Laboratory for Life science

Tehrān, Iran

Laboratory for Life science

Tehrān, Iran
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Arzi L.,University of Tehran | Keyhani J.,Laboratory for Life science | Keyhani E.,Laboratory for Life science
Acta Horticulturae | Year: 2010

Satureja hortensis, an annual herb belonging to the Lamiaceae family, has been used since Antiquity in the Mediterranean and Indo-Iranian regions as culinary herb as well as medicinal plant. Although the medicinal value of S. hortensis has been recognized for thousands of years by tradition, and recently by modern experimental procedures, information on the basic aspects of the physiology and enzymology of the plant is only scarcely available. In this study, flavocytochrome b2 activity was investigated in an extract obtained from S. hortensis leaves. Flavocytochrome b2, or L-(+)-lactate ferricytochrome c oxidoreductase (EC.1.1.2.3), is involved in respiration and energy production. It transfers electrons from lactate to cytochrome c (as a physiological electron acceptor) or to other electron acceptors such as potassium ferricyanide. Flavocytochrome b2 activity was measured in the extract by following spectrophotometrically, at 420 nm, the reduction of potassium ferricyanide in the presence of lactate and EDTA. PH activity profile exhibited one peak at 9.5 and a shoulder at 8.0. Kinetics parameters, determined at pH optima were as follows: a) with lactate as the varied substrate, apparent Km and Vmax were, respectively, 2.6 mM and 0.5 μM.min-1.mg prot-1 at pH 8.0, and 6 mM and 0.95 μM.min-1.mg prot-1 at pH 9.5; b) with potassium ferricyanide as the varied substrate, apparent Km and Vmax were, respectively, 23 μM and 0.3 μM.min-1.mg prot-1 at pH 8.0, and 40 μM and 0.65 μM.min-1.mg prot-1 at pH 9.5. Malate was a competitive inhibitor of the activity with an IC50 of 13 mM at pH 8.0 and 27 mM at pH 9.5. Electrophoresis of the extract in polyacrylamide gel under non-denaturing conditions revealed two bands after activity staining in the presence of lactate and tetrazolium blue. Data suggest that at least two isoenzymes of flavocytochrome b2 are present in S. hortensis leaves.


Keyhani J.,Laboratory for Life science | Keyhani E.,Laboratory for Life science
Acta Horticulturae | Year: 2010

Enzymes are of critical importance in maintaining a species and protecting it against various forms of environmental stresses. In particular, the formation of reactive oxygen species and ensuing oxidative stress is a constant menace for any organism. Plants are equipped with a battery of antioxidative stress enzymes, including peroxidases that fulfill a variety of functions while scavenging H2O2. In this study, peroxidase activities were identified in an extract obtained from Thymus citriodorus roots by monitoring spectrophotometrically the H2O2-mediated oxidation of either o-dianisidine (at 460 nm), guaiacol (at 470 nm) or ferulic acid (at 310 nm) (lignin peroxidase) in the presence of extract aliquots. Assays were performed at pH 4 for o-dianisidine, 6 for guaiacol and 5 for ferulic acid oxidation, using extinction coefficients of, respectively, 11.3, 26.6 and 8.68 mM-1.cm-1. With o-dianisidine as the reducing substrate, apparent Km, Vmax and catalytic efficiency were, respectively, 0.65 ±0.06 mM, 0.17 ±0.007 mM.min-1.mg prot-1 and 0.27 ±0.01 min-1.mg prot-1 for odianisidine and 0.5 ±0.05 mM, 0.255 ±0.005 mM.min -1.mg prot-1 and 0.5 min-1.mg prot-1 for H2O2; with guaiacol, the kinetics parameters were, respectively, 4.5 ±3 mM, 0.6 ±0.06 mM.min-1. mg prot-1 and 0.13 ±0.002 min-1.mg prot-1 for guaiacol and 0.9 ±0.2 mM, 0.52 ±0.04 mM.min-1.mg prot-1 and 0.58 ±0.04 min-1.mg prot-1 for H2O2; with ferulic acid, kinetics parameters were, respectively, 0.07 ±0.01 mM, 0.9 ±0.09 mM.min-1. mg prot-1 and 13 ±1 min-1.mg prot-1 for ferulic acid and 0.025 ±0.005 mM, 0.7 ±0.01 mM.min-1.mg prot-1 and 28 ±3 min-1.mg prot-1 for H2O2. The peroxidatic activities were sensitive to KCN, with IC50 of 0.12 ± 0.02 μM for o-dianisidine, 0.75 ±0.05 μM for guaiacol and 0.6 ± 0.05 μM for ferulic acid. Results point out the predominance of lignin peroxidase activity over o-dianisidine and guaiacol peroxidases.

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