Method of obtaining fluorescent derivatives of catecholamines and their metabolites by method of derivatisation
SUBSTANCE: invention relates to a novel method of obtaining fluorescent catecholamines selected from dopamine and adrenalin, and their metabolites, selected from homovanillic and vanillin-mandelic acids, by a method of derivation. The compounds can be used as highly sensitive and selective markers for the determination of various diseases. The method of derivation includes oxidation of the initial compounds and their interaction with amines that form condensed structures in a medium of the CAPS-buffer solution or glycin - KOH 0.2 mM hydrogen peroxide in the presence of horseradish peroxidase as a catalyst. The process in preferably carried out in a 0.1 M buffer solution with the concentration of horseradish peroxidase 0.01-1 mcM; concentration of hydrogen peroxide - 100 mcM, amine concentration - 0.1-33 mM; concentration of catecholamines and metabolites - 0.03-1 mcM.
EFFECT: method is simple and producible as it does not require higher temperature and is realised in a water solution.
2 cl, 2 dwg, 3 ex
The invention relates to medicine and can be used for highly sensitive and selective detection of markers of various diseases.
The problem with this definition is an important task of modern chemical analysis. Currently the most common markers of various diseases are physiologically active substances, such as catecholamines (CA) (dopamine (DA), adrenaline (AD)), and their main metabolites (gomovanilinovoi and vanillylmandelic acid (GVK and CMC, respectively)). The possibility of determination of these compounds in biological fluids and human tissues at levels mu concentrations and lower (according to studies in related biochemical analysis fields) allows you to diagnose various diseases in its earliest stages.
A significant problem in the determination of trace quantities of CA and their metabolites in real biological objects occurs when they contain components absorbing in the visible region of the spectrum and thereby interfering matrix effects. To improve not only the sensitivity of the method to determine these compounds (at the level of mu concentrations), but also selectivity and promising technique, which consists in the translation of the definition of short wavelength in the visible region of the spectrum (λex 340-355 and λem470-485 nm), where there are no matrix effects of biological objects by derivatization of SPACECRAFT and their metabolites organic molecules. Furthermore, the resulting compounds have significantly more intense fluorescence than the original-defined compounds, which increases not only the selectivity but also the sensitivity analysis.
Known method for the derivatization of SPACECRAFT and their metabolites 0,1 M diphenylethylenediamine (DEAD) in the presence of potassium ferrocyanide, catalyzing the oxidation of the dissolved oxygen molecules KA up to KA-o-quinones, which in turn interact with derivatization reagent (DEAD) by Michael's reaction (N. Nohta, A. Mitsui, Y. Ohkura. High-performance liquid chromatographic determination of urinary catecholamines by direct pre-column fluorescence derivatization with 1,2-diphenylethylenediamine. J. Chromatogr. 380 (1986) 229-231.). The end product derivatization with KA are DEAD 2-phenyl(4,5-dihydropyrrolo)[2,3-f]benzoxazole derivatives, having a more intense fluorescence than the KA and their metabolites in the visible region of the spectrum. The process takes place in a mixture of buffer solution and methanol: 0.3 M CAPS - CON with a pH of 10 as methanol (30:70 vol.%). Oxidation with air oxygen in the presence as catalyst of a 20 mm solution of ferrocyanide K3[Fe(CN)6], at temperature T=50°C for 20 min.
However, the low adaptability of this process is due to�the selection of toxic solvent 70%. methanol, the need for temperature control of the reaction mixture at an elevated temperature of 50°C, the duration time of obtaining derivatives of catecholamines and their metabolites necessary intensity, which is 100 Rel. units.
Known adopted as a prototype (K. V. of Contributors. New aspects of native and immobilized horseradish peroxidase for the determination of inhibitors and substrates. Author's abstract on competition of degree of candidate of chemical Sciences, majoring in Analytical chemistry 02.00.02. Moscow. 2010. 22 p) derivatization method of SPACECRAFT and their metabolites 0.5 M benzylamine (BA) or diphenylethylenediamine (DEAD) in micellar and aqueous-organic medium in the presence of peroxidase from horseradish roots, catalyzing the oxidation by hydrogen peroxide molecules to KA up to KA-o-quinones, which in turn interact with derivatization reagent, which was used benzylamine (BA) and diphenylethylenediamine (DEAD) by Michael's reaction.
The process takes place in 5 mm solution of CTMA in 0.1 M CAPS - KOH pH 11 (derivateservlet agent - BA), or 0.5 M buffer solution glycine - KOH, DMSO (70:30 vol.%, derivateservlet agent - DED), the hydrogen peroxide concentration of 1 mm, t=30 min (less time 5 min derivatization was achieved only for adrenaline), T=37°C.
The end product derivatization KA BA or DEAD are 2-phenyl(4,5-DIH�droprole)[2,3-f]benzoxazole derivatives, having in the visible region of the spectrum more intense fluorescence than the KA and their metabolites.
However, this method has the same drawbacks as the above analog: low manufacturability of this process due to the presence of toxic solvent 30 vol.% DMSO and incubation of the reaction mixture at an elevated temperature of 37°C.
The present invention solves the problem of increasing the efficiency of formation of fluorescent derivatives of catecholamine while improving the manufacturability of the process.
The problem is solved by a method of producing a fluorescent derivatives of catecholamines and their metabolites derivatization method, comprising the oxidation of parent compounds and their processing forming a condensed structure of amines, in the presence of a catalyst - peroxidase from horseradish roots. The novelty in this case is that the oxidation of parent compounds catalyzed by horseradish peroxidase, carried out in the presence of 0.1 M CAPS buffer solution (pH 11) and 100 μm solution of hydrogen peroxide. The increase in 5 times the concentration of the used buffer solution and a 10 times reduction in the concentration of hydrogen peroxide in comparison with the prototype leads to the stabilization of the biocatalyst - horseradish peroxidase and reduced denaturing step n� oxidant enzyme - peroxide moderadores carried out in aqueous solution at room temperature, i.e. increases the efficiency of peroxidase in the claimed process derivatization of catecholamines and their metabolites by means of amines, which allows to obtain fluorescent derivatives of the same intensity (100 Rel. units), but room temperature (without temperature control) and in the aquatic environment (in the absence of organic solvents and surfactants, which reduce the durability of devices and consumables to the emergence of environmental problems).
The most intense signal is obtained when carrying out the process in 0.1 M CAPS buffer solution at a concentration of horseradish peroxidase is 0.01 - 1 μm; the concentration of hydrogen peroxide is 100 μm, the concentration derivatizing agent amine is 0.1 to 33 mm; the concentration of catecholamines and metabolites - 0.03 to 1 micron.
The highest intensity of the fluorescence signal obtained using as amines benzylamine, diphenylethylenediamine or o-phenylenediamine. Last for derivatization stated catecholamines and metabolites were not applied.
The technical result of the invention thus consists not only in reducing the reaction time from 30 to 5 min, but also the improvement of the technological process due to exception of prolonged incubation of the reaction mixture at a value� temperature in the presence of toxic solvents (methanol, DMSO).
Selection oxidative systems by varying the nature of the biocatalyst and oxidizer (horseradish peroxidase - hydrogen peroxide, mushroom tyrosinase is the oxygen of the air, laccase - oxygen) showed that only the selected authors of the present invention in the inventive system conditions combines high technology process with high yield obtained by the derivatization products.
Analysis of the known technical solutions allows to make a conclusion that the invention is not known from the prior art that demonstrates its compliance with the criterion of "novelty."
The essence of the present invention for professionals not obvious from the prior art, which allows to make a conclusion about its compliance with the criterion of "inventive step".
The possibility of carrying out of the method on the traditional equipment with the achievement of the objectives testifies to the invention, the criterion of "industrial applicability".
Fig. 1 shows absorption spectra of HELL (1), HELL-o-quinone product of the peroxidase oxidation of HELL (2) and 2-phenyl-4,5-dihydropyrrole-2-hydroxy-3-methyl-benzoxadiazole - product derivatization HELL with BA (3), (garden - 30 μm, the reaction time is 5 min).
Fig. 2 shows the mass spectrum of the products of derivatization of adrenaline with a BA in optimum conditions of the reaction.
These examples suggest, but do not limit the claimed invention.
Example 1. The method of obtaining 2-phenyl-4,5-dihydropyrrole-2-hydroxy-3-methyl-benzoxazole, a fluorescent derivative of catecholamines by enzymatic derivatization HELL with BA
As a derivative of catecholamines was taken adrenaline (AD), as derivatizing agent benzylamine (BA).
The reaction derivatization HELL with asthma was performed by the following method: in the glass vial was sequentially introduced to 2.7 ml of 0.1 M CAPS buffer solution-KOH (pH of 11.0), 0.100 ml of a 0.1 M solution of BA (33 mm), 0.100 ml of a 30 μm solution of peroxidase (1 μm), a solution of AD (30-300 nm) and 0.100 ml of 3 mm solution of H2O2(0.1 mm). The reaction mixture was mixed thoroughly, transferred to a quartz cuvette and measured the intensity of fluorescence fluorimetry method. After 5 minutes of the start of the reaction (λex=350 nm, λem=495 nm). These results indicate that the increase in the fluorescence intensity at about 1.5 times through one and the same time after start of the reaction compared to the ways derivatization of catecholamines and their metabolites in the presence of an inorganic catalyst.
The identity of composition of the products of derivatization of catecholamines on the example of adrenaline (AD) in the claimed conditions and according to the method prototype (used with�Itanium as a catalyst of potassium ferrocyanide) was monitored by the methods of spectrophotometry(Fig. 1+ and liquid chromatography / mass spectrometry.)
From the absorption spectra it is seen that in the case of peroxidase oxidation of adrenaline that belongs to a class of catecholamines, there is a maximum absorption at 300 nm corresponding to the products of its oxidation, the molecules of which contain o-heneidy fragment. In the spectrum of absorption of the products of derivatization HELL in the presence of potassium ferrocyanide has a maximum at 350 nm corresponding to the wavelength of fluorescence excitation of derivatized HELL. The obtained spectral data provide indirect evidence about the identity of the products of derivatization HELL, obtained in the presence of HRP and potassium ferrocyanide.
For a more rigorous proof of identity of the reaction products derivatization KA, obtained in the presence of HRP and potassium ferrocyanide, studied the mass spectra of the products of derivatization of adrenaline with BA (Fig. 2). The data obtained are strictly in line with literature data (Justin P. Pennington, Christian Schoneich, John F. Stobaugh. Selective Fluorogenic Derivatization with Isotopic Coding of Catechols and 2-Amino Phenols with Benzylamine: A Chemical Basis for the Relative Determination of 3-Hydroxy-tyrosine and 3-Nitro-tyrosine Peptides.Chromatographia November 2007, Volume 66, Issue 9-10, pp. 649-659, K. B. Of Contributors. New aspects of native and immobilized horseradish peroxidase for the determination of inhibitors and substrates. Author's abstract on competition of degree of candidate of chemical Sciences, majoring in Analytical�th chemistry 02.00.02. Moscow. 2010. 22 p).
On the mass spectrum shows peaks of molecular ions, confirming the identity of the final products of enzymatic and non-enzymatic (method prototype) derivatization (last described in the literature).
Example 2. The method of obtaining 2-phenyl-4,5-dihydropyrrolo-benzoxazole, a fluorescent derivative of catecholamines on the example of enzymatic derivatization YES with DEAD.
As a derivative of catecholamines was taken dopamine (DA), as derivatizing agent - diphenylethylenediamine (DEAD).
The derivatization reaction with DEAD was performed by the following method: in the glass vial was sequentially injected with 2.7 ml of 0.5 M buffer solution, glycine-KOH (pH 8.0), 0,010 ml of a 0.1 M solution of DEAD (33 mm), 0.100 ml of a 30 μm solution of peroxidase (1 μm), the solution is YES (30-300 nm) and 0.100 ml of 3 mm solution of H2O2(100 μm). The reaction mixture was mixed thoroughly, transferred to a quartz cuvette and measured the fluorescence intensity after 5 min from the beginning of reaction (λex=350 nm, λem=495 nm).
Example 3. The method of obtaining 2-phenyl-benzoxazol-6-acetic acid, a derivative of catecholamine metabolites, for example, enzymatic derivatization with GVK BA.
As a derivative of catecholamine metabolites was taken gomovanilinovoi acid (GVK), as derive�tiirough agent benzylamine (BA).
The derivatization reaction with GVK BA conducted by the following method: in the glass vial was sequentially introduced to 2.7 ml of 0.1 M CAPS buffer solution-KOH (pH of 11.0), 0.100 ml of a 0.1 M solution of BA (33 mm), 0.100 ml of a 30 μm solution of peroxidase (1 μm), the solution GVK (0.1 to 10 μm in the system) and 0.100 ml of 3 mm solution of H2O2(100 μm). The reaction mixture was mixed thoroughly, transferred to a quartz cuvette and measured the fluorescence intensity after 5 min from the beginning of reaction (λex=350 nm, λem=430 nm).
As can be seen from the above examples, the present invention solves the problem of obtaining fluorescent derivatives of catecholamines and their metabolites, as well as more intensely fluorescent derivative in less time while improving the manufacturability of the process by reducing the reaction time (from 30 to 5 min), non-heating and temperature control, the use of toxic or corrosive organic solvents.
1. A method of producing a fluorescent derivative of a catecholamine selected from dopamine and epinephrine, and their metabolites selected from homovanillic and vanillylmandelic acids, derivatization method, comprising the oxidation of parent compounds and their interaction with forming a condensed structure of amines in the presence of a catalyst, ex�featuring the what derivatization compounds is carried out in the presence of CAPS buffer solution or glycine-KOH 0.1 mm hydrogen peroxide using the catalyst of horseradish peroxidase.
2. A method according to claim 1, characterized in that the process is carried out in a 0.1 M buffer solution at a concentration of horseradish peroxidase is 0.01 - 1 μm; the concentration of hydrogen peroxide is 100 μm, the concentration of amine is 0.1 to 33 mm; the concentration of catecholamines and metabolites of 0.03-1 μm.
SUBSTANCE: membrane-bound red cell haemoglobin by giant combined diffusion (GCD) spectroscopy is analysed by the use of nanostructured coatings in the form of ring silver nanostructures having the hierarchical structure. Silver ring rims consists of connected micron-scale porous silver aggregates, the surface of which comprises rounded silver nanoparticles 2-100nm embedded into a matrix. The red cell immobilisation time on the nanostructured coatings makes 5-40 minutes, and GCD spectra are generated by green laser at wave length 514 or 532 nm.
EFFECT: invention enables diagnosing membrane-bound haemoglobin in undamaged red cells by the giant combined diffusion.
4 cl, 4 ex, 1 dwg
FIELD: measurement equipment.
SUBSTANCE: group of inventions relates to measurement and control of presence of hydrophobic pollutants. The invention presents a version of a monitoring method of presence of one or more types of hydrophobic pollutants during paper manufacture, which involves the following: a. obtaining a fluid medium sample from the specified paper manufacturing process; b. measurement of turbidity of the specified fluid medium sample; c. selection of a hydrophobic colouring agent capable of interaction with the specified pollutants in the specified fluid medium and fluorescence in the specified fluid medium; d. addition of the specified colouring agent to the specified fluid medium and exposure during the time required for interaction of the specified colouring agent with the specified pollutants in the specified fluid medium; e. measurement of colouring agent fluorescence in the specified fluid medium; and f. setting of correlation between fluorescence of the colouring agent and concentration of the specified pollutants; with that, if turbidity measured at stage (b) is more than 2000 nephelometric turbidity units (NTU), then the specified sample is diluted or separated additionally prior to addition of the specified colouring agent at stage (d) and measurement of fluorescence.
EFFECT: invention presents a measurement method of efficiency of one or more chemical reagents reducing the number of one or more hydrophobic pollutants during paper manufacture Quick, accurate and reliable monitoring is achieved.
20 cl, 3 dwg, 3 ex
FIELD: physics, control.
SUBSTANCE: invention is intended for monitoring a plurality of discrete fluorescence signals, particularly for DNA sequencing using fluorescently labelled nucleotides. The detector (118) comprises a plurality of pixels (130) for individually detecting said fluorescence signals from the plurality of fluorescent signal sources (104), wherein each pixel (130) comprises a predetermined number of at least two detection elements (D1, Dn) for detecting a received fluorescent signal and for generating detection signals, as well as a signal conversion circuit (140) for receiving said detection signals from said at least two detection elements (D1, Dn) and for generating a pixel output signal indicating which of said at least two detection elements (D1, Dn) generated the strongest detection signal.
EFFECT: monitoring a plurality of discrete fluorescence signals.
15 cl, 7 dwg
SUBSTANCE: invention relates to chemistry of organometallic compounds, particularly to alkynyl phosphine gold-copper complexes which dissociate in a solution to form ions. The gold-copper alkynyl phosphine complexes are capable of forming covalent conjugates with proteins, thereby turning into a water-soluble form, exhibit luminescent properties and can be used as labels for fluorescence microscopy and in luminescent analysis.
EFFECT: improved properties of the compounds.
5 dwg, 4 tbl, 3 ex
SUBSTANCE: apparatus comprises a fluorescent-reflective spectrometer, which includes an illumination system and spectrometric system connected to a Y-shaped fibre-optic probe. The apparatus is further provided with two channels, one of which intended for supplying liquid to the investigated organ to wash off blood and is connected to a pump, and the other channel is intended for sucking the liquid and blood from the investigated organ and is connected to a pump. Both channels and the distal end of the fibre-optic probe are placed in a ferrule to form a fibre-optic probe. The ferrule is in the form of a metal cylinder with a socket at the end which is adjacent to the investigated organ.
EFFECT: high accuracy and consistency of measurement results, enabling investigation of the heart within the body.
7 cl, 5 dwg
SUBSTANCE: invention relates to biotechnology and represents method of spectral analysis of fluorescent properties of DNA nucleotide successions. Claimed invention can be applied for genetic diagnostics, research of mitogenetic irradiation of cells, study of coding hereditary and proliferative information. Method includes preparation of water solutions of dyes of basic fluorescence colours. Background fluorescence of dye is measured in respective detection channels by means of fluorescent detector FDG-001. DNA sample is added to dye solution in quantity 150-200 ng. Fluorescence of dye solutions is measured. Results of analysis are registered in form of presentation of values in conventional units of positive or negative increase of fluorescence of dyes relative to their initial background before addition of DNA sample in form of building columnar diagrams or curves of signal growth dynamics in time. Results of dye fluorescence growth are interpreted.
EFFECT: claimed invention makes it possible to determine nucleotide rebuildings of telomeric DNA ends, point mutations, polymorphisms of gens, chromosome rebuildings, and change of karyotype or cell genome.
15 cl, 16 dwg, 1 tbl, 6 ex
SUBSTANCE: invention relates to the field of molecular biology and biochemistry. A device consists of a light source, the radiation of which is directed on a transparent substrate with oligonucleotides immobilised on its surface and a system of detecting the intensity of light, which passed through the substrate, located under it. The substrate contains at least two zones, with a layer of oligonucleotides, non-specific to the nucleotide sequence under study, being immobilised on the surface of one of them, and a layer of nucleotides, specific to the nucleotide sequence under study, being immobilised on the surface of the other zone. The detection system contains at least two photosensitive independent sections, each of which is illuminated by the radiation, which passed through only one zone.
EFFECT: device makes it possible to carry out the qualitative and quantitative analysis of the nucleotide sequences, increases the accuracy of the identification of the nucleotide sequences.
3 cl, 5 ex
FIELD: measurement equipment.
SUBSTANCE: invention relates to a measuring method of variations of fluorescence intensity of voltage-sensitive fluorochrome depending on a potential gradient or ionic strength, which involves addition to the voltage-sensitive fluorochrome of an ionising compound to induce variation of potential or ionic strength, as well as addition of vitamin E and/or cholesterol to increase variation of potential or ionic strength as to voltage-sensitive fluorochrome. Besides, the invention relates to a potential measuring method of actions of cultivated cardiomyocytes.
EFFECT: invention provides measurement of fluorescence intensity of voltage-sensitive fluorochromes or voltage-dependent quantitative variations of their fluorescence intensity without using any such materials (membrane carriers), as cells or two-layer lipidic liposomes.
10 cl, 3 ex, 5 dwg
SUBSTANCE: invention can be used when determining content of benzene, toluene and xylene (BTX) vapour in urban air, air in residential facilities, chemical laboratories, filling stations and oil processing enterprises, in gas emissions of industrial plants. The method of determining concentration of benzene, toluene and xylene vapour in a gaseous mixture comprises placing material containing boron difluoride dibenzoylmethane (BF2DBM) fluorophore or a methyl- or methoxy derivative thereof into the gas mixture, illuminating the material with light in the wavelength range of 355-400 nm and measuring fluorescence intensity of the material in the wavelength range of 400-550 nm. Unlike the existing method, measurement is carried out on not less than two spectral channels, wherein the number of channels is selected not less than the number of determined components in the mixture plus one. The measured values are then used to calculate relative intensity of spectra of the fluorophore and exciplexes thereof with benzene, toluene and xylene. Concentration of benzene, toluene and xylene is then determined from the ratio of intensities of the corresponding exciplex to the intensity of BF2DBM.
EFFECT: simultaneous continuous selective measurement of benzene, toluene and xylene in gaseous mixtures in a wide range of concentrations with short reaction time.
2 cl, 4 dwg, 1 tbl
SUBSTANCE: invention relates to the field of biochemistry. Claimed is a method of evaluating cell viability in a microbioreactor by means of an optical light guide. The method includes the placement of cells into a membrane compartment of a replaceable cell unit of the microbioreactor, application of a working solution of a vital dye, introduction of the dye into the microbioreactor compartment. After the introduction incubation of the cells in the vital dye solution and removal of the vital dye solution, which has not bound with the cells, are performed. Removal is performed by the replacement of the incubation solution with a growth medium, which does not contain dye. The optical light guide, connected to a spectrometer, is brought into contact with an optically transparent material with the replaceable cell unit under the membrane compartment of the microbioreactor. After that, the support spectrum of a fluorescent signal is measured as an integral of the intensity of fluorescence on the membrane compartment of the microbioreactor, in which the cells to be analysed are absent. Also measured is the spectrum of the fluorescent signal as the integral of the intensity of fluorescence on the membrane compartment of the microbioreactor with the analysed cells. After that, the support spectrum of the fluorescent signal for the membrane compartment of the microbioreactor without the cells to be analysed is subtracted from the obtained spectrum of the fluorescent signal for the membrane compartment with the analysed cells. The quantity of the viable cells in the membrane compartment of the microbioreactor is calculated on the basis of the obtained value of the fluorescence signal intensity.
EFFECT: invention makes it possible to quickly determine viability of the cells under an impact of influencing factors in a real time mode in the microbioreactor.
6 cl, 3 dwg, 5 tbl, 5 ex
SUBSTANCE: invention pertains to new compounds with general formula (I) , and their salts used in pharmacology, and their hydra as well as others, where W represents or and R3, R7, R16, R17, R20, R21 and R21 are identical or different and each of them represents a hydrogen atom or assumes other values, given in the formula of invention. The invention also relates to pharmaceutical compositions and medicinal preparations based on these compounds, cultures, used for obtaining them, methods of inhibition and treatment and use.
EFFECT: formula (I) can be used as medicinal preparations for curing solid malignant tumours.
43 cl, 4 tbl, 60 ex
SUBSTANCE: invented group pertains to biotechnologies; in particular, to the method of obtaining macrolide compound 11107D with formula (II) and to Streptomyces sp. FERM BP-8551, Mortierella sp. FERM BP-8547, Mortierella sp. FERM BP-8548 and Micromonosporaceae FERM BP-8550 strains. The initial compound 11107B with formula (I) undergoes biotransformation to the target compound with formula (II) through incubation in the presence of a strain, capable of converting macrolide compound 11107B into 11107D substance and a Mortierella, Streptomyces type or Micromonosporaceae family, or a mycelium cultivated strain sample. The target substance 11107D is separated from the reaction medium.
EFFECT: increased anti-tumour activity and stability of the compound.
8 cl, 9 tbl, 10 ex
SUBSTANCE: medium contains, in mass percent: 9.0 of glucose, 3.0 of corn-steep extract, 0.1 of the twice-substituted ammonium phosphate, 0.05-0.2 of cystine, 50.0-85.0 of the water extract from soybean prepared on basis of 7% suspension of the fat-free soybean flour as a nutritious substrate, up to 10.0 of water.
EFFECT: provides increased efficiency of coproporphyrin III biosynthesis.
FIELD: biotechnology, biochemistry, enzymes, microbiology.
SUBSTANCE: invention proposes a method for microbiological oxidation of N-, O- or S-heterocyclic mono- or multinuclear aromatic compounds. The method involves culturing the recombinant microorganism that expresses cytochrome P-450-dependent monooxygenase BM-3 from Bacillus megaterium with amino acid sequence represented in SEQ ID NO:2 that comprises at least one functional mutation in region 86-88 and, if necessary, at least one functional mutation in one of regions 73-82, 172-224, 39-43, 48-52, 67-170, 300-335 and 352-356. The prepared oxidation product is isolated from medium, Invention provides carrying out oxidation of organic compounds with enhanced degree of effectiveness.
EFFECT: enhanced effectiveness of enzymes.
17 cl, 1 tbl, 7 ex
SUBSTANCE: liposomes are used as a matrix for activated ferment - horseradish peroxidase. To 5 mg of horseradish peroxidase oxygenated with a periodate method there added is 1 ml of liposome suspension in 0.01 M solution of carbonate-bicarbonate buffer at pH 9.5, and subjected to ultrasonic treatment during 1 min. Then it is incubated for 1 hour; immobilised with immunoglobulins in concentration of 5 mg during 2 hours at the temperature of 22±4°C; stabilised with 5 mg of sodium borane with further gel-chromatographic cleaning.
EFFECT: invention allows obtaining liposomal-immunoperoxidase conjugate for indication of infectious agents in an immunoenzymometric analysis and increasing service life of a preparation up to 6 years.
1 tbl, 3 ex
SUBSTANCE: crushed biomass of horseradish root is soaked in 0.1 M sodium phosphate buffer solution pH 7.0, with preliminary blown nitrogen in presence of 5 mcM hemin solution and 5 mcM calcium chloride. The extract is separated by decantation followed by filtration and concentrating by ultrafiltration through ultrafilters with a pore size of less than 30 kilodalton. The enzyme extract is saturated with ammonium sulfate to 35% saturation and applied to a column with phenyl-sepharose after extensive washing of the buffer with sulfate, the active fractions are removed with a gradient of ammonium sulfate (35%-0%) and increasing the pH to 8.0. The enzyme is purified by gel filtration on Toyopearl HW55F, dialysed and dried lyophilisation.
EFFECT: use of hemin-containing buffers at a stage of extraction and gel filtration enables to obtain highly active enzyme with high acceleration of enzyme production process and improving its catalytic characteristics and stability.
7 cl, 1 tbl