科研之友
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摘要
Luciferases are now widely used in studies of molecular genetics (gene-reporters), in biochemical assays in wide screening of chemical contamination of the environment (ecology), in genetic engineering works (selection), etc. The high sensitivity and ease of detection of the light signal with a help of luminometer or scintillation counter, a direct proportionality between the amount of the enzyme luciferase and intensity of bioluminescence in a wide range (up to ten orders of magnitude), the ability to measure with equal success the enzyme activity in vitro and in vivo, without the destruction of the cells, and other benefits determine the application of luciferases of the bacterial genes is (genes lux) and eukaryotic (genes luc) origin in various genetic and biochemical tests, such as the search and analysis of the promoter and regulatory regions of DNA, by screening of DNA-tropic compounds, by measurement of ATP and other trace. Lux - biosensors are widely used in studies of genetic engineering and biotechnology, as well as for the detection of toxic agents (environmental monitoring).([1-3]) Previously, we have shown that the derivatives of hemin (DH) have expressed peroxidase activity,([6]) and they are able to catalyse the oxidation of the reduced biological substrates (NADH) by hydrogen peroxide and by a model lipophilic tert-butyl hydroperoxide. In continuation of these studies the DH were demonstrated to catalyze the oxidation of polyunsaturated fatty acids (PUFAs) by oxygen. In this case, the calculated and indirect experimental methods with sufficient reliability the PUFA hydroperoxides were shown to participate in the chain oxidation catalysed by DH.([7])
To develop this work the studies of creation of the best synthetic methods of amino acid and peptide DH are under way in our laboratory.([8.9])
Among the synthesized DH there were identified substances such as conjugates of hemin with esters of amino acids, which have strong antibacterial activity against Gram-positive bacteria, including drug-resistant strains of Staphylococcus aureus. Ability of some of the synthesized DH to model the peroxidase enzyme functions, to increase the lipid peroxidation in the lipid membrane may determine the antimicrobial activity of DH.([10])
Considering the presence of bacteria lipids containing PUFAs in the cell membranes, we have undertook the experiment, the purpose of which was to confirm the probability of involvement of reactive oxygen species (peroxides) in the interaction of DH with live bacteria, which can lead to the destruction of its membrane.
Here, the degree of oxidative stress was determined by the known method,([11]) using the bacterium E. coli K-12, containing hybrid plasmids, which are embedded in the genes of luminous bacteria and encoding luciferase and the reductase, which are highly sensitive to hydrogen peroxide (lux - biosensors).
The threshold sensitivity of the biosensor with PkatG to hydrogen peroxide is about 5.10(-6) M, which is about 10 times higher than the intracellular concentration of hydrogen peroxide (5.10(-7) M), formed in the process of breathing.([11])
As a result it was shown that the interaction of DH with live bacterium E. coli K-12 is accompanied by accumulation of peroxides. Thus, after 18 hours the concentration of peroxide was significant and greater than 10(-3) M, in some cases even at submicromolar concentrations. Amino acid derivatives Hemin 1, 2 and 3 were synthesized according to previously described methods.([8]) Thus, when using lux - biosensor the oxidative stress as a part of DH antibacterial mechanism was confirmed at the interaction the living bacteria, what is in accordance with the catalytic activity at the oxidation of organic substrates by DH, identified earlier by physical and chemical methods.
- 出版日期2013
