Bioluminescence assay for cell viability.

Theoretical aspects of the adenosine triphosphate bioluminescence assay based on the use of the firefly luciferin-luciferase system are considered, as well as its application for assessing cell viability in microbiology, sanitation, medicine, and ecology. Various approaches for the analysis of individual or mixed cultures of microorganisms are presented, and capabilities of the method for investigation of biological processes in live cells including necrosis, apoptosis, as well as for investigation of the dynamics of metabolism are described.

Site-directed mutagenesis of cysteine residues of Luciola mingrelica firefly luciferase.

Single mutants (C62S, C62V, C86S, C146S, C164S), double mutants (C62/146S, C62/164S, C86/146S, C146/164S), and triple mutant C62/146/164S of the Luciola mingrelica firefly luciferase carrying C-terminal His(6)-tag were obtained on the basis of plasmid pETL7 by site-directed mutagenesis. Bioluminescence and fluorescence spectra were not altered by the introduced mutations. In the case of mutants C86S, C86/146S, C62/164S, and the triple mutant C62/146/164S, the K(m)(ATP) and K(m)(LH)(2) values were increased by a factor of ~1.5-1.9. Their expression level, specific activity, and thermal stability were significantly decreased. The other mutations had almost no effect on the K(m)(ATP) and K(m)(LH)(2) values, specific activity, and thermal stability of the enzyme. Thermal stability of the C146S mutant was increased by a factor of ~2 and 1.3 at 37 and 42°C, respectively. The possible mechanism of the influence of these mutations on properties and structure of the enzyme is discussed.

[Rapid bioluminescent antibiotic susceptibility assay].

Rapid testing of pathogen susceptibility to antibiotics is of great practical value for rational chemotherapy of pyoinflammatory deseases and postoperative complications of microbial etiology. The standard microbiological methods, i.e., the disk diffusion method and the method of serial dilutions are labour- and time-consuming (not less than 18-36 hours). The method of the authors is based on measuring bioluminescence resulting from interaction of adenosine-5'-triphosphate (ATP) and ATP reagent, a standard reaction mixture of firefly luciferase (an enzyme) and luciferin. The bioluminescence intensity is proportional to the ATP concentration in the reaction mixture and the ATP concentration is proportional to the number of the pathogen viable cells in the sample. The bioluminescence intensity value in the pathogen suspension aliquots with and without (control) the antibiotic were compared after the incubation for 5 hours and the coefficient of the microbial cell growth inhibition was calculated. Satisfactory correlation (R2 > 88%) of the results of the bioluminescent assay and the assay with the disk diffusion method and the method of serial dilutions was observed.

Chemical modification of the epsilon-amino groups of lysine residues in horseradish peroxidase and its effect on the catalytic properties and thermostability of the enzyme.

Chemical modification of horseradish peroxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7) (isoenzyme C) by anhydrides of mono- and dicarboxylic acids and picryl sulfonic acid has been performed. The effect of the modification on the catalytic activity, absorption and circular dichroism spectra of peroxidase has been studied. Rate constants of irreversible thermoinactivation (kin) for the native and modified peroxidase at 56--80 degrees C have been measured. The effective values of the thermodynamic activation parameters of thermoinactivation, delta H not equal to and delta S not equal to, have been also determined. A relationship between the number of modified epsilon-amino groups of lysine residues and the nature of the modifier on the one hand, and the conformation and thermostability of the enzyme on the other, is discussed. It has been shown that it is the degree of modification, rather than the nature of the modifier, that produces the major effect on the macromolecular conformation and the thermostability of the enzyme after modification. The conclusion is drawn that the thermostability of the modified enzyme increases due to the decrease of the conformational mobility in the protein moiety around the heme.

The protoporphyrin-apoperoxidase complex as a horseradish peroxidase analog. A fluorimetric study of the heme pocket.

Similarity of the protein tertiary structures of the native horseradish peroxidase (donor: hydrogen-peroxide oxidoreductase, EC 1.11.1.7) and protoporphyrin-apoperoxidase complex has been shown on the basis of identity of the tryptophan fluorescence parameter at pH 2.0-8.0 and of the circular dichroism spectra of the two proteins. Absorption and fluorescence spectra have been obtained for protoporphyrin in the complex in the pH range 7.0-1.6. A shift in the apparent pK by 4 units has been observed for protonation of the protoporphyrin pyrrolic ring in the complex. From this shift, the dielectric constant has been evaluated for the heme pocket of the peroxidase (approx. 20). Fluorescence quantum yield of protoporphyrin in the complex increased with pH decreasing from 5.0 to 3.5, whereas the spectrum pattern and fluorescence lifetime did not change. The ions, I- and [Fe(CN)6]-4, peroxidase substrates, did not quench the protoporphyrin fluorescence in the complex at about neutral pH, whereas the quenching markedly enhanced with lowering pH. The bimolecular constant for the I- -quenching of the porphyrin fluorescence on the complex showed a pH-dependence similar to that of the bimolecular rate constant for the reaction of peroxidase compound I with I-. Mechanism for I- oxidation at an acid pH in the presence of peroxidase has been proposed.

Luciferase from the east European firefly Luciola mingrelica: cloning and nucleotide sequence of the cDNA, overexpression in Escherichia coli and purification of the enzyme.

We have cloned cDNA encoding luciferase in Luciola mingrelica, fireflies living near the Black Sea in southern Russia, and obtained high level expression of the cloned sequences in Escherichia coli. The nucleotide sequences of two isolated clones were determined; five single base differences were observed, but none resulted in a change in the encoded amino acid residue. The cDNA encoded a protein of 548 amino acid residues. The overall amino acid sequence identity with the luciferase from Photinus pyralis, the North American firefly, was 67%, while comparison of the L. mingrelica luciferase with L. cruciata and L. lateralis, both indigenous to Japan, showed about 80% of the residues were strictly conserved. A novel overexpression system which employs the regulatory genes of the luminous bacterium Vibrio fischeri allowed growth of cultures to high cell density and high luciferase content, facilitating purification of the enzyme. Luciferase was purified to homogeneity in good yield from lysates of recombinant E. coli by ammonium sulfate fractionation and chromatography on columns of DEAE Sephadex and Blue Sepharose. The physicochemical properties of the luciferases from the available recombinant sources are significantly different and should allow detailed investigations into the mechanism of the bioluminescence reaction and the physical basis of the differences in the color of light emitted from the various enzymes.

Kinetics of the inactivation of the protein-lipid complex, firefly luciferase, by sodium deoxycholate and its reactivation by phosphatidylcholine.

Firefly luciferase has been shown to be a protein-lipid complex. Phospholipids and neutral lipids bound to luciferase have been identified. Sodium deoxycholate rapidly inactivated the enzyme, but an excess of phosphatidylcholine recovered luciferase activity. From the kinetics of inactivation and reactivation, a mechanism for interaction of the enzyme with detergents and phospholipids has been proposed. The substrates ATP and Mg2+ stabilized luciferase during delipidation.

Synthesis and pathway of Luciola mingrelica firefly luciferase in Xenopus laevis frog oocytes and in cell-free systems.

Poly (A+)RNA was isolated from the lanterns of adult fireflies of L. mingrelica. The poly (A+)RNA was translated in a cell-free translation mixture from rabbit reticulocyte and from Krebs II mouse ascites cells and in Xenopus laevis frog oocytes. The synthesis of the enzymatically active firefly luciferase was demonstrated in all systems. In the cell-free systems a maximal quantity of luciferase is synthesized during the first 1-2 h, then the decreasing activity of luciferase is observed. The optimal concentration of mRNA for translation in the mouse ascites cell extract was found. The kinetics of the synthesis of the luciferase, its pathway and stability in X. laevis oocytes was studied. It was observed that luciferase is secreted from oocytes into the incubation medium.

Bioluminescent assay for human lymphocyte blast transformation.

One of the basic tests of in vitro evaluation of immune cell functional activity is a proliferative response of lymphocytes on the action of external stimuli such as mitogenic lectines, antigens, etc. We compared two methods used to assess the lymphocyte functional status. (1) [3H]thymidine incorporation and (2) bioluminescence for determination of intracellular ATP in blast cells. Comparison has been done for healthy donors and patients with proven low immunological status. The proposed bioluminescent method for evaluation of the proliferative response was shown to be sensitive enough for diagnostic purposes. This method allows one to process a large number of samples at the same time and correlates highly with the radionuclide test use hazardous radioactive materials.

Fluorescent properties of firefly luciferases and their complexes with luciferin.

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.

Quenching of tryptophan fluorescence of firefly luciferase by substrates.

The interaction of firefly luciferase with substrates (luciferin and MgATP) by steady-state and time-resolved fluorescence is studied. The efficient quenching of tryptophan fluorescence of the active enzyme takes place upon its binding with substrates. In the presence of ATP the quenching is of dynamic type and is caused by structural changes in the protein molecule upon ATP binding. A model is proposed in which the complex has smaller fluorescence quantum yield than the free enzyme because of partial quenching of tryptophan fluorescence by the new microenvironment. Quenching of tryptophan fluorescence by luciferin due to the efficient energy transfer from tryptophan to luciferin is discussed. The calculated distance between Trp-419 and luciferin for the L. mingrelica luciferase in the enzyme-substrate complex is less than 12 A.

Immobilized bacterial luciferase and its applications.

This review discusses the properties of the bioluminescent bacterial system as well as the methods for immobilization of bacterial luciferases and for their co-immobilization with other enzymes. The analytical systems using immobilized bacterial luciferases and their applications in analytical biochemistry and biotechnology have been described.

Intracellular ATP in a glucosephosphate isomerase mutant of Saccharomyces cerevisiae.

The degree of ATP depletion caused by glucose in a glucosephosphate isomerase-deficient strain of Saccharomyces cerevisiae was determined. Even in the presence of a sugar normally fermentable by the mutant, the addition of glucose can decrease the intracellular ATP, depending on the competition of the sugars for transport and subsequent phosphorylation. For both parent and mutant cells, a correlation exists between the calculated velocity of ATP formation or ATP consumption during the utilization of different concentrations of sugars and the experimental intracellular ATP level. For initially resting yeast cells, a rate increase of 35 mumol per min per g ATP was calculated to increase the intracellular level of this nucleotide by 1 mumol per g cell mass.

[Inhibition of horseradish peroxidase by N-ethylamide of o-sulfobenzoylacetic acid]. / Ingibirovanie peroksidazy khrena N-étilamidom o-sul'fobenzoiluksusnoikisloty.

The carboxylic groups of horseradish peroxidase were modified by 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate by the Koshland method. The catalytic properties of the native and modified peroxidase were studied in the presence of N-ethylamide of o-sulfobenzoylacetic acid (EASBA) at pH 5.0-7.5. In the oxidation of o-dianisidine, EASBA is a competitive inhibitor of the carbidiimide-modified peroxidase, and it increases both K(m) and Vm in the case of the native enzyme. These data show that at least one of the carboxylic groups modified with carbodiimide is located at the area of the peroxidase active site.

[The ATP content of the neutrophils and whole blood in the inhabitants of regions in the Altai Territory subjected to nuclear testing exposure]. / Soderzhanie ATF v neitrofilakh i tsel'noi krovi u zhitelei raionov Altaiskogo kraia, podvergshikhsia vozdeistviiu iadernykh ispytanii.

The bioluminescent method was used in the studies of the influence of ionizing irradiation and/or xenobiotics on the content of ATP in RBC and neutrophils of rats, and in whole blood and neutrophils of 80 examined women of Altai Region exposed to ionizing radiation during a series of nuclear tests in Semipalatinsk in 1949-1965. Deviations from the normal ATP content were measured with due to account of the natural variability of a given metabolite. For rats, deviations from the content of ATP in erythrocytes were short-term, those in neutrophils were long-term. For people, a statistically significant increase in the content of ATP in neutrophils as compared to the control was observed. A non-linear correlation between the content of ATP in neutrophils and the calculated dose of radiation was observed. An increase in the ATP content in whole blood, with regard to the control, was not statistically significant for all groups of examined persons.

[The bioanalytical uses of the luciferase from fireflies (a review)]. / Bioanaliticheskie primeneniia liutsiferazy svetliakov (obzor).

Main principles of the bioluminescent microassay with the use of firefly luciferase are considered. Literature data and own experimental results of the author on application of luciferase in microbiology, clinical biochemistry, and express assays for antibiotic sensitivity and resistance to biocorrosion are generalized. New approaches to the use of luciferase for the detection of enzyme labels in EIA and for the nonradioactive detection of DNA probes are discussed.

[Immobilized luciferase of Luciola mingrelica fireflies. The kinetic properties and thermostability of luciferase immobilized on cellulose films]. / Immobilizovannaia liutsiferaza svetliakov Luciola mingrelica. Kineticheskie svoistva i termostabil'nost' liutsiferazy, immobilizovannoi na tselliuloznykh plenkakh.

Luciferase of fireflies Luciola mingrelica was immobilized on cellulose films activated by cyanuric chloride or sodium periodate. Kinetic properties and the contribution of diffusional obstacles to the kinetics of the immobilized enzyme were examined. External and internal diffusion were found to influence the kinetic parameters. The stability of the enzyme was investigated at 25 degrees C and pH 7.8. Thermoactivation of the immobilized enzyme was shown to proceed in two stages: fast and slow. Dithiotreitol and cystein stabilized the enzyme at the fast stage while salt supplements at both stages. The fast thermoinactivation stage was apparently associated with the oxidation of luciferase SH-groups. It is demonstrated that the immobilized enzyme of Luciola mingrelica can be employed to measure ATP traces with the detection limit 0.1 mM. The enzyme immobilized on cellulose films can be used repeatedly.

[Comparative evaluation of methods of intracellular ATP extraction from different types of microorganisms for bioluminescent determinationof microbial cells]. / Sravnenie metodov ekstraktsii vnutrikletochnogo ATF mikroorganizmov razlichnogo tipa dlia bioliuminestsentnogo opredeleniia kletok mikro organizma.

The efficiency of dimethyl sulfoxide (DMSO), trichloroacetic acid (TCA), and cetyltrimethylammonium bromide (CTAB) as extractants of intracellular ATP from various microorganisms was compared in bioluminescent measurements of microbial cell concentrations. Extraction with CTAB was found to provide an approximately ten times higher sensitivity of the bioluninescent assay of microbial cells than extraction with DMSO or TCA. In Gram-positive bacteria and yeasts, the ATP concentration in the extract was a linear function of the microbial suspension density only within a cell concentration range of D600 0.02-3.5 in the three types of tested extracts. In Gram-negative bacteria, a significant deviation from the linear dependence between ATP concentration and microbial suspension density was observed in CTAB extracts at D600 > 1.

[Bioluminescent method of determining picomolar amounts of nicotinamide-adenine dinucleotide using an immobilized extract of the luminescent bacterium Beneckea harveyi]. / Bioliuminestsentyi metod opredeleniia nikotinamidadenindinukleotida s pomoshch'iu svetiashchikhsia bakterii Beneckea harveyi.

The luminous bacteria Beneckea Harveyi were immobilized on BrCN-sepharose and cellulose films activated with cyanuric chloride. Preparations with high luciferase and FMN-reductase activities were obtained, which showed no background luminescence without NADH being added. The storage conditions for the preparations obtained were optimized, and their kinetic parameters and thermostability were studied. Standard curves for NADH determining within the concentration range 1 nM-1 microM were plotted with the detection level of 1 picomol NADH. The preparations are very promising for bioluminescent assay due to their high activity, simple production, high stability during storage and a possibility for the repeated use.

[Bioluminescent methods of analysis in microbiology]. / Bioliuminestsentnye metody analiza v mikrobiologii.

The prospects for application of bioluminescent ATP-metry in microbiology are considered. A bioluminescent assay is proposed to analyse biomass by measuring the content of intracellular ATP by means of immobilized firefly luciferase after ATP extraction with dimethylsulfoxide. The assay can be used for plotting the growth curves of microorganisms and for determining the sensitivity of microorganisms to antibiotics. The detection limit of the assay is 700 cells per ml of the measured solution.