9 research outputs found
Bioluminescent System of Luminous Bacteria for Detection of Microbial Contamination
Microbial contamination is usually analyzed using luciferin-luciferase system of fireflies by the detection of adenosine-5β-triphosphate (ATP). There is an opportunity to assess the bacterial contamination of various objects based on a quantitative analysis of other nucleotides. In the present study, a bioluminescent enzyme system of luminous bacteria NADH:FMN-oxidoreductase (Red) and luciferase (BLuc) was investigated to understand if it can be used for quantitative measurements of bacterial cells by nicotinamide adenine dinucleotide (NADH) and flavin mononucleotide (FMN) detection. To increase the sensitivity of bioluminescent system to FMN and NADH, optimization of assay conditions was performed by varying enzymes and substrates concentrations. The lowest limits of detection were 1.2 nM FMN and 0.1 pM NADH. Escherichia coli cells were used as a model bacterial sample. FMN and NADH extraction was made by destructing cell membrane by ultrasonication. Cell suspension was added into the reaction mixture instead of FMN and NADH, and light intensity depended on number of bacterial cells in the reaction mixture. Centrifugation of sonicated sample as an additional step of sample preparation did not improve the sensitivity of method. The experimental results showed that Red and BLuc system could detect at least 800 thousand bacterial cells mL-1 by determining concentration of NADH extracted from lysed cells, while 3.9 million cells mL-1 can be detected by determining concentration of FM
Toxicity of Different Types of Surfactants via Cellular and Enzymatic Assay Systems
Surfactants have a widespread occurrence, not only as household detergents, but also in their application in industry and medicine. There are numerous bioassays for assessing surfactant toxicity, but investigations of their impact on biological systems at the molecular level are still needed. In this paper, luminous marine bacteria and their coupled NAD(P)H:FMN-oxidoreductase + luciferase (Red + Luc) enzyme system was applied to examine the effects of different types of surfactants, including cationic cetyltrimethylammonium bromide (CTAB), non-ionic polyoxyethylene 20 sorbitan monooleate (Tween 80) and anionic sodium lauryl sulfate (SLS), and to assess whether the Red + Luc enzyme system can be used as a more sensitive indicator of toxicity. It was shown that the greatest inhibitory effect of the surfactants on the activity of luminous bacteria and the Red + Luc enzyme system was in the presence of SLS samples. The calculated IC50 and EC50 values of SLS were 10β5 M and 10β2 M for the enzymatic and cellular assay systems, respectively. The results highlight the benefits of using the enzymatic assay system in ecotoxicology as a tool for revealing surfactant effects on intracellular proteins if the cellular membrane is damaged under a long-term exposure period in the presence of the surfactants. For this purpose, the bioluminescent enzyme-inhibition-based assay could be used as an advanced research tool for the evaluation of surfactant toxicity at the molecular level of living organisms due to its technical simplicity and rapid response time
ΠΠΈΠΎΠ»ΡΠΌΠΈΠ½Π΅ΡΡΠ΅Π½ΡΠ½Π°Ρ ΡΠΈΡΡΠ΅ΠΌΠ° ΡΠ²Π΅ΡΡΡΠΈΡ ΡΡ Π±Π°ΠΊΡΠ΅ΡΠΈΠΉ Π΄Π»Ρ Π°Π½Π°Π»ΠΈΠ·Π° ΠΌΠΈΠΊΡΠΎΠ±Π½ΠΎΠ³ΠΎ Π·Π°Π³ΡΡΠ·Π½Π΅Π½ΠΈΡ
Microbial contamination is usually analyzed using luciferin-luciferase system of fireflies by the detection
of adenosine-5β-triphosphate (ATP). There is an opportunity to assess the bacterial contamination
of various objects based on a quantitative analysis of other nucleotides. In the present study, a
bioluminescent enzyme system of luminous bacteria NADH:FMN-oxidoreductase (Red) and luciferase
(BLuc) was investigated to understand if it can be used for quantitative measurements of bacterial
cells by nicotinamide adenine dinucleotide (NADH) and flavin mononucleotide (FMN) detection. To
increase the sensitivity of bioluminescent system to FMN and NADH, optimization of assay conditions
was performed by varying enzymes and substrates concentrations. The lowest limits of detection were
1.2 nM FMN and 0.1 pM NADH. Escherichia coli cells were used as a model bacterial sample. FMN
and NADH extraction was made by destructing cell membrane by ultrasonication. Cell suspension
was added into the reaction mixture instead of FMN and NADH, and light intensity depended on
number of bacterial cells in the reaction mixture. Centrifugation of sonicated sample as an additional
step of sample preparation did not improve the sensitivity of method. The experimental results showed
that Red and BLuc system could detect at least 800 thousand bacterial cells mL-1 by determining
concentration of NADH extracted from lysed cells, while 3.9 million cells mL-1 can be detected by
determining concentration of FMNΠΠ»Ρ Π°Π½Π°Π»ΠΈΠ·Π° ΠΌΠΈΠΊΡΠΎΠ±Π½ΠΎΠ³ΠΎ Π·Π°Π³ΡΡΠ·Π½Π΅Π½ΠΈΡ ΡΠΈΡΠΎΠΊΠΎ ΠΈΡΠΏΠΎΠ»ΡΠ·ΡΠ΅ΡΡΡ Π±ΠΈΠΎΠ»ΡΠΌΠΈΠ½Π΅ΡΡΠ΅Π½ΡΠ½ΡΠΉ ΠΌΠ΅ΡΠΎΠ΄,
ΠΎΡΠ½ΠΎΠ²Π°Π½Π½ΡΠΉ Π½Π° ΠΎΠΏΡΠ΅Π΄Π΅Π»Π΅Π½ΠΈΠΈ Π°Π΄Π΅Π½ΠΎΠ·ΠΈΠ½-5β-ΡΡΠΈΡΠΎΡΡΠ°ΡΠ° (ΠΠ’Π ) Ρ ΠΏΠΎΠΌΠΎΡΡΡ Π»ΡΡΠΈΡΠ΅ΡΠΈΠ½-
Π»ΡΡΠΈΡΠ΅ΡΠ°Π·Π½ΠΎΠΉ ΡΠΈΡΡΠ΅ΠΌΡ ΡΠ²Π΅ΡΠ»ΡΠΊΠΎΠ². Π‘ΡΡΠ΅ΡΡΠ²ΡΠ΅Ρ ΠΏΡΠΈΠ½ΡΠΈΠΏΠΈΠ°Π»ΡΠ½Π°Ρ Π²ΠΎΠ·ΠΌΠΎΠΆΠ½ΠΎΡΡΡ ΠΎΡΠ΅Π½ΠΊΠΈ ΡΡΠ΅ΠΏΠ΅Π½ΠΈ
Π±Π°ΠΊΡΠ΅ΡΠΈΠ°Π»ΡΠ½ΠΎΠΉ ΠΎΠ±ΡΠ΅ΠΌΠ΅Π½Π΅Π½Π½ΠΎΡΡΠΈ ΡΠ°Π·Π»ΠΈΡΠ½ΡΡ
ΠΎΠ±ΡΠ΅ΠΊΡΠΎΠ² ΠΈΡΡ
ΠΎΠ΄Ρ ΠΈΠ· ΠΊΠΎΠ»ΠΈΡΠ΅ΡΡΠ²Π΅Π½Π½ΠΎΠ³ΠΎ Π°Π½Π°Π»ΠΈΠ·Π°
Π΄ΡΡΠ³ΠΈΡ
Π½ΡΠΊΠ»Π΅ΠΎΡΠΈΠ΄ΠΎΠ². Π ΡΠ°Π±ΠΎΡΠ΅ ΠΈΡΡΠ»Π΅Π΄ΠΎΠ²Π°Π½Π° Π²ΠΎΠ·ΠΌΠΎΠΆΠ½ΠΎΡΡΡ ΠΏΡΠΈΠΌΠ΅Π½Π΅Π½ΠΈΡ Π±ΠΈΠΎΠ»ΡΠΌΠΈΠ½Π΅ΡΡΠ΅Π½ΡΠ½ΠΎΠΉ
ΡΠΈΡΡΠ΅ΠΌΡ ΡΠ²Π΅ΡΡΡΠΈΡ
ΡΡ Π±Π°ΠΊΡΠ΅ΡΠΈΠΉ NADH:FMN-ΠΎΠΊΡΠΈΠ΄ΠΎΡΠ΅Π΄ΡΠΊΡΠ°Π·Π° (Red) ΠΈ Π»ΡΡΠΈΡΠ΅ΡΠ°Π·Π° (BLuc)
Π΄Π»Ρ ΠΊΠΎΠ»ΠΈΡΠ΅ΡΡΠ²Π΅Π½Π½ΠΎΠ³ΠΎ Π°Π½Π°Π»ΠΈΠ·Π° Π±Π°ΠΊΡΠ΅ΡΠΈΠ°Π»ΡΠ½ΡΡ
ΠΊΠ»Π΅ΡΠΎΠΊ ΠΏΡΡΠ΅ΠΌ ΠΎΠΏΡΠ΅Π΄Π΅Π»Π΅Π½ΠΈΡ ΠΊΠΎΠ»ΠΈΡΠ΅ΡΡΠ²Π°
Π½ΠΈΠΊΠΎΡΠΈΠ½Π°ΠΌΠΈΠ΄Π°Π΄Π΅Π½ΠΈΠ½Π΄ΠΈΠ½ΡΠΊΠ»Π΅ΠΎΡΠΈΠ΄Π° (NADH) ΠΈ ΡΠ»Π°Π²ΠΈΠ½ΠΌΠΎΠ½ΠΎΠ½ΡΠΊΠ»Π΅ΠΎΡΠΈΠ΄Π° (FMN) Π² ΠΎΠ±ΡΠ°Π·ΡΠ΅. ΠΠ»Ρ
ΡΠ²Π΅Π»ΠΈΡΠ΅Π½ΠΈΡ ΡΡΠ²ΡΡΠ²ΠΈΡΠ΅Π»ΡΠ½ΠΎΡΡΠΈ Π±ΠΈΠΎΠ»ΡΠΌΠΈΠ½Π΅ΡΡΠ΅Π½ΡΠ½ΠΎΠΉ ΡΠΈΡΡΠ΅ΠΌΡ ΠΊ FMN ΠΈ NADH ΠΎΡΡΡΠ΅ΡΡΠ²Π»Π΅Π½Π°
ΠΎΠΏΡΠΈΠΌΠΈΠ·Π°ΡΠΈΡ ΡΡΠ»ΠΎΠ²ΠΈΠΉ ΠΏΡΠΎΠ²Π΅Π΄Π΅Π½ΠΈΡ Π°Π½Π°Π»ΠΈΠ·Π° ΠΏΡΡΠ΅ΠΌ ΠΏΠΎΠ΄Π±ΠΎΡΠ° ΠΊΠΎΠ½ΡΠ΅Π½ΡΡΠ°ΡΠΈΠΉ ΡΠ΅ΡΠΌΠ΅Π½ΡΠΎΠ² ΠΈ
ΡΡΠ±ΡΡΡΠ°ΡΠΎΠ² Π² ΡΠ΅Π°ΠΊΡΠΈΠΎΠ½Π½ΠΎΠΉ ΡΠΌΠ΅ΡΠΈ. ΠΠ°ΠΊΡΠΈΠΌΠ°Π»ΡΠ½Π°Ρ ΡΡΠ²ΡΡΠ²ΠΈΡΠ΅Π»ΡΠ½ΠΎΡΡΡ Π±ΠΈΡΠ΅ΡΠΌΠ΅Π½ΡΠ½ΠΎΠΉ ΡΠΈΡΡΠ΅ΠΌΡ
Red + BLuc ΡΠΎΡΡΠ°Π²ΠΈΠ»Π° 1,2 Π½Π FMN ΠΈ 0,1 ΠΏΠ NADH. ΠΠ»Ρ ΠΏΡΠΎΠ²Π΅Π΄Π΅Π½ΠΈΡ ΠΊΠΎΠ»ΠΈΡΠ΅ΡΡΠ²Π΅Π½Π½ΠΎΠ³ΠΎ Π°Π½Π°Π»ΠΈΠ·Π°
Π±Π°ΠΊΡΠ΅ΡΠΈΠ°Π»ΡΠ½ΡΡ
ΠΊΠ»Π΅ΡΠΎΠΊ ΠΈΡΠΏΠΎΠ»ΡΠ·ΠΎΠ²Π°Π»ΠΈ ΠΌΠΎΠ΄Π΅Π»ΡΠ½ΡΠΉ Π±Π°ΠΊΡΠ΅ΡΠΈΠ°Π»ΡΠ½ΡΠΉ ΠΎΠ±ΡΠ°Π·Π΅Ρ β ΠΊΡΠ»ΡΡΡΡΡ Escherichia
coli. ΠΠΊΡΡΡΠ°Π³ΠΈΡΠΎΠ²Π°Π½ΠΈΠ΅ FMN ΠΈ NADH ΠΏΡΠΎΠ²ΠΎΠ΄ΠΈΠ»ΠΈ ΠΏΡΡΠ΅ΠΌ ΡΠ°Π·ΡΡΡΠ΅Π½ΠΈΡ ΠΊΠ»Π΅ΡΠΎΡΠ½ΠΎΠΉ ΠΌΠ΅ΠΌΠ±ΡΠ°Π½Ρ
ΡΠ»ΡΡΡΠ°Π·Π²ΡΠΊΠΎΠ²ΡΠΌ Π΄Π΅Π·ΠΈΠ½ΡΠ΅Π³ΡΠ°ΡΠΎΡΠΎΠΌ. ΠΠ»Π΅ΡΠΎΡΠ½ΡΡ ΡΡΡΠΏΠ΅Π½Π·ΠΈΡ Π΄ΠΎΠ±Π°Π²Π»ΡΠ»ΠΈ Π² ΡΠ΅Π°ΠΊΡΠΈΠΎΠ½Π½ΡΡ ΡΠΌΠ΅ΡΡ
Π²ΠΌΠ΅ΡΡΠΎ ΡΠ°ΡΡΠ²ΠΎΡΠ° FMN ΠΈΠ»ΠΈ NADH, ΠΏΡΠΈ ΡΡΠΎΠΌ ΠΈΠ½ΡΠ΅Π½ΡΠΈΠ²Π½ΠΎΡΡΡ ΡΠ²Π΅ΡΠ΅Π½ΠΈΡ Π±ΠΈΠΎΠ»ΡΠΌΠΈΠ½Π΅ΡΡΠ΅Π½ΡΠ½ΠΎΠΉ
ΡΠΈΡΡΠ΅ΠΌΡ Π·Π°Π²ΠΈΡΠ΅Π»Π° ΠΎΡ ΠΊΠΎΠ»ΠΈΡΠ΅ΡΡΠ²Π° Π±Π°ΠΊΡΠ΅ΡΠΈΠ°Π»ΡΠ½ΡΡ
ΠΊΠ»Π΅ΡΠΎΠΊ Π² ΡΠ΅Π°ΠΊΡΠΈΠΎΠ½Π½ΠΎΠΉ ΡΠΌΠ΅ΡΠΈ. ΠΠ²Π΅Π΄Π΅Π½ΠΈΠ΅ Π²
ΠΏΡΠΎΠ±ΠΎΠΏΠΎΠ΄Π³ΠΎΡΠΎΠ²ΠΊΡ Π΄ΠΎΠΏΠΎΠ»Π½ΠΈΡΠ΅Π»ΡΠ½ΠΎΠΉ ΠΏΡΠΎΡΠ΅Π΄ΡΡΡ ΡΠ΅Π½ΡΡΠΈΡΡΠ³ΠΈΡΠΎΠ²Π°Π½ΠΈΡ ΠΎΠ±ΡΠ°Π·ΡΠ°, ΠΏΠΎΠ΄Π²Π΅ΡΠ³Π½ΡΡΠΎΠ³ΠΎ
ΡΠ»ΡΡΡΠ°Π·Π²ΡΠΊΠΎΠ²ΠΎΠΉ ΠΎΠ±ΡΠ°Π±ΠΎΡΠΊΠ΅, Π½Π΅ ΠΏΡΠΈΠ²Π΅Π»ΠΎ ΠΊ ΡΠ²Π΅Π»ΠΈΡΠ΅Π½ΠΈΡ ΡΡΠ²ΡΡΠ²ΠΈΡΠ΅Π»ΡΠ½ΠΎΡΡΠΈ ΠΌΠ΅ΡΠΎΠ΄Π°. Π’Π°ΠΊΠΈΠΌ ΠΎΠ±ΡΠ°Π·ΠΎΠΌ,
Π±ΡΠ»ΠΎ ΠΏΠΎΠΊΠ°Π·Π°Π½ΠΎ, ΡΡΠΎ ΡΡΠ²ΡΡΠ²ΠΈΡΠ΅Π»ΡΠ½ΠΎΡΡΠΈ Π±ΠΈΡΠ΅ΡΠΌΠ΅Π½ΡΠ½ΠΎΠΉ ΡΠΈΡΡΠ΅ΠΌΡ Red + BLuc Π΄ΠΎΡΡΠ°ΡΠΎΡΠ½ΠΎ Π΄Π»Ρ
ΠΎΠΏΡΠ΅Π΄Π΅Π»Π΅Π½ΠΈΡ Π½Π΅ ΠΌΠ΅Π½Π΅Π΅ 800 ΡΡΡ Π±Π°ΠΊΡΠ΅ΡΠΈΠ°Π»ΡΠ½ΡΡ
ΠΊΠ»Π΅ΡΠΎΠΊ Π² ΠΌΠΈΠ»Π»ΠΈΠ»ΠΈΡΡΠ΅ ΠΏΡΡΠ΅ΠΌ ΡΠΊΡΡΡΠ°Π³ΠΈΡΠΎΠ²Π°Π½ΠΈΡ
NADH ΠΈΠ· ΡΠ°Π·ΡΡΡΠ΅Π½Π½ΡΡ
ΠΊΠ»Π΅ΡΠΎΠΊ. ΠΡΠΈ Π°Π½Π°Π»ΠΈΠ·Π΅, ΠΎΡΠ½ΠΎΠ²Π°Π½Π½ΠΎΠΌ Π½Π° ΠΎΠΏΡΠ΅Π΄Π΅Π»Π΅Π½ΠΈΠΈ ΠΊΠΎΠ½ΡΠ΅Π½ΡΡΠ°ΡΠΈΠΈ FMN Π²
Π±Π°ΠΊΡΠ΅ΡΠΈΠ°Π»ΡΠ½ΠΎΠΌ ΠΎΠ±ΡΠ°Π·ΡΠ΅, ΡΡΠ²ΡΡΠ²ΠΈΡΠ΅Π»ΡΠ½ΠΎΡΡΡ ΠΌΠ΅ΡΠΎΠ΄Π° ΡΠΎΡΡΠ°Π²ΠΈΠ»Π° 3,9 ΠΌΠ»Π½ ΠΊΠ»Π΅ΡΠΎΠΊ Π½Π° ΠΌΠΈΠ»Π»ΠΈΠ»ΠΈΡ
ΠΠΈΠΎΠ»ΡΠΌΠΈΠ½Π΅ΡΡΠ΅Π½ΡΠ½Π°Ρ ΡΠΈΡΡΠ΅ΠΌΠ° ΡΠ²Π΅ΡΡΡΠΈΡ ΡΡ Π±Π°ΠΊΡΠ΅ΡΠΈΠΉ Π΄Π»Ρ Π°Π½Π°Π»ΠΈΠ·Π° ΠΌΠΈΠΊΡΠΎΠ±Π½ΠΎΠ³ΠΎ Π·Π°Π³ΡΡΠ·Π½Π΅Π½ΠΈΡ
Microbial contamination is usually analyzed using luciferin-luciferase system of fireflies by the detection
of adenosine-5β-triphosphate (ATP). There is an opportunity to assess the bacterial contamination
of various objects based on a quantitative analysis of other nucleotides. In the present study, a
bioluminescent enzyme system of luminous bacteria NADH:FMN-oxidoreductase (Red) and luciferase
(BLuc) was investigated to understand if it can be used for quantitative measurements of bacterial
cells by nicotinamide adenine dinucleotide (NADH) and flavin mononucleotide (FMN) detection. To
increase the sensitivity of bioluminescent system to FMN and NADH, optimization of assay conditions
was performed by varying enzymes and substrates concentrations. The lowest limits of detection were
1.2 nM FMN and 0.1 pM NADH. Escherichia coli cells were used as a model bacterial sample. FMN
and NADH extraction was made by destructing cell membrane by ultrasonication. Cell suspension
was added into the reaction mixture instead of FMN and NADH, and light intensity depended on
number of bacterial cells in the reaction mixture. Centrifugation of sonicated sample as an additional
step of sample preparation did not improve the sensitivity of method. The experimental results showed
that Red and BLuc system could detect at least 800 thousand bacterial cells mL-1 by determining
concentration of NADH extracted from lysed cells, while 3.9 million cells mL-1 can be detected by
determining concentration of FMNΠΠ»Ρ Π°Π½Π°Π»ΠΈΠ·Π° ΠΌΠΈΠΊΡΠΎΠ±Π½ΠΎΠ³ΠΎ Π·Π°Π³ΡΡΠ·Π½Π΅Π½ΠΈΡ ΡΠΈΡΠΎΠΊΠΎ ΠΈΡΠΏΠΎΠ»ΡΠ·ΡΠ΅ΡΡΡ Π±ΠΈΠΎΠ»ΡΠΌΠΈΠ½Π΅ΡΡΠ΅Π½ΡΠ½ΡΠΉ ΠΌΠ΅ΡΠΎΠ΄,
ΠΎΡΠ½ΠΎΠ²Π°Π½Π½ΡΠΉ Π½Π° ΠΎΠΏΡΠ΅Π΄Π΅Π»Π΅Π½ΠΈΠΈ Π°Π΄Π΅Π½ΠΎΠ·ΠΈΠ½-5β-ΡΡΠΈΡΠΎΡΡΠ°ΡΠ° (ΠΠ’Π ) Ρ ΠΏΠΎΠΌΠΎΡΡΡ Π»ΡΡΠΈΡΠ΅ΡΠΈΠ½-
Π»ΡΡΠΈΡΠ΅ΡΠ°Π·Π½ΠΎΠΉ ΡΠΈΡΡΠ΅ΠΌΡ ΡΠ²Π΅ΡΠ»ΡΠΊΠΎΠ². Π‘ΡΡΠ΅ΡΡΠ²ΡΠ΅Ρ ΠΏΡΠΈΠ½ΡΠΈΠΏΠΈΠ°Π»ΡΠ½Π°Ρ Π²ΠΎΠ·ΠΌΠΎΠΆΠ½ΠΎΡΡΡ ΠΎΡΠ΅Π½ΠΊΠΈ ΡΡΠ΅ΠΏΠ΅Π½ΠΈ
Π±Π°ΠΊΡΠ΅ΡΠΈΠ°Π»ΡΠ½ΠΎΠΉ ΠΎΠ±ΡΠ΅ΠΌΠ΅Π½Π΅Π½Π½ΠΎΡΡΠΈ ΡΠ°Π·Π»ΠΈΡΠ½ΡΡ
ΠΎΠ±ΡΠ΅ΠΊΡΠΎΠ² ΠΈΡΡ
ΠΎΠ΄Ρ ΠΈΠ· ΠΊΠΎΠ»ΠΈΡΠ΅ΡΡΠ²Π΅Π½Π½ΠΎΠ³ΠΎ Π°Π½Π°Π»ΠΈΠ·Π°
Π΄ΡΡΠ³ΠΈΡ
Π½ΡΠΊΠ»Π΅ΠΎΡΠΈΠ΄ΠΎΠ². Π ΡΠ°Π±ΠΎΡΠ΅ ΠΈΡΡΠ»Π΅Π΄ΠΎΠ²Π°Π½Π° Π²ΠΎΠ·ΠΌΠΎΠΆΠ½ΠΎΡΡΡ ΠΏΡΠΈΠΌΠ΅Π½Π΅Π½ΠΈΡ Π±ΠΈΠΎΠ»ΡΠΌΠΈΠ½Π΅ΡΡΠ΅Π½ΡΠ½ΠΎΠΉ
ΡΠΈΡΡΠ΅ΠΌΡ ΡΠ²Π΅ΡΡΡΠΈΡ
ΡΡ Π±Π°ΠΊΡΠ΅ΡΠΈΠΉ NADH:FMN-ΠΎΠΊΡΠΈΠ΄ΠΎΡΠ΅Π΄ΡΠΊΡΠ°Π·Π° (Red) ΠΈ Π»ΡΡΠΈΡΠ΅ΡΠ°Π·Π° (BLuc)
Π΄Π»Ρ ΠΊΠΎΠ»ΠΈΡΠ΅ΡΡΠ²Π΅Π½Π½ΠΎΠ³ΠΎ Π°Π½Π°Π»ΠΈΠ·Π° Π±Π°ΠΊΡΠ΅ΡΠΈΠ°Π»ΡΠ½ΡΡ
ΠΊΠ»Π΅ΡΠΎΠΊ ΠΏΡΡΠ΅ΠΌ ΠΎΠΏΡΠ΅Π΄Π΅Π»Π΅Π½ΠΈΡ ΠΊΠΎΠ»ΠΈΡΠ΅ΡΡΠ²Π°
Π½ΠΈΠΊΠΎΡΠΈΠ½Π°ΠΌΠΈΠ΄Π°Π΄Π΅Π½ΠΈΠ½Π΄ΠΈΠ½ΡΠΊΠ»Π΅ΠΎΡΠΈΠ΄Π° (NADH) ΠΈ ΡΠ»Π°Π²ΠΈΠ½ΠΌΠΎΠ½ΠΎΠ½ΡΠΊΠ»Π΅ΠΎΡΠΈΠ΄Π° (FMN) Π² ΠΎΠ±ΡΠ°Π·ΡΠ΅. ΠΠ»Ρ
ΡΠ²Π΅Π»ΠΈΡΠ΅Π½ΠΈΡ ΡΡΠ²ΡΡΠ²ΠΈΡΠ΅Π»ΡΠ½ΠΎΡΡΠΈ Π±ΠΈΠΎΠ»ΡΠΌΠΈΠ½Π΅ΡΡΠ΅Π½ΡΠ½ΠΎΠΉ ΡΠΈΡΡΠ΅ΠΌΡ ΠΊ FMN ΠΈ NADH ΠΎΡΡΡΠ΅ΡΡΠ²Π»Π΅Π½Π°
ΠΎΠΏΡΠΈΠΌΠΈΠ·Π°ΡΠΈΡ ΡΡΠ»ΠΎΠ²ΠΈΠΉ ΠΏΡΠΎΠ²Π΅Π΄Π΅Π½ΠΈΡ Π°Π½Π°Π»ΠΈΠ·Π° ΠΏΡΡΠ΅ΠΌ ΠΏΠΎΠ΄Π±ΠΎΡΠ° ΠΊΠΎΠ½ΡΠ΅Π½ΡΡΠ°ΡΠΈΠΉ ΡΠ΅ΡΠΌΠ΅Π½ΡΠΎΠ² ΠΈ
ΡΡΠ±ΡΡΡΠ°ΡΠΎΠ² Π² ΡΠ΅Π°ΠΊΡΠΈΠΎΠ½Π½ΠΎΠΉ ΡΠΌΠ΅ΡΠΈ. ΠΠ°ΠΊΡΠΈΠΌΠ°Π»ΡΠ½Π°Ρ ΡΡΠ²ΡΡΠ²ΠΈΡΠ΅Π»ΡΠ½ΠΎΡΡΡ Π±ΠΈΡΠ΅ΡΠΌΠ΅Π½ΡΠ½ΠΎΠΉ ΡΠΈΡΡΠ΅ΠΌΡ
Red + BLuc ΡΠΎΡΡΠ°Π²ΠΈΠ»Π° 1,2 Π½Π FMN ΠΈ 0,1 ΠΏΠ NADH. ΠΠ»Ρ ΠΏΡΠΎΠ²Π΅Π΄Π΅Π½ΠΈΡ ΠΊΠΎΠ»ΠΈΡΠ΅ΡΡΠ²Π΅Π½Π½ΠΎΠ³ΠΎ Π°Π½Π°Π»ΠΈΠ·Π°
Π±Π°ΠΊΡΠ΅ΡΠΈΠ°Π»ΡΠ½ΡΡ
ΠΊΠ»Π΅ΡΠΎΠΊ ΠΈΡΠΏΠΎΠ»ΡΠ·ΠΎΠ²Π°Π»ΠΈ ΠΌΠΎΠ΄Π΅Π»ΡΠ½ΡΠΉ Π±Π°ΠΊΡΠ΅ΡΠΈΠ°Π»ΡΠ½ΡΠΉ ΠΎΠ±ΡΠ°Π·Π΅Ρ β ΠΊΡΠ»ΡΡΡΡΡ Escherichia
coli. ΠΠΊΡΡΡΠ°Π³ΠΈΡΠΎΠ²Π°Π½ΠΈΠ΅ FMN ΠΈ NADH ΠΏΡΠΎΠ²ΠΎΠ΄ΠΈΠ»ΠΈ ΠΏΡΡΠ΅ΠΌ ΡΠ°Π·ΡΡΡΠ΅Π½ΠΈΡ ΠΊΠ»Π΅ΡΠΎΡΠ½ΠΎΠΉ ΠΌΠ΅ΠΌΠ±ΡΠ°Π½Ρ
ΡΠ»ΡΡΡΠ°Π·Π²ΡΠΊΠΎΠ²ΡΠΌ Π΄Π΅Π·ΠΈΠ½ΡΠ΅Π³ΡΠ°ΡΠΎΡΠΎΠΌ. ΠΠ»Π΅ΡΠΎΡΠ½ΡΡ ΡΡΡΠΏΠ΅Π½Π·ΠΈΡ Π΄ΠΎΠ±Π°Π²Π»ΡΠ»ΠΈ Π² ΡΠ΅Π°ΠΊΡΠΈΠΎΠ½Π½ΡΡ ΡΠΌΠ΅ΡΡ
Π²ΠΌΠ΅ΡΡΠΎ ΡΠ°ΡΡΠ²ΠΎΡΠ° FMN ΠΈΠ»ΠΈ NADH, ΠΏΡΠΈ ΡΡΠΎΠΌ ΠΈΠ½ΡΠ΅Π½ΡΠΈΠ²Π½ΠΎΡΡΡ ΡΠ²Π΅ΡΠ΅Π½ΠΈΡ Π±ΠΈΠΎΠ»ΡΠΌΠΈΠ½Π΅ΡΡΠ΅Π½ΡΠ½ΠΎΠΉ
ΡΠΈΡΡΠ΅ΠΌΡ Π·Π°Π²ΠΈΡΠ΅Π»Π° ΠΎΡ ΠΊΠΎΠ»ΠΈΡΠ΅ΡΡΠ²Π° Π±Π°ΠΊΡΠ΅ΡΠΈΠ°Π»ΡΠ½ΡΡ
ΠΊΠ»Π΅ΡΠΎΠΊ Π² ΡΠ΅Π°ΠΊΡΠΈΠΎΠ½Π½ΠΎΠΉ ΡΠΌΠ΅ΡΠΈ. ΠΠ²Π΅Π΄Π΅Π½ΠΈΠ΅ Π²
ΠΏΡΠΎΠ±ΠΎΠΏΠΎΠ΄Π³ΠΎΡΠΎΠ²ΠΊΡ Π΄ΠΎΠΏΠΎΠ»Π½ΠΈΡΠ΅Π»ΡΠ½ΠΎΠΉ ΠΏΡΠΎΡΠ΅Π΄ΡΡΡ ΡΠ΅Π½ΡΡΠΈΡΡΠ³ΠΈΡΠΎΠ²Π°Π½ΠΈΡ ΠΎΠ±ΡΠ°Π·ΡΠ°, ΠΏΠΎΠ΄Π²Π΅ΡΠ³Π½ΡΡΠΎΠ³ΠΎ
ΡΠ»ΡΡΡΠ°Π·Π²ΡΠΊΠΎΠ²ΠΎΠΉ ΠΎΠ±ΡΠ°Π±ΠΎΡΠΊΠ΅, Π½Π΅ ΠΏΡΠΈΠ²Π΅Π»ΠΎ ΠΊ ΡΠ²Π΅Π»ΠΈΡΠ΅Π½ΠΈΡ ΡΡΠ²ΡΡΠ²ΠΈΡΠ΅Π»ΡΠ½ΠΎΡΡΠΈ ΠΌΠ΅ΡΠΎΠ΄Π°. Π’Π°ΠΊΠΈΠΌ ΠΎΠ±ΡΠ°Π·ΠΎΠΌ,
Π±ΡΠ»ΠΎ ΠΏΠΎΠΊΠ°Π·Π°Π½ΠΎ, ΡΡΠΎ ΡΡΠ²ΡΡΠ²ΠΈΡΠ΅Π»ΡΠ½ΠΎΡΡΠΈ Π±ΠΈΡΠ΅ΡΠΌΠ΅Π½ΡΠ½ΠΎΠΉ ΡΠΈΡΡΠ΅ΠΌΡ Red + BLuc Π΄ΠΎΡΡΠ°ΡΠΎΡΠ½ΠΎ Π΄Π»Ρ
ΠΎΠΏΡΠ΅Π΄Π΅Π»Π΅Π½ΠΈΡ Π½Π΅ ΠΌΠ΅Π½Π΅Π΅ 800 ΡΡΡ Π±Π°ΠΊΡΠ΅ΡΠΈΠ°Π»ΡΠ½ΡΡ
ΠΊΠ»Π΅ΡΠΎΠΊ Π² ΠΌΠΈΠ»Π»ΠΈΠ»ΠΈΡΡΠ΅ ΠΏΡΡΠ΅ΠΌ ΡΠΊΡΡΡΠ°Π³ΠΈΡΠΎΠ²Π°Π½ΠΈΡ
NADH ΠΈΠ· ΡΠ°Π·ΡΡΡΠ΅Π½Π½ΡΡ
ΠΊΠ»Π΅ΡΠΎΠΊ. ΠΡΠΈ Π°Π½Π°Π»ΠΈΠ·Π΅, ΠΎΡΠ½ΠΎΠ²Π°Π½Π½ΠΎΠΌ Π½Π° ΠΎΠΏΡΠ΅Π΄Π΅Π»Π΅Π½ΠΈΠΈ ΠΊΠΎΠ½ΡΠ΅Π½ΡΡΠ°ΡΠΈΠΈ FMN Π²
Π±Π°ΠΊΡΠ΅ΡΠΈΠ°Π»ΡΠ½ΠΎΠΌ ΠΎΠ±ΡΠ°Π·ΡΠ΅, ΡΡΠ²ΡΡΠ²ΠΈΡΠ΅Π»ΡΠ½ΠΎΡΡΡ ΠΌΠ΅ΡΠΎΠ΄Π° ΡΠΎΡΡΠ°Π²ΠΈΠ»Π° 3,9 ΠΌΠ»Π½ ΠΊΠ»Π΅ΡΠΎΠΊ Π½Π° ΠΌΠΈΠ»Π»ΠΈΠ»ΠΈΡ
The Effects of Commercial Pesticide Formulations on the Function of In Vitro and In Vivo Assay Systems: A Comparative Analysis
Pesticides are commonly used in agriculture and are an important factor of food security for humankind. However, the overuse of pesticides can harm non-target organisms, and, thus, it is vital to comprehensively study their effects on the different metabolic pathways of living organisms. In the present study, enzyme-inhibition-based assays have been used to investigate the effects of commercial pesticide formulations on the key enzymes of the organisms, which catalyze a wide variety of metabolic reactions (protein catabolism, lactic acid fermentation, alcohol metabolism, the conduction of nerve impulses, etc.). Assay conditions have been optimized, and the limitations of the methods used in the study, which are related to the choice of the solvent for commercial pesticide formulations and optical effects occurring when commercial pesticide formulations are mixed with solutions of enzymes and substrates of assay systems, have been revealed. The effects of commercial pesticide formulations on simple chemoenzymatic assay systems (single-enzyme reactions) have been compared to their effects on complex multicomponent molecular systems (multi-enzyme reactions) and organisms (luminescent bacterium). The in vitro assay systems have shown higher sensitivity to pesticide exposure than the in vivo assay system. The sensitivity of the in vitro assay systems increases with the elongation of the chain of conjugated chemoenzymatic reactions. The effects exerted by commercial pesticide formulations with the same active ingredient but produced by different manufacturers on assay system functions have been found to differ from each other
Enzyme Inhibition-Based Assay to Estimate the Contribution of Formulants to the Effect of Commercial Pesticide Formulations
Pesticides can affect the health of individual organisms and the function of the entire ecosystem. Therefore, thorough assessment of the risks associated with the use of pesticides is a high-priority task. An enzyme inhibition-based assay is used in this study as a convenient and quick tool to study the effects of pesticides at the molecular level. The contribution of formulants to toxicological properties of the pesticide formulations has been studied by analyzing effects of 7 active ingredients of pesticides (AIas) and 10 commercial formulations based on them (AIfs) on the function of a wide range of enzyme assay systems differing in complexity (single-, coupled, and three-enzyme assay systems). Results have been compared with the effects of AIas and AIfs on bioluminescence of the luminous bacterium Photobacterium phosphoreum. Mostly, AIfs produce a considerably stronger inhibitory effect on the activity of enzyme assay systems and bioluminescence of the luminous bacterium than AIas, which confirms the contribution of formulants to toxicological properties of the pesticide formulation. Results of the current study demonstrate that “inert” ingredients are not ecotoxicologically safe and can considerably augment the inhibitory effect of pesticide formulations; therefore, their use should be controlled more strictly. Circular dichroism and fluorescence spectra of the enzymes used for assays do not show any changes in the protein structure in the presence of commercial pesticide formulations during the assay procedure. This finding suggests that pesticides produce the inhibitory effect on enzymes through other mechanisms
Enzyme Inhibition-Based Assay to Estimate the Contribution of Formulants to the Effect of Commercial Pesticide Formulations
Pesticides can affect the health of individual organisms and the function of the entire ecosystem. Therefore, thorough assessment of the risks associated with the use of pesticides is a high-priority task. An enzyme inhibition-based assay is used in this study as a convenient and quick tool to study the effects of pesticides at the molecular level. The contribution of formulants to toxicological properties of the pesticide formulations has been studied by analyzing effects of 7 active ingredients of pesticides (AIas) and 10 commercial formulations based on them (AIfs) on the function of a wide range of enzyme assay systems differing in complexity (single-, coupled, and three-enzyme assay systems). Results have been compared with the effects of AIas and AIfs on bioluminescence of the luminous bacterium Photobacterium phosphoreum. Mostly, AIfs produce a considerably stronger inhibitory effect on the activity of enzyme assay systems and bioluminescence of the luminous bacterium than AIas, which confirms the contribution of formulants to toxicological properties of the pesticide formulation. Results of the current study demonstrate that βinertβ ingredients are not ecotoxicologically safe and can considerably augment the inhibitory effect of pesticide formulations; therefore, their use should be controlled more strictly. Circular dichroism and fluorescence spectra of the enzymes used for assays do not show any changes in the protein structure in the presence of commercial pesticide formulations during the assay procedure. This finding suggests that pesticides produce the inhibitory effect on enzymes through other mechanisms