Method of determining peroxidase activity and substrate mixture for determination of peroxiadse activity

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

 

The invention relates to biotechnology and can be used to create analytic sets, based on the application of reaction-enhanced chemiluminescence catalyzed by peroxidase (A.Roda, M. Guardigli, Analytical chemiluminescence and bioluminescence: latest achievements and new horizons // Analyt. Bioanal. Chem. 2012. Vol.402, P.69-76; C.A.Marquette, L.J.Blum, Chemiluminescent enzyme immunoassays: a review ofbioanalytical applications // Bioanalysis. 2009, Vol.1, P.1259-1269; U.S. patent No. 0192736. 2002). Determination of peroxidase activity is most often used in biochemical and immunoassay kits for the determination of a variety of biologically active substances.

Peroxidase activity in most cases is determined by using the colorimetric method using chromogenic substrates. Although there are different substrates of peroxidase, the most popular of these is 3,3',5,5'-tetramethylbenzidine. However, in cases where the lower detection limit and the extension of the working range of the assay uses chemiluminescent method for determining the activity of peroxidase.

Based chemiluminescent method for the determination of peroxidase activity is the oxidation reaction lyuminola hydrogen peroxide flowing in an alkaline environment. It is known that the peroxidase isolated from the roots of horseradish (HRP), is a light catalyst for this reaction (G.H.G. Thorpe, L.J. Kricka // Meth. Enzymol. 1986. Vol.13. Part C. R-353). The soybean peroxidase (PS), although it is a more active catalyst in relation to the lyuminola (I.S.Alpeeva, I.Yu.Sakharov // J. Agric. Food Chem. 2005. Vol.53. P.5784-5788), and this enzyme exhibits low catalytic efficiency for its practical application.

Previously it was found that a wide range of compounds, including derivatives of 6-hydroxybenzothiazole, substituted phenols, Naftali and anilines, capable of accelerating the reaction of the peroxidase-catalyzed oxidation lyuminola (G.H.G.Thorpe, L.J.Kricka, E.Gillespie, R.Moseley, R.Amess, N.Buggett, T.P.Whietehead // Anal. Biochem. 1985. Vol.145. P.96-100; T.J.N.Carter, C.J.Groucutt, R.A.W.Stott, G.H.G.Thorpe, T.P.Whitehead // Europ. patent 87959, 1982; G.H.G.Thorpe, L.J.Kricka, R.Moseley, T.P.Whietehead // Clin. Chem. 1985. Vol.31. P.1335-1341). Substrate-amplifier and luminal when the joint oxidation exhibit synergism, which is expressed in auditively the observed signal and signals for the individual oxidation of substrates. Enhancing chemiluminescence can reach several hundred times and depends on the nature of the substrate amplifier, reagent concentrations and reaction conditions (G.H.G.Thorpe, L.J.Kricka // Meth. Enzymol. 1986. Vol.133. Part C. R). The greatest enhancing effect has 3-(10'-phenothiazinyl)propane-1-sulfonate sodium (FTPS), used in conjunction with 4-aminopyridine (E.Marzocchi, S.Grilla, L.Della Ciana, L.Prodi, M.Mirasoli, A.Roda // Anal. Biochem. 2008. Vol.377. P.189-194; M.M.Vdovenko, L.Delia Ciana, I.Yu.Sakharov // Anal. Biochem. 2009. Vol.392. P.54-58; U.S. patent No. 7855287, 2010; U.S. patent No. 0053200, 2011)./p>

In the study of reaction-enhanced chemiluminescence (J.Lind, G.Merenyi, ..Eriksen // J. Amer. Chem. Soc. 1983. Vol.105. P.7655; S.B.Vlasenko, A.A.Arefyev, A.D.Klimov, B.B.Kirn, E.L.Gorovits, A.P.Osipov, E.M.Gavrilova, A.M.Yegorov // J. Biolum. Chemilum. 1989. Vol.4. P.164; P.M.Easton, A.C.Simmonds, A.Rakishev, A.M.Egorov, L.P.Candeas // J. Amer. Chem. Soc. 1996. Vol.118. P.6619) was postulated the following mechanism of co oxidation lyuminola and amplifier. At the first stage flows through a series of enzymatic reactions oxidation of the amplifier in accordance with the "ping-pong" mechanism:

E+H2O2⇒EI

EI+SH⇒EII+S

EII+SH⇒E+S,

where SH - substrate-power, S- radical oxidation product of the substrate amplifier, E, EI, EII - various forms of peroxidase.

In further flows through a series of enzymatic reactions, such as oxidation by radicals amplifier molecules anion lyuminola, education diazepinone lyuminola when interacting radicals lyuminola with oxygen or in the process of disproportionation of radicals lyuminola, education peroxide lyuminola when interacting radicals lyuminola with superoxide radicals or the interaction of diazepinone lyuminola with the anion of hydrogen peroxide and the decomposition of peroxide lyuminola nitrogen and 3-aminophthalate located in the electronically excited state. Transition 3-aminophthalate in the base state is accompanied by emission of kV the NTA light. These reactions are presented below in the form of chemical equations:

S+AH-⇒SH+A•-

A•-+O2⇒A+O2•-

A•-+O2•-+H+⇒AO2H-

2A•-⇒A+AH-

A+HO2-⇒AO2H-

AO2H-⇒3-AP+N2+H+hν

where an-- anionic form lyuminola in an alkaline environment. And•-- radical oxidation product lyuminola And - diazepine lyuminola, AO2H-peroxide lyuminola and 3-AR - 3-aminophthalate.

Although the use of reaction-enhanced chemiluminescence has allowed us to develop a highly sensitive method for the determination of peroxidase activity, yet in some cases bioanalytical practice requires increased sensitivity of this method of analysis.

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

The problem is solved in that in the method for determining the activity of peroxidases, comprising preparing a substrate of a mixture of buffer solution, lyuminola, hydrogen peroxide, 4-aminopyridine and amplifier, introduction to substrate mixture of peroxidase with the subsequent registration of the intensity of the resulting fluorescence, according to the invention as an amplifier using N-carboxypropyl the derivative fenotiazina, if the pH of the buffer solution is 7.9-9.0.

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

Buffer solution 10-125 mm

Luminal 0.05-8 mm

Hydrogen peroxide 0.05-8 mm

4-aminopyridines 0.1-10 mm

N-carboxypropanoyl fenotiazina 0.1-10 mm,

at the same time as N-carboxyprothrombin fenotiazina use N-carboxypeptidases, or N-(carboxymethyl)phenothiazines, or N-(2-carboxyethyl)phenothiazines, or N-(3-carboxypropyl)phenotiazine, and as a 4-aminopyridine use 4-morpholinopropan, or 4-dimethylaminopyridine, or 4-pyrrolidinedione.

Thus, the method of determining the activity of peroxidases with chemiluminescent detection provides for the reaction of enzymatic oxidation lyuminola hydrogen peroxide in the presence of 4-aminopyridine, peroxidases and amplifier, where the amplifier using N-carboxypropanoyl fenotiazina. As a 4-aminopyridine preferably used 4-morpholinopropan, 4-dimethylaminopyridine and 4-pyrrolidinedione.

The use of N-carboxyprothrombin fenotiazina as the amplifier instead of 3-(10'-phenothiazinyl)-Propp-1-sulfonate used in the method prototype for chemiluminescent determine the possible activity of peroxidase, helped to dramatically improve the sensitivity of the developed method of analysis.

At present there is no theoretical framework that could predict the emergence of new high-efficiency amplifiers peroxidase-dependent chemiluminescence. Therefore the solution of the problem associated with conducting scientific research and experimental work, which resulted in the discovery that N-carboxypropanoyl fenotiazina are high-performance amplifiers. The proof of "non-obviousness" of the solution found is also the discovery of the fact that others investigated the phenothiazines, namely promethazine, hloratsizin, nonahlazin, ethacyzin and perphenazine, did not possess the power amplifier peroxidase-dependent chemiluminescence.

The inventive method for determining the activity of peroxidases includes preparation of substrate mixture, the introduction of the substrate in a mixture of peroxidase with the subsequent registration of the intensity of the resulting fluorescence. When this substrate mixture has the following composition components final concentration:

Buffer solution 10-125 mm

Luminal 0.05-8 mm

Hydrogen peroxide 0.05-8 mm

4-aminopyridines 0.1-10 mm

N-carboxypropanoyl fenotiazina 0.1-10 mm.

The best result is achieved when the pH of a buffer solution selected is the first of the interval values of 7.9 and 9.0, when using as N-carboxyprothrombin fenotiazina N-carboxypeptidases, or N-(carboxymethyl)fenotiazina, or N-(2-carboxyethyl)fenotiazina, or N-(3-carboxypropyl)fenotiazina, and when used as a 4-aminopyridine - 4-morpholinopropan or 4-dimethylaminopyridine or 4-pyrrolidinedione.

In the process of implementation of the method the following reactions occur:

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

b) reaction of Compound I with N-carboxypropanoyl fenotiazina with the formation of cation-radical derivatives fenotiazina;

C) reaction of the cation-radical derivatives fenotiazina with lyuminola with the formation of 3-aminophthalate and generation of chemiluminescence.

The use of N-carboxyprothrombin fenotiazina as amplifiers in response enzymatic oxidation lyuminola hydrogen peroxide allows the determination of peroxidase activity of plants with high sensitivity.

The invention is illustrated by the following examples.

Example No. 1.

In 100 mm Tris buffer solution, pH 8.3 was added to the luminal, hydrogen peroxide, N-(2-carboxyethyl)phenothiazines and 4-morpholinopropan in concentrations of 1, 3, 5.2 and 9.3 mm, respectively. To initiate the enzymatic reaction to the prepared substrate mixture was added horseradish peroxidase (control e is speriment enzyme was not added), and then using a luminometer recorded the intensity of the resulting fluorescence. The sensitivity of the method for the determination of peroxidase was 525150 conventional unit of luminous intensity /PM peroxidase (sensitivity method for determination of 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 was added to the luminal, hydrogen peroxide, N-(2-carboxyethyl)phenothiazines and 4-morpholinopropan in concentrations of 0.5, 3, 4.2 and 7.5 mm, respectively. To initiate the enzymatic reaction to the prepared substrate mixture was added horseradish peroxidase (in the control experiment, the enzyme was not added), and then using a luminometer recorded the intensity of the resulting fluorescence. The sensitivity of the method for the determination of peroxidase in the working range was 573000 conventional unit of luminous intensity /PM peroxidase.

Example No. 3.

In 100 mm Tris buffer solution, pH 8.3 was added to the luminal, hydrogen peroxide, N-(carboxymethyl)phenothiazines and 4-morpholinopropan in concentrations of 1, 3, 5.2 and 9.3 mm, respectively. To initiate the enzymatic reaction to the prepared substrate mixture was added horseradish peroxidase (in the control experiment, the enzyme was not added), and then using a luminometer recorded the intensity of the resulting fluorescence. Sensibility the method of determination of peroxidase in the working range was $ 467100 light intensity /PM peroxidase.

Example No. 4.

In 100 mm Tris buffer solution, pH 8.3 was added to the luminal, hydrogen peroxide, N-carboxypeptidases and 4-morpholinopropan in concentrations of 1, 3, 5.2 and 9.3 mm, respectively. To initiate the enzymatic reaction to the prepared substrate mixture was added horseradish peroxidase (in the control experiment, the enzyme was not added), and then using a luminometer recorded the intensity of the resulting fluorescence. The sensitivity of the method for the determination of peroxidase in the working range was 325050 conventional unit of luminous intensity /PM peroxidase.

Example No. 5.

In 100 mm Tris buffer solution, pH 8.3 was added to the luminal, hydrogen peroxide, N-(3-carboxypropyl)phenothiazines and 4-morpholinopropan in concentrations of 1, 3, 5.2 and 9.3 mm, respectively. To initiate the enzymatic reaction to the prepared substrate mixture was added horseradish peroxidase (in the control experiment, the enzyme was not added), and then using a luminometer recorded the intensity of the resulting fluorescence. The sensitivity of the method for the determination of peroxidase in the working range was 455300 conventional unit of luminous intensity /PM peroxidase.

Example No. 6.

In 100 mm Tris buffer solution, pH 8.3 was added to the luminal, hydrogen peroxide, N-(2-carboxyethyl)phenothiazines and 4-dimethylaminopyridine in concentrations of 1, 3, 5.2 and .3 mm, respectively. To initiate the enzymatic reaction to the prepared substrate mixture was added horseradish peroxidase (in the control experiment, the enzyme was not added), and then using a luminometer recorded the intensity of the resulting fluorescence. The sensitivity of the method for the determination of peroxidase in the working range was 499400 conventional unit of luminous intensity /PM peroxidase.

Example No. 7.

In 100 mm Tris buffer solution, pH 8.3 was added to the luminal, hydrogen peroxide, N-(2-carboxyethyl)phenothiazines and 4-pyrrolidinone in concentrations of 1, 3, 5.2 and 9.3 mm, respectively. To initiate the enzymatic reaction to the prepared substrate mixture was added horseradish peroxidase (in the control experiment, the enzyme was not added), and then using a luminometer recorded the intensity of the resulting fluorescence. The sensitivity of the method for the determination of peroxidase in the working range was 471800 conventional unit of luminous intensity /PM peroxidase.

Example No. 8.

In 100 mm Tris buffer solution, pH 8.3 was added to the luminal, hydrogen peroxide, N-(2-carboxyethyl)phenothiazines and 4-morpholinopropan in concentrations of 1, 3, 5.2 and 9.3 mm, respectively. To initiate the enzymatic reaction to the prepared substrate mixture was added to the soybean peroxidase (in the control experiment, the enzyme was not added), and then using a luminometer re who was interaval the intensity of the resulting fluorescence. The sensitivity of the method for the determination of peroxidase in the working range was 475900 conventional unit of luminous intensity /PM peroxidase.

Example No. 9 (comparative).

In 50 mm Tris buffer solution, pH 8.3 was added to the luminal, hydrogen peroxide, sodium salt 3-(10'-phenothiazinyl)-propane-1-sulfonate and 4-morpholinopropan in 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 to the prepared substrate mixture was added horseradish peroxidase (in the control experiment, the enzyme was not added), and then using a luminometer recorded the intensity of the resulting fluorescence. The sensitivity of the method for the determination of peroxidase in the working range was 56625 conventional unit of luminous intensity/PM peroxidase.

Thus, on the basis of the above examples was made clear conclusion that the substitution in the reaction solution of 3-(10'-phenothiazinyl)-propane-1-sulfonate on M-carboxypropanoyl fenotiazina leads to a sharp increase in the sensitivity of the method chemiluminescent determination of peroxidase.

1. The method for determining the activity of peroxidases, comprising preparing a substrate of a mixture of buffer solution, lyuminola, hydrogen peroxide, 4-amino is of eidinow and amplifier, introduction in the substrate a mixture of peroxidase with the subsequent registration of the intensity of the resulting fluorescence, characterized in that as the amplifier using N-carboxypeptidases, or N-(carboxymethyl)phenothiazines, or N-(2-carboxyethyl)phenothiazines in the following ratio of the components identified in final concentrations:

Buffer solution10-125 mm
Luminal0.05-8 mm
Hydrogen peroxide0.05-8 mm
4-aminopyridines0.1-10 mm
N-carboxypeptidases or
N-(carboxymethyl)phenothiazines or
N-(2-carboxyethyl)phenothiazines0.1-10 mm

if the pH buffer solution of 7.9-9.0.

2. The method according to claim 1, characterized in that as a 4-aminopyridine use 4-morpholinopropan, or 4-dimethylaminopyridine, or 4-pyrrolidinedione.

3. Substrate mixture for determining the activity of peroxidases, including buffer solution, luminal, hydrogen peroxide, 4-aminopyridines and power, great is connected with the fact, what power it contains N-carboxypeptidases, or N-(carboxymethyl)phenothiazines, or N-(2-carboxyethyl)phenothiazines in the following ratio of the components identified in final concentrations:

Buffer solution10-125 mm
Luminal0.05-8 mm
Hydrogen peroxide0.05-8 mm
4-aminopyridines0.1-10 mm
N-carboxypeptidases or
N-(carboxymethyl)phenothiazines or
N-(2-carboxyethyl)phenothiazines0.1-10 mm

if the pH buffer solution of 7.9-9.0.

4. Substrate mixture according to claim 3, characterized in that as a 4-aminopyridine use 4-morpholinopropan, or 4-dimethylaminopyridine, or 4-pyrrolidinedione.



 

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FIELD: medicine.

SUBSTANCE: invention relates to field of biotechnology. Claimed is method of obtaining 4-hydroxy-L-leucine or its salt by method of enzymatic conversion of L-leucine or its salt in presence of bacterial deoxygenase, selected from group, which consists of dioxygenases with amino acid sequence SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 22, SEQ ID NO: 54, SEQ ID NO: 62 or their versions. Described is method of obtaining 4-hydroxy-L-leucine or its salt in L-leucine-containing medium in presence of first bacterium, transformed with DNA molecule, encoding dioxygenase, which consists of dioxygenases with amino acid sequence SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 22, SEQ ID NO: 54, SEQ ID NO: 62 or their versions. Described is bacterium - producent of 4-hydroxy-L-leucine or its salt, transformed with DNA molecule, encoding bacterial dioxygenase, selected from group, consisting of dioxygenases with amino acid sequence SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 22, SEQ ID NO: 54, SEQ ID NO: 62 or their versions in medium, containing L-leucine or its salt.

EFFECT: invention makes it possible to obtain 4-hydroxy-L-leucine or its salt with high degree of efficiency.

9 cl, 24 dwg, 5 tbl, 11 ex

FIELD: genetic engineering, biotechnology, biochemistry, agriculture, food industry, medicine.

SUBSTANCE: invention relates to the transformation of plant with nucleic acid encoding enzyme Δ6-desaturase in C. elegans that results to preparing a plant with enhanced content of gamma-linolenic acid and resistance to cold. Desaturase extracted from the plant can be used for preparing a drug used for treatment of disorder in body associated with deficiency of gamma-linolenic acid in it.

EFFECT: valuable biological properties of genes and desaturases.

36 cl, 9 dwg, 2 ex

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