Characterization of peroxidase and catalase enzymes from the plant Galium aparine L.

Some oxygen species (reactive oxygen species, ROS) essential for life can be extremely harmful to human health. ROS is mostly produced by free radicals and is chemically more reactive than molecular oxygen. In case free radicals cannot be destroyed; ROS can cause events such as the death of cells by breaking down proteins in the cell membrane, DNA fragmentation and becoming open to mutations, cancer, aging and weakening of the immune system. The body develops defense mechanisms to prevent the formation of ROS and the damage they cause. These defense systems found in all living organisms are called antioxidant defense systems, briefly antioxidants. Plant tissues also contain various antioxidant enzymes such as catalase (CAT), peroxidase (POD), superoxide dismutase (SOD) and phenolic compounds to protect their cells under stress conditions by eliminating ROS. All parts of plants contain natural antioxidants in the form of carotenoids, phenols, vitamins and flavonoids. The antioxidants in plants act as peroxide scavengers, free radical scavengers and enzyme inhibitors. In this way, they are not affected by free radicals. Enzymes are biocatalysts that are in protein structure and carry out biochemical reactions. Peroxidase enzyme ((POD) EC 1.11.1.7) is an oxidoreductase group enzyme that catalyzes the oxidation of many substrates such as flavonoids, phenols, sulfonoids, aromatic amines and pyrogallol in the presence of hydrogen peroxide. POD is an enzyme found in the cell wall of plants, animals, bacteria and fungi, and is also essential for plant growth. It has been reported that POD plays an important role in important functions such as plant defense mechanisms, regulation of indolacetic acid in fruits and vegetables, hormone activity and lignin biosynthesis. POD is abundant in plant foods. In plants, POD is found in flowers, stems, leaves, cell nuclei, cell membranes, ribosomes, and cell walls. POD enzymes catalyze the oxidation of inorganic and organic substrates and the dehydrogenation of hydroquinones, phenols, hydroquinoid amines and other aromatic compounds using H2O2. Examples of catalytic molecules; guaiacol, o-dianisidine, pyrogallol, azo dye derivatives, o phenylenediamine can be counted. The another antioxidant enzyme catalase ((CAT), H2O2 oxidoreductase, E.C.1.11.1.6) is very common in nature. It has high catalytic activity in almost all aerobic microorganisms, plant and animal cells. It belongs to the class of hydroperoxidases, a catalase oxidoreductase group. Hydroperoxidases use hydrogen peroxide and organic peroxides as substrates. It catalyzes cellular hydrogen peroxide to water and oxygen. Catalase enzyme is also used in industrial applications sucha as textile, food, medicine, etc. For examplei, catalase enzyme is used in contact lens hygiene and lens disinfection with H2O2 solutions. A catalase enzyme solution reliably removes H2O2 before contact lens reuse. UV rays accelerate the formation of free radicals. Free radicals damage the skin and cause deterioration of skin quality. Antioxidant molecules exert a variety of effects and reduce skin damage caused by oxidation. Yogurt grass (Galium aparine) is a climbing plant that also grows in North America, Europe and Asia. Galium aparine is traditionally used to coagulate milk because of its chemical composition. Therefore, it is known as "Yogurt grass" in public. The active ingredients of G. aparine are alkanes, flavonoids, anthraquinones, polyphenolic acids, tannins, iridoids and vitamin C. It has been used in the treatment of lymph nodes, tonsil foci, jaundice, cancer, fever and leukemia. In this study, peroxidase and catalase enzymes from Galium aparine L. (Yogurt grass) plant grown in Sakarya, Turkey were purified and their kinetic properties and activities against some inhibitors were determined. The collected plant was stored at -20 ℃ until extraction. Enzyme isolation procedures were performed at +4 ℃. Enzymes were extracted bye using 0.1 M phosphate buffer (pH 7.0) containing ascorbic acid, PVP and Triton X-100. Yogurt grass peroxidase and catalase enzymes were purified by three-phase separation technique (TPP) after izolation and molecular weights of enzymes were determined by SDS-PAGE method. The molecular weight of peroxidase enzyme was found to be 65 kDa, and the molecular weight of catalase enzyme was found to be 50 kDa. Peroxidase enzyme activity was determined by measuring absorbance at 420 nm for 60 seconds using 4-methyl catechol and H2O2 substrates. Catalase enzyme activity was determined by observing the absorbance decrease of the H2O2 substrate for 180 seconds at 240 nm. The Vmax and Km values of the peroxidase enzyme were determined as 0.00238 EU/min and 1.47mM, respectively. Vmax and Km values of catalase enzyme were determined as 0.0007 EU/min and 6.41 mM. The peroxidase enzyme substrate specificity was determined with pyrogallol, o-phenylenediamine, gallic acid, caffeic acid, 4-methylcatechol substrates in the presence of H2O2 and and the most relevant substrate of the G. aparine peroxidase enzyme was determined as the pyrogallol substrate with a Km value of 1.086 mM. Optimum pH was determined as 6.5 for POD enzyme and 8 for CAT enzyme. G. apirine peroxidase and optimum temperature determination was made for G. aparine catalase. The optimum temperature for peroxidase and catalase enzymes was found to be 30 ℃. Sodium azide and thiourea were used as inhibitors in the inhibition experiment for G. aparine peroxidase and catalase. It was concluded that both inhibitors inhibited POD and CAT enzymes. In order to determine the amount of inhibitor that halves the enzyme activities, firstly the % relative activities of the enzymes were calculated and the IC50 values were determined with the help of graphics. The IC50 value calculated by the effect of the thiourea inhibitor on the peroxidase enzyme activity was calculated as 1.18 mM, and the IC50 value with the sodium azide inhibitor was calculated as 2.05 mM. The IC50 value was determined as 0.85 mM as a result of inhibition of the catalase enzyme by thiourea, and as 0.97 mM as a result of sodium azide inhibition. The effects of Fe(III), Cu(II), Mn(II), Co(II), Ni(II), Ba(II), Ca(II), Pb(II), Hg(II) ) and Cd(II) metals on G. aparine peroxidase and catalase enzymes were also investigated. Metal solutions at 3 different concentrations, 0.5-1 and 5 mM, were prepared and their effects on the enzymes were measured. Inhibition rates of metals were determined by calculating their % residual activity of enzymes. The results showed that the peroxidase enzyme was inhibited by 47.18% at 0.5 mM concentration, 84.67 % at 1 mM concentration and 80 % at 5 mM Hg(II). It was measured that the catalase enzyme was inhibited by 40 % with Pb(II) at 0.5 mM concentration and 52 % by Cd(II) at 1 mM concentration. Additionally, the solvent effects on G. aparine peroxidase and catalase enzyme activities were tested. Acetone,ethanol, DMSO, t-butanol and DMF were chosen as solvents. Solutions of 5 %, 10 %, 15 %, 20 %, 25 % and 30 % (v/v) of the solvents were incubated with POD and CAT enzymes. Activity measurement was performed at 420 nm for peroxidase and at 240 nm for catalase. Comparison was made by calculating the percentage of remaining activity of each enzyme. When the solvent effect on the peroxidase enzyme was examined, it was determined that the addition of 30 % ethanol was the solvent that inhibited the enzyme activity the most. When the solvent effect on the cayalase enzyme was examined, it was determined that the 30 % DMF addition was the solvent that inhibited the enzyme activity the most. In both enzymes, 30% was determined as the highest inhibitory value for all solvents. In addition to all tests, salt tolerance test was applied to the peroxidase and catalase enzymes. For this test, NaCl solutions at 1, 2, 3, 4, 5 mM concentrations were prepared. First, the enzymes were incubated separately for 2 hours with the salt solutions at the specified concentrations, then, the activity of the enzymes was determined. According to the results obtained, it was observed that the activities of the enzymes decreased with increasing salt concentration. It was determined that G. aparine peroxidase showed more salt tolerance than G. aparine catalase enzyme. For the ionic strength effect of the peroxidase and catalase enzymes, 1, 2, 3, 4 and 5 mM concentration NaCl solutions were prepared and the enzyme activity measurements were performed. It was observed that the G. aparine peroxidase enzyme showed the highest activity with 3 mM NaCl solution. But, the enzyme was inhibited by 42% at 1 mM salt concentration. It was observed that the catalase enzyme had the highest activity at 3mM salt concentration, but its activity decreased to 40 % at 5 mM salt concentration.