Catalase and superoxide dismutase activities in Desulfovibrio desulfuricans ІМV К-6 under the influence of high concentrations of zinc chloride

Desulfovibrio spp. are mesophilic, Gram-negative, rod-shaped sulfate-reducing bacteria that produce hydrogen sulfide as a terminal by-product of their metabolic activity. These bacteria are strictly anaerobic microorganisms and presented in anoxic environments where are high sulfate- iones concentrations. Desulfovibrio species can widely vary in aerotolerance: from extreme anaerobes to species that retain viability for a long time under aerobic conditions. Protection against toxic compounds (for example, heavy metals) in aerobes and facultative anaerobes is provided by enzymes of antioxidative defense, the most important of them being superoxide dismutase (SOD) and catalase. Zinc ion (Zn2+) is an essential cofactor required by numerous metal enzymes and is important for structural and regulatory systems in bacterial cells. Zn2+ is a trace element: bacterial cells require only very small quantities. Zinc ion also possesses biologically dangerous properties since they catalyse generation of reactive oxygen species. The investigation of zinc chloride influence on the growth of Desulfovibrio desulfuricans ІМV К-6 demonstrated that low concentrations Zn2+ (0.5–2.5 mM) have not essential inhibitory influence towards biomass accumulation and dissimilatory sulfate reduction. The aim of our work was to study superoxide dismutase and catalase activities in sulfate reducing bacteria D. desulfuricans ІМV К-6 under the influence of high concentrations of zinc chloride. Bacteria were cultivated in the modified Kravtsov-Sorokin's liquid medium for ten days at +30°C under anaerobic conditions. Zinc chloride was added into the growth medium in concentrations 2–16 mM. Before bacteria seeding in the medium, 0.05 ml/l of sterile solution of Na2S×9H2O (1%) was added. The sterile 10N solution of NaOH (0.9 ml/l) in the medium was used to provide the optimal pH (7.2). The tubes were brim-filled with medium and closed to provide anaerobic conditions. Metal salt was not added into the control. Biomass was determined by the photoelectrocolorimetric method. After the second, fourth, sixth, eighth and tenth day of bacterial growth cells were disintegrated on the ultrasonic homogenizer at 22 kHz at 4 °C and cell-free extracts were obtained. Protein concentration in cell-free extract was determined by Lowry method. The catalase activity was determined spectrophotometrically by the concentration of fermented hydrogen peroxide. Specific catalase activity was determined by the reaction of hydrogen peroxide with ammonium molybdate. Specific superoxide dismutase activity was determined by the degree of inhibition of the quercetin oxidation reaction. Quercetin concentration was measured spectrophotometrically. Statistical calculation of results was carried out by application of the Origin and Excel programs. It was discovered that the level of biomass accumulation by D. desulfuricans IMV K-6 changed depending on the zinc ions concentration in the medium. The highest biomass (1.55±0.07 g/l) has been observed on the eighth day in the medium without zinc chloride. At the increase of Zn2+ concentration, a decline in biomass is marked, that, probably, is a consequence of the effect of this metal on the processes of cellular cycle. Low concentrations of zinc chloride (2–4 mM) caused an increase of the superoxide dismutase activity in D. desulfuricans IMV K-6 compared to the control. The highest superoxide dismutase activity has been observed on the fourth day in the control sample and under the influence of 2–4 mM zinc chloride. An increase of Zn2+ concentration in the cultivation medium to 6−16 mM led to a decrease of superoxide dismutase activity in D. desulfuricans IMV K-6 compared to the control. Catalase activity decreased according to the increase of zinc chloride concentration, which indicates, perhaps, the formation of a metal-enzyme complex and the replacement of ferrum (II) ions by zinc in the active centre of catalase.