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1.
Biofizika ; 56(3): 389-95, 2011.
Article in Russian | MEDLINE | ID: mdl-21786690

ABSTRACT

Two novel FRET-pairs: Tb3+ -binding peptide-DsRed2 and Tb3+ -binding peptide-TagRFP have been produced based on the terbium-binding peptide and the red fluorescent proteins DsRed2 and TagRFP. Two induction-resonance energy transfer processes in both FRET-pairs have been registered, from tryptophan of the terbium-binding peptide to Tb3+ and from sensitized Tb3+ to the acceptor, the chromophore, DsRed2 or TagRFP. The lifetimes of terbium in the presence and absence of the acceptor have been determined. It has been shown that the lifetime of Tb3+ in the presence of 150 mM NaCl decreases in both FRET-pairs. The efficiency of fluorescent resonance transfer from Tb3+ to the acceptor proteins is 28 and 35% for Tb3+ -binding peptide-DsRed2 and Tb3+ -binding peptide-TagRFP, respectively.


Subject(s)
Fluorescence Resonance Energy Transfer , Luminescent Proteins/chemistry , Peptides/chemistry , Terbium/chemistry
2.
Prikl Biokhim Mikrobiol ; 46(2): 166-71, 2010.
Article in Russian | MEDLINE | ID: mdl-20391759

ABSTRACT

The genetically encoded FRET-pair was developed on the basis of terbium-binding peptide and red fluorescent protein DsRed2. To study fluorescence resonance energy transfer within FRET-pair, the gene-engineered construction was obtained, where sequences of terbium-binding peptide and red fluorescent protein DsRed2 were fused in single reading frame. The expression of this construction in strain E. coli BL21 (DE3) was studied and conditions of synthesis, isolation and purification of recombinant protein were optimized. The hydrodynamic radius of hybrid protein was determined by the method of dynamic light scattering. Energy transfer between sensitized terbium and red fluorescent protein was confirmed by the methods of phosporescent spectroscopy. The obtained FRET-pair can be used both for studies in vitro and as a reporter in living cells.


Subject(s)
Fluorescence Resonance Energy Transfer/methods , Fluorescent Dyes/metabolism , Luminescent Proteins/metabolism , Terbium/metabolism , Energy Transfer , Escherichia coli/genetics , Escherichia coli/metabolism , Luminescent Proteins/chemistry , Luminescent Proteins/genetics , Models, Molecular , Plasmids , Protein Binding , Protein Conformation , Protein Engineering , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Terbium/chemistry , Red Fluorescent Protein
3.
Biomed Khim ; 49(1): 70-9, 2003.
Article in Russian | MEDLINE | ID: mdl-14569875

ABSTRACT

The determination of thyroxin (T4) is a basic confirmative test for congenital hypothyroidism. 70% cases of this period inborn disease are taking asymptomatic course. We developed immunofluorescent T4-assay in dried blood spots with anti-T4 monoclonal antibody and europium chelate (dianhydride of diethylentriaminipentaacetic acid (DA-DTPA)). This method requires DELFIA Plate Fluorometer 1232 (Wallac, Finland) or its sub modifications. Panel of monoclonal antibodies for T4 has been obtained. Type/subtype (IgG2b, IgG1), affinity constants (10(7)-10(8) M-1), cross reactivity to homologous structures (0-2%) were determined. Stable clones with high affinity and viability were selected for the development of the assay. Conjugates of thyroxin and europium chelate were synthesized. Inclusion of sodium salicylate (2 mg/ml) and EDTA (2 mM) into the buffer reduced the nonspecific signal. Limit for T4 detection (T4 standarts) was 10 nM with not more than 15% variation coefficient. Accuracy was estimated by Bio-Rad Lipochek Immunoassay Plus Control Kit. Obtained results were within control confidence interval.


Subject(s)
Blood Specimen Collection , Europium , Hypothyroidism/diagnosis , Neonatal Screening/methods , Thyroxine/blood , Antibodies, Monoclonal , Buffers , Congenital Hypothyroidism , Fluorescent Antibody Technique , Humans , Immunoenzyme Techniques , Infant, Newborn , Sensitivity and Specificity , Thyroxine/immunology , Time Factors
4.
Bioorg Khim ; 28(4): 303-7, 2002.
Article in Russian | MEDLINE | ID: mdl-12197386

ABSTRACT

Site-directed mutagenesis was used to study the structural basis of color diversity of fluorescent proteins by the example of two closely related proteins from one organism (coral polyp Zoanthus sp.), one of which produces green and the other, yellow fluorescence. As a result, the following conversions of emission colors were performed: from yellow to green, from yellow to a dual color (yellow and green), and from green to yellow. The saltatory character of the spectral transitions and the manifestation of the dual-color fluorescence suggest that chemically different fluorophores are responsible for the green and yellow fluorescence. The simultaneous presence of three residues, Gly63, Lys65, and Asp68, is necessary for the efficient formation of the yellow rather than green fluorophore. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2002, vol. 28, no. 4; see also http://www.maik.ru.


Subject(s)
Cnidaria/chemistry , Luminescent Proteins/chemistry , Amino Acid Sequence , Animals , Color , Molecular Sequence Data , Mutagenesis, Site-Directed , Spectrometry, Fluorescence
5.
Biosci Rep ; 20(1): 21-30, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10888408

ABSTRACT

Fluorescence of luciferases from Luciola mingrelica (single tryptophan residue, Trp-419) and Photinus pyralis (two tryptophan residues, Trp-417, Trp-426) was studied. Analysis of quenching of tryptophan fluorescence showed that the tryptophan residue conserved in all luciferases is not accessible for charged quenchers, which is explained by the presence of positively and negatively charged amino acid residues in the close vicinity to it. An effective energy transfer from tryptophan to luciferin was observed during quenching of tryptophan fluorescence of both luciferases with luciferin. From the data on the energy transfer, the distance between the luciferin molecule and Trp-417 (419) in the luciferin luciferase complex was calculated: 11-15 A for P. pyralis and 12-17 A for L. mingrelica luciferases. The role of the conserved Trp residue in the catalysis is discussed.


Subject(s)
Firefly Luciferin/chemistry , Luciferases/chemistry , Amino Acids/metabolism , Animals , Binding Sites , Coleoptera/enzymology , Firefly Luciferin/metabolism , Kinetics , Luciferases/metabolism , Models, Chemical , Models, Molecular , Protein Binding , Recombinant Proteins/chemistry , Spectrometry, Fluorescence , Tryptophan/chemistry , Tryptophan/metabolism
6.
Biochemistry (Mosc) ; 64(10): 1097-103, 1999 Oct.
Article in English | MEDLINE | ID: mdl-10561553

ABSTRACT

Quenching of tryptophan fluorescence of Luciola mingrelica (single tryptophan residue, Trp-419) and Photinus pyralis (two tryptophan residues, Trp-417 and Trp-426) luciferases with different quenchers (I-, Cs+, acrylamide) was studied. The conserved Trp-417(419) residue was shown to be not accessible to charged particles, and positively and negatively charged amino acid residues are located in close vicinity to it. We found previously unreported effective energy transfer from this tryptophan to luciferin during the quenching of the tryptophan fluorescence. The distance between the luciferin molecule and Trp-417(419) was calculated: 11-15 and 12-17 A for P. pyralis and L. mingrelica luciferases, respectively. The role of the conserved Trp residue in the catalysis is discussed. ATP and AMP are also quenchers of the tryptophan fluorescence of the luciferases. In this case, an allosteric mechanism of the interaction of Trp-417(419) with an excess of ATP (AMP) is proposed.


Subject(s)
Coleoptera/enzymology , Luciferases/metabolism , Tryptophan/metabolism , Adenosine Monophosphate/metabolism , Adenosine Triphosphate/metabolism , Allosteric Regulation , Animals , Catalysis , Fluorescence , Luciferases/chemistry , Models, Molecular , Substrate Specificity
7.
Vestn Ross Akad Med Nauk ; (8): 15-20, 1999.
Article in Russian | MEDLINE | ID: mdl-10487118

ABSTRACT

The next revolution in in-vitro bioaffinity assays will be associated with the minimization of the liquid phase of an experiment and with the extremely rapid detection of a large number of samples. The ultra detection unit is one of the essential elements of such devices. The fluorescence detection systems are most promising. The fluorescence detection limit of europium ions is as low as 10(-15) M in the complex with beta-diketone. The authors' approach to designing new highly effective chelates is to synthesize a stable chelate wherein several molecules of beta-diketones are properly oriented and chemically fixed. The molecular mechanic calculation of three dimensional structure of different Eu chelates should be used in optimizing the strategy of chemical synthesis of new compounds. New chelates can be efficiently used for the development of a new approach to measuring distances equal to over 10 nm in the biological systems. Pesticides also represent challenging targets because the levels of compounds are to be detects in minor concentrations, thus the time-resolution fluorescence assay may have some advantages due to its high sensitivity. DNA-based microbiochips are the most promising area in the practical use of a new chelate. A novel europium chelate is a key compound for the development of new methods for studying biological and chemical objects, as well as new diagnostic systems based on a basically new instrumental base that is characterized by extremely high miniaturization.


Subject(s)
Environmental Monitoring/methods , Fluoroimmunoassay/methods , Chelating Agents , Containment of Biohazards , DNA Probes , Europium , Hazardous Substances/analysis , Humans , Indicators and Reagents , Luminescent Measurements
8.
Bioorg Khim ; 24(1): 64-71, 1998 Jan.
Article in Russian | MEDLINE | ID: mdl-9551204

ABSTRACT

Conjugates of cobalt and aluminum phthalocyanines with monoclonal antibodies were synthesized using an AOT/n-octane reversed micellar system or water-organic mixtures with a low content of organic solvent as media. The effect of the degree of hydration of the micelles and the concentration of phthalocyanines on the composition of conjugates was studied. The immune activity of the resulting conjugates in comparison to that of native antibodies was evaluated. The catalytic activity of free cobalt phthalocyanines and their antibody conjugates was studied in the reaction of ascorbic acid oxidation.


Subject(s)
Antibodies, Monoclonal/chemistry , Immunoconjugates/chemistry , Indoles/chemistry , Organometallic Compounds/chemistry , Aluminum/chemistry , Ascorbic Acid/chemistry , Cobalt/chemistry , Dioctyl Sulfosuccinic Acid/chemistry , Immunoconjugates/isolation & purification , Micelles , Octanes/chemistry , Ovalbumin , Oxidation-Reduction , Solvents/chemistry , Spectrometry, Fluorescence , Water/chemistry
9.
Prikl Biokhim Mikrobiol ; 27(5): 772-7, 1991.
Article in Russian | MEDLINE | ID: mdl-1775450

ABSTRACT

Digitoxin at concentrations up to 5 x 10(-10) M (therapeutic concentrations are 2 x 10(-8) M) can be reliable measured by a fluoroimmunometric method with coproporphyrin as a tracer. The use of nylon filters with immobilized digitoxin to remove an excess of labelled antibodies increases reliability and reduces the time of measurements, and thus simplifies the assay.


Subject(s)
Digitoxin/blood , Chromatography, Ion Exchange , Coproporphyrins , Fluorescent Antibody Technique , Humans , Immune Sera
10.
Biomed Sci ; 2(1): 63-7, 1991.
Article in English | MEDLINE | ID: mdl-1912248

ABSTRACT

A flow-injection method for the determination of glucose in serum is presented. It is based on the enzymatic measurement of oxygen consumption detected via oxygen quenching of the luminescence of certain metalloporphyrins. Phosphorescent water-soluble Pt2+ and Pd(2+)-porphyrins have been characterized by luminescence spectroscopy and decay-time measurements in various buffers, and found to be suitable for oxygen detection in biological systems. A new method for the flow-injection analysis of glucose has been developed based on the use of a column of immobilized glucose oxidase and the indicators Pt(2+)-coproporphyrin III and Pd(2+)-coproporphyrin I. The system has been optimized for glucose determination in aqueous samples and in whole serum with the 0.5-200 mM glucose range. Twenty assays can be performed in an hour, and the system has potential for commercial development with biotechnological and medical applications.


Subject(s)
Blood Glucose/analysis , Humans , In Vitro Techniques , Luminescent Measurements , Metalloporphyrins , Oxygen Consumption , Reference Standards
11.
Biomed Sci ; 2(5): 536-9, 1991.
Article in English | MEDLINE | ID: mdl-1840843

ABSTRACT

A fibre-optic oxygen sensor is described which is based on an oxygen-sensitive luminescent film made from platinum octaethylporphyrin and polystyrene. The luminescence and quenching characteristics of such films were studied for their use in a fibre-optic oxygen biosensor. A prototype oxygen sensor was made from this material, and was tested in aqueous solutions and in the gaseous phase at physiological oxygen concentrations. Measurements of luminescence intensity and decay time were employed to determine oxygen concentration from luminescence quenching. The main working characteristics of this prototype oxygen sensor were studied.


Subject(s)
Biosensing Techniques , Fiber Optic Technology/instrumentation , Oxygen/analysis , Luminescent Measurements , Polystyrenes
12.
Biull Eksp Biol Med ; 109(4): 349-51, 1990 Apr.
Article in Russian | MEDLINE | ID: mdl-2386825

ABSTRACT

The effective photogeneration of singlet molecular oxygen (1O2) by porphyrins (coproporphyrin I; 2,4-bi (alpha-methoxyethyl) deuteroporphyrin IX and cyclopanten-coproporphyrin I) conjugated with antibodies (mouse monoclonal IgG and IgM and human gamma-globulin) have been observed with the direct luminescence method of 1O2 detection. Absolute quantum yields of 1O2 formation by the conjugates have been determined. The data suggest that porphyrin-antibody conjugates are promising for the use as drugs in photodynamic tumor treatment.


Subject(s)
Immunotoxins/pharmacology , Oxygen , Photochemotherapy , Porphyrins/pharmacology , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/therapeutic use , Humans , Immunotoxins/therapeutic use , Laser Therapy , Mice , Photochemistry , Porphyrins/therapeutic use , Singlet Oxygen , Solutions , Water
13.
Biomed Sci ; 1(4): 401-6, 1990 Apr.
Article in English | MEDLINE | ID: mdl-2133059

ABSTRACT

The photodynamic action of a number of carboxylic porphyrins and their covalent conjugates with specific monoclonal IgM antibodies on a human lung adenocarcinoma cell line was studied. All the conjugates showed strong phototoxicity which did not correspond with the phototoxicity of the free porphyrins. Absolute quantum yields of singlet oxygen photogeneration by the porphyrins and their conjugates were obtained and compared with the phototoxicity of the compounds.


Subject(s)
Antibodies, Neoplasm/administration & dosage , Oxygen/metabolism , Photochemotherapy/methods , Porphyrins/pharmacology , Radiation-Sensitizing Agents/pharmacology , Tumor Cells, Cultured/drug effects , Adenocarcinoma/immunology , Adenocarcinoma/pathology , Antigens, Neoplasm/immunology , Humans , Immunoglobulin M/administration & dosage , Lung Neoplasms/immunology , Lung Neoplasms/pathology , Oxidation-Reduction , Porphyrins/administration & dosage , Porphyrins/radiation effects , Radiation-Sensitizing Agents/administration & dosage , Singlet Oxygen , Structure-Activity Relationship
14.
Vopr Virusol ; 35(2): 115-7, 1990.
Article in Russian | MEDLINE | ID: mdl-2202151

ABSTRACT

Monoclonal antibodies, epidemic strains of influenza A and B viruses, conjugates were studied by fluorescence immunoassay with temporary resolution using equipment of different companies. Differences and specificity of monoclonal antibodies to influenza A and B viruses were demonstrated. The highest sensitivity of the method with the test system used was determined, being 20 ng/ml of viral protein. The method was shown to be useful for influenza virus detection both in solutions containing purified virions and in virus-containing allantoic preparations. It was established that virological studies could be carried out using conjugates and knock-down microplates of the national make alongside with foreign ones.


Subject(s)
Influenza A virus/immunology , Influenza B virus/immunology , Antibodies, Monoclonal , Antibodies, Viral/analysis , Antibody Specificity/immunology , Evaluation Studies as Topic , Fluorescent Antibody Technique/instrumentation , Viral Core Proteins/immunology
15.
FEBS Lett ; 259(2): 335-7, 1990 Jan 01.
Article in English | MEDLINE | ID: mdl-2294023

ABSTRACT

The 34-mer oligodeoxynucleotide was shown to be selectively modified at the G17 position upon photoirradiation in the presence of complementary 17-mer oligodeoxynucleotide bearing Pd(II)-coproporphyrin I covalently linked to the 5'-end phosphate group.


Subject(s)
Coproporphyrins , Oligodeoxyribonucleotides , Palladium , Porphyrins , Autoradiography , Base Sequence , Molecular Sequence Data , Oligodeoxyribonucleotides/chemical synthesis , Phosphorus Radioisotopes , Photochemistry
17.
Prikl Biokhim Mikrobiol ; 25(5): 618-23, 1989.
Article in Russian | MEDLINE | ID: mdl-2602344

ABSTRACT

The chromatographic behaviour of tetra- and octacarboxylic porphyrins and tetra-(p-sulphophenyl)-porphin was being studied by HPLC using Protein Pak and TSK-type gel permeation columns. The mobility of the porphyrins on the carriers depended on the ionic strength of the eluating buffer Tris-HCl. The conditions for separation of the porphyrins and their protein conjugates were optimized. The separation was performed under mild conditions with minimum denaturation of conjugates. The purification technique can be used to separate porphyrin conjugates with various proteins. The technique can be also used in conventional column chromatography on Toyopearl supports.


Subject(s)
Fluorescent Dyes/analysis , Porphyrins/analysis , Proteins/analysis , Chromatography, High Pressure Liquid , Immunoglobulin G/analysis , Solubility
18.
Prikl Biokhim Mikrobiol ; 25(4): 548-57, 1989.
Article in Russian | MEDLINE | ID: mdl-2682597

ABSTRACT

The synthesis of protein conjugates with the new high-efficient fluorescent labile coproporphyrin-I was optimized. A number of conjugates of monoclonal antibodies with different coproporphyrin-I content were synthesized, and their spectral properties were studied in water and micellar solutions, i.e. adsorption, excitation and emission spectra, fluorescence quantum yields, fluorescence pH-dependences. The binding constants of coproporphyrin-I and its protein conjugates with serum albumin were determined. The antibodies labelled with coproporphyrin-I retain the functional activity and photochemically stable in water solutions. The sensitivity of fluorometric detection of coproporphyrin-I and its conjugates with proteins is more than 10 times greater than in case of FITC.


Subject(s)
Coproporphyrins/immunology , Immunoglobulin G/immunology , Porphyrins/immunology , Animals , Antibodies, Monoclonal , Cattle , Chromatography, Liquid , Coproporphyrins/isolation & purification , Coproporphyrins/metabolism , Fluorescent Antibody Technique , Humans , Hydrogen-Ion Concentration , Immunoglobulin G/isolation & purification , Photochemistry , Serum Albumin/metabolism , Serum Albumin, Bovine/metabolism , Spectrometry, Fluorescence
20.
Vopr Med Khim ; 34(5): 116-21, 1988.
Article in Russian | MEDLINE | ID: mdl-3064408

ABSTRACT

Recently developed solid-phase immunofluorescent assay with temporal distinction involved a newly produced procedure for binding of of the boron group label with antibodies via modified polymer, which is able to bind with antibodies across the avidin bridge. The assay developed is more simple than the widely used procedures, it exhibits high sensitivity being superior as compared with a corresponding immunoenzyme assay by a decimal order.


Subject(s)
Europium , Fluorescent Antibody Technique , Polymers , Animals , Immunoglobulin G/analysis , Mice , Rabbits
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