grilla, L. Delia Ciana, L.Prodi, M. Mirasoli, A. Roda // Anal. Biochem. 2008. Vol.377. P.189-194 ) To initiate the enzymatic reaction, horseradish peroxidase was added to the prepared substrate mixture (in the control experiment, the enzyme was not added), and then the intensity of the formed luminescence was recorded using a luminometer. The sensitivity of the method for determining peroxidase in the working range was 56625 cu luminescence intensities / pm peroxidase.

Thus, on the basis of the above examples, an unequivocal conclusion was made that the replacement of 3- (10'-phenothiazinyl) propane-1-sulfonate in the reaction solution with M-carboxyphenothiazine derivatives leads to a sharp increase in the sensitivity of the method of chemiluminescent determination of peroxidases.

Claims (4)

1. A method for determining the activity of peroxidases, including the preparation of a substrate mixture consisting of a buffer solution, luminol, hydrogen peroxide, 4-aminopyridines and an amplifier, introducing peroxidases into the substrate mixture, followed by recording the intensity of the generated glow, characterized in that N- is used as an amplifier carboxyphenothiazine, or N- (carboxymethyl) phenothiazine, or N- (2-carboxyethyl) phenothiazine in the following ratio of components indicated in final concentrations:
Buffer solution 10-125 mm Luminol 0.05-8 mm Hydrogen peroxide 0.05-8 mm 4-aminopyridines 0.1-10 mm N-carboxyphenothiazine or N- (carboxymethyl) phenothiazine or N- (2-carboxyethyl) phenothiazine 0.1-10 mm,
while the pH of the buffer solution is 7.9-9.0. 2. The method according to claim 1, characterized in that 4-morpholinopyridine, or 4-dimethylaminopyridine, or 4-pyrrolidinopyridine is used as 4-aminopyridines. 3. A substrate mixture for determining the activity of peroxidases, including a buffer solution, luminol, hydrogen peroxide, 4-aminopyridines and an amplifier, characterized in that it contains N-carboxyphenothiazine, or N- (carboxymethyl) phenothiazine, or N- (2 β-carboxyethyl) phenothiazine in the following ratio of components indicated in final concentrations:
Buffer solution 10-125 mm Luminol 0.05-8 mm Hydrogen peroxide 0.05-8 mm 4-aminopyridines 0.1-10 mm N-carboxyphenothiazine or N- (carboxymethyl) phenothiazine or N- (2-carboxyethyl) phenothiazine 0.1-10 mm,
while the pH of the buffer solution is 7.9-9.0. 4. The substrate mixture according to claim 3, characterized in that 4-morpholinopyridine or 4-dimethylaminopyridine or 4-pyrrolidinopyridine is used as 4-aminopyridines.