The key objective of this study was to investigate the effect of germination on biochemical and enzymatic antioxidant activities of Trifolium alexandrinum seeds. The T. alexandrinum was chosen for GST purification and characterization due to its highest GST activity and antioxidant capacity, beside its economical importance. Seeds of T. alexandrinum were germinated for 6 days. Changes in glutathione S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glutathione peroxidase (GPx) in germinating and dormant seeds of T. alexandrinum were evaluated. Also the changes in the total phenolic and flavonoid contents as well as the antioxidant capacities were monitored for 6 days of germination using spectrophotometeric methods. Simple reproducible procedures for the purification of T. alexandrinum GST from both dormant and 6 days germinated seeds were established using DEAE-Sepharose and Sephadex G-100 columns chromatography. The results showed the presence of three isoenzymes for both dormant and germinated seeds designated as GST1, GST2 and GST3. The major GST of the dormant seed was GST2 while for the germinated one was GST3. Dormant GST2 was expressed as a heterodimer with molecular weight of 27.5 and 28.5 KDa while germinated GST3 was expressed as a homodimer with molecular weight of 27 KDa. Characterization of both dormant GST1 & GST2 and germinated GST2 and GST3 including optimum pH, kinetic parameters [kmGSH of 1.17±0.39, 1.22±0.13, 0.84±0.19, 0.96±0.24mM, respectively and kmCDNB of 0.65±0.095, 0.57±0.11, 0.59±0.14, 1.02±0.18 mM, respectively]. The substrate selectivity using different electrophilic compounds and inhibitor effects were carried out. In opposite to other isoenzymes, germinated GST3 exhibited enzymatic activity towards ethacrynic acid with specific activity of 0.073±0.005 µmol/min/mg protein while cibacron blue was the most potent inhibitor for these isoenzymes.
| Published in | American Journal of Life Sciences (Volume 3, Issue 4) |
| DOI | 10.11648/j.ajls.20150304.12 |
| Page(s) | 257-267 |
| Creative Commons |
This is an Open Access article, distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium or format, provided the original work is properly cited. |
| Copyright |
Copyright © The Author(s), 2015. Published by Science Publishing Group |
Antioxidant Enzymes, Inhibition, Kinetic Parameters, Seed Germination, Trifolium alexandrinum
| [1] | Ma ZL and Gao KS. Spiral breakage and photoinhibition of Arthrospira platensis (Cyanophyta) caused by accumulation of reactive oxygen species under solar radiation. Environ Exp Bot 2010; 68: 208-13. |
| [2] | Ribeiro DA and Assis GF. Expression of placental glutathione S-transferase in rat tongue mucosa exposed to cigarette smoke. J Mol Histol. 2008; 39:115-9. |
| [3] | Edwards R, Dixon DP, Walbot V. Plant glutathione S-transferases: enzymes with multiple functions in sickness and in health. Trends Plant Sci. 2000; 5:193- 8. |
| [4] | Kampranis SC, Damianova R, Atallah M, Toby G, Kondi G, Tsichlis PN, Makris AM. A novel plant glutathione S-transferase/peroxidase suppresses Bax Lethality in yeast. J Biol Chem. 2000; 38: 29207 - 16. |
| [5] | Cummins I, Wortley DJ, Sabbadin F, He Z, Coxon CR, Straker HE, et al. Key role for a glutathione transferase in multiple-herbicide resistance in grass weeds. PNAS. 2015; 110: 5812-17. |
| [6] | Fulkerson WJ, Neal JS, Clark CF, Horadagoda A, Nandra KS, Barchia I. Nutritive value of forage species grown in the warm temperate climate of Australia for dairy cows: grasses and legumes. Livest Sci. 2006; 107: 253- 64. |
| [7] | Mohamed KM, Hassanean HA, Ohtani K, Kasai R, Yamasaki K. Chalcanol glucosides from seeds of Trifolium alexandrinum. Phytochem. 2000; 53: 401- 4. |
| [8] | Shah AS, Ahmed M, Alkreathy HM, Khan MR, Khan RA, Khan S. Phytochemical screening and protective effects of Trifolium alexandrinum (L.) against free radical-induced stress in rats. Food Sci. & Nutri. 2014; 2: 751- 7. |
| [9] | Butnariu M. An analysis of Sorghum halepense behavior in presence of tropane alkaloids from Datura stramonium extracts. Chem Cent J. 2012; 6: 1-7. |
| [10] | Fransisca L, Park HK, Feng H. E. coli o157: H7 population reduction from alfalfa seeds with malic acid and thiamine dilauryl sulfate and quality evaluation of the resulting sprouts. Food Sci J. 2012; 77: 121– 6. |
| [11] | Gaunii A, Pribac G, Grozea L, Gaitin D, Samfira L. Design of optimal solvent for extraction of bio-active ingredients from six varieties of Medicago sativa. Chem. Central J..2012; 6: 123-30. |
| [12] | Lopez-Amoros ML, Hernandez T, Estrella I. Effect of germination on legume phenolic compounds and their antioxidant activity. J Food Comp Anal. 2006; 19: 277- 83. |
| [13] | Cadenas E. Basic mechanisms of antioxidant activity. Biofactors. 1997; 6: 391– 7. |
| [14] | Valko M, Leibfritz D, Moncol J, Cronin M T D, Mazur M, Telser J. Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol. 2007; 39: 44–8. |
| [15] | Singleton VL and Rossi JA. Colorimetry of total phenolics with phosphomolybdic-phospho-tungstic acid reagents. Am j Enol Viticult. 1965; 16: 144- 58. |
| [16] | Woisky R, Salatino A. Analysis of propolis, some parameters and procedure for chemical quality control. J Agricul Res. 1998; 37: 99-105. |
| [17] | Blois M S. Antioxidant determination by the use of stable free radical. Nature. 1985; 181:1199- 200. |
| [18] | Oberg B and Philipson L. Gel filtration of nucleic acids on sphere-condenesd agarose. Arch Biochem Biophys. 1967; 119: 504- 9. |
| [19] | Bradford M M. A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principal of protein-dye-binding. Anal Biochem.1976; 72: 248- 54. |
| [20] | Laemmli U K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970; 227: 680- 5. |
| [21] | Habig W H, Pabst M J, Jakoby W B. Glutathione S-Transferase. J Biol Chem.1974; 249: 7130- 9. |
| [22] | Lawrence R A and Burk R F. Glutathione peroxidase activity in selenium-deficient rat liver. Biochem Biophys Res Commun. 1976; 71: 952- 8. |
| [23] | Ivarsson Y, Mackey AJ, Edalat M, Pearson WR, Mannervik B. Identification of residues in glutathione transferase capable of driving functional diverstification in evolution: a novel approach to protein redesign. J Biol Chem. 2003; 278: 8733- 8. |
| [24] | Zanetti G. Rabbit liver glutathione reductase, purification and properties. Arch Biochem Biophys. 1979; 198: 241- 6. |
| [25] | Weinhold L C, Ahmad S, Pardini R S. Insect glutathione S-transferase: A predictor of allelochemical and oxidative stress. Comp Biochem Physiol. 1990; 95 B: 355- 63. |
| [26] | Aebi H. Catalase in vitro. Methods in Enzymol. 1984; 105:121- 6. |
| [27] | Nishikimi M, Roa N A, Yogi K. Measurement of superoxide dismutase. Biochem Biophys Res Commun. 1972; 46: 849- 54. |
| [28] | Yalcin S, Jensson H, Mannervik B. A set of inhibitors for discrimination between the basic isozymes of glutathione transferase in rat liver. Biochem Biophys Res Commun. 1983; 114: 829- 34. |
| [29] | Koldziejczyk-Czepas J, Nowak P, Kowalska I, Stochmal A. Biological activity of clovers-free radical scavenging ability and antioxidant action of six Trifolium species. Pharmaceu Biol. 2014; 52: 1308- 14. |
| [30] | Oleszek W and Stochmal A. Triterpene saponins and flavonoids in the seeds of Trifolium species. Phytochem. 2002; 61:165–70. |
| [31] | Sabudak T and Guler N. Trifolium L. A review on its phytochemical and pharmacological profile. Phytother Res.2009; 23: 439- 46. |
| [32] | Vadivel V, Stuetz W, Scherbaum V, Biesalski K H. Total free phenolic content and health relevant functionality of Indian wild legume grains: Effect of indigenous processing methods. J Food Comp Anal. 2011; 24: 935- 43. |
| [33] | Lin P Y and Lai H M. Bioactive compounds in legumes and their germinated products. J Agricul Food Chem. 2006; 54: 3807- 14. |
| [34] | Cevallos-Casals B A and Cisneros-Zevallos L. Impact of germination on phenolic content and antioxidant activity of 13 edible seed species. Food Chem. 2010; 119: 1485- 90. |
| [35] | Scandalios J. Molecular genetics of superoxide dismutases in plants. In: Scandalios J, editor. Oxidative stress and the molecular biology of antioxidant defenses. New York: Cold Spring Harbor Laboratory Press; 1997; pp: 527– 68. |
| [36] | Bailly C, Leymarie J, Lehner A, Rousseau C D, Corbineau F. Catalase activity and expression in de¬veloping sunflower seeds as related to drying. J. Exp. Bot. 2004 ; 55: 475- 83. |
| [37] | Lehner A, Mamadou N, Poels P, Come D, Bailly C, Corbineau F. Changes in soluble carbohydrates, lipid peroxidation and antioxidant enzyme activities in the embryo during ageing in wheat grains. J. Cereal Sci. 2008; 47: 555- 65. |
| [38] | Randhir R., Lin Y T, Shetty K. Stimulation and phenolics, antioxidant and antimicrobial activities in dark germinated mung bean sprouts in response to peptide and phytochemical elicitors. Process Biochem. 2004; 39: 637- 47. |
| [39] | Rohman M M, Suzuki T, Fujita M. Identification of a glutathione S-transferase inhibitor in onion bulb (Allium cepa L.). Aust J Crop Sci. 2009; 3: 28-36. |
| [40] | Gronwald J W, Plaisance KL. Isolation and characterization of GST isozymes from sorghum. Plant Physiol. 1998; 117: 877- 92 |
| [41] | Buono D D, Scarponi L, Espen L. Glutathione S-transferases in Festuca arundinacea: Identification, characterization and inducibility by safener benoxacor. Phytochem. 2007; 68: 2614- 24. |
| [42] | Cho H Y, Lee H J, Kong K H. A Phi class glutathione S-transferase from Oryza sativa (OsGSTF5): molecular cloning, expression and biochemical characteristics. Biochem. Mol. Biol. 2007; 40: 511-516. |
| [43] | Park H-J, Cho H-Y, Kong K-H. Purification and biochemical properties of glutathione S-transferase from Lactuca sativa. J. Biochem Mol Biol. 2005; 38: 232-237. |
| [44] | Cummins I, O’Hagan D, Jablonkai I, Cole D J, Hehn A, Werck-Reichhart D, Edwards R,, Cloning R. Characterization and regulation of a family of Phi class glutathione transferases from wheat. Plant Mol Biol. 2003; 52: 591-603. |
| [45] | Benekos K, Kissoudis C, Nianiou-Obeidat I, Labrou N, Madesis P, Kalamaki M, et al. Overexpression of a specific soybean GmGSTU4 isoenzyme improves diphenyl ether and chloroacetanilide herbicide tolerance of transgenic tobacco plants. J Biotechnol.. 2010; 150: 195-201. |
| [46] | Gyamfi M A, Aniya Y, Ohtani I I, Shinno E. Inhibition of glutathione S-transferase by thonningianin A, isolated from the African medicinal herb, Thonningia sanguinea in vitro. Food Chem Toxicol. 2004; 42: 1401- 8. |
| [47] | Jo H J, Lee J J, Kong K H. A plant-specific tau class glutathione S-transferase from Oryza sativa with very high activity against 1-chloro-2,4-dinitrobenzene and chloroacetanilide herbicides. Pest Biochem Physiol. 2011; 101: 265- 9. |
APA Style
Ragaa Reda Hamed, Ehab Mostafa Mohamed Ali, Abeer Shokeer, Rasha Awni Guneidy, Abdul Aziz Mohamad Gad. (2015). Biochemical Changes of Glutathione S-Transferase Activity During the Germination of Trifolium alexandrinum. American Journal of Life Sciences, 3(4), 257-267. https://doi.org/10.11648/j.ajls.20150304.12
ACS Style
Ragaa Reda Hamed; Ehab Mostafa Mohamed Ali; Abeer Shokeer; Rasha Awni Guneidy; Abdul Aziz Mohamad Gad. Biochemical Changes of Glutathione S-Transferase Activity During the Germination of Trifolium alexandrinum. Am. J. Life Sci. 2015, 3(4), 257-267. doi: 10.11648/j.ajls.20150304.12
AMA Style
Ragaa Reda Hamed, Ehab Mostafa Mohamed Ali, Abeer Shokeer, Rasha Awni Guneidy, Abdul Aziz Mohamad Gad. Biochemical Changes of Glutathione S-Transferase Activity During the Germination of Trifolium alexandrinum. Am J Life Sci. 2015;3(4):257-267. doi: 10.11648/j.ajls.20150304.12
Share This Article
Twitter
Linked In
Facebook
Copy
Download@article{10.11648/j.ajls.20150304.12,
author = {Ragaa Reda Hamed and Ehab Mostafa Mohamed Ali and Abeer Shokeer and Rasha Awni Guneidy and Abdul Aziz Mohamad Gad},
title = {Biochemical Changes of Glutathione S-Transferase Activity During the Germination of Trifolium alexandrinum},
journal = {American Journal of Life Sciences},
volume = {3},
number = {4},
pages = {257-267},
doi = {10.11648/j.ajls.20150304.12},
url = {https://doi.org/10.11648/j.ajls.20150304.12},
eprint = {https://article.sciencepublishinggroup.com/pdf/10.11648.j.ajls.20150304.12},
abstract = {The key objective of this study was to investigate the effect of germination on biochemical and enzymatic antioxidant activities of Trifolium alexandrinum seeds. The T. alexandrinum was chosen for GST purification and characterization due to its highest GST activity and antioxidant capacity, beside its economical importance. Seeds of T. alexandrinum were germinated for 6 days. Changes in glutathione S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glutathione peroxidase (GPx) in germinating and dormant seeds of T. alexandrinum were evaluated. Also the changes in the total phenolic and flavonoid contents as well as the antioxidant capacities were monitored for 6 days of germination using spectrophotometeric methods. Simple reproducible procedures for the purification of T. alexandrinum GST from both dormant and 6 days germinated seeds were established using DEAE-Sepharose and Sephadex G-100 columns chromatography. The results showed the presence of three isoenzymes for both dormant and germinated seeds designated as GST1, GST2 and GST3. The major GST of the dormant seed was GST2 while for the germinated one was GST3. Dormant GST2 was expressed as a heterodimer with molecular weight of 27.5 and 28.5 KDa while germinated GST3 was expressed as a homodimer with molecular weight of 27 KDa. Characterization of both dormant GST1 & GST2 and germinated GST2 and GST3 including optimum pH, kinetic parameters [kmGSH of 1.17±0.39, 1.22±0.13, 0.84±0.19, 0.96±0.24mM, respectively and kmCDNB of 0.65±0.095, 0.57±0.11, 0.59±0.14, 1.02±0.18 mM, respectively]. The substrate selectivity using different electrophilic compounds and inhibitor effects were carried out. In opposite to other isoenzymes, germinated GST3 exhibited enzymatic activity towards ethacrynic acid with specific activity of 0.073±0.005 µmol/min/mg protein while cibacron blue was the most potent inhibitor for these isoenzymes.},
year = {2015}
}
TY - JOUR T1 - Biochemical Changes of Glutathione S-Transferase Activity During the Germination of Trifolium alexandrinum AU - Ragaa Reda Hamed AU - Ehab Mostafa Mohamed Ali AU - Abeer Shokeer AU - Rasha Awni Guneidy AU - Abdul Aziz Mohamad Gad Y1 - 2015/06/19 PY - 2015 N1 - https://doi.org/10.11648/j.ajls.20150304.12 DO - 10.11648/j.ajls.20150304.12 T2 - American Journal of Life Sciences JF - American Journal of Life Sciences JO - American Journal of Life Sciences SP - 257 EP - 267 PB - Science Publishing Group SN - 2328-5737 UR - https://doi.org/10.11648/j.ajls.20150304.12 AB - The key objective of this study was to investigate the effect of germination on biochemical and enzymatic antioxidant activities of Trifolium alexandrinum seeds. The T. alexandrinum was chosen for GST purification and characterization due to its highest GST activity and antioxidant capacity, beside its economical importance. Seeds of T. alexandrinum were germinated for 6 days. Changes in glutathione S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glutathione peroxidase (GPx) in germinating and dormant seeds of T. alexandrinum were evaluated. Also the changes in the total phenolic and flavonoid contents as well as the antioxidant capacities were monitored for 6 days of germination using spectrophotometeric methods. Simple reproducible procedures for the purification of T. alexandrinum GST from both dormant and 6 days germinated seeds were established using DEAE-Sepharose and Sephadex G-100 columns chromatography. The results showed the presence of three isoenzymes for both dormant and germinated seeds designated as GST1, GST2 and GST3. The major GST of the dormant seed was GST2 while for the germinated one was GST3. Dormant GST2 was expressed as a heterodimer with molecular weight of 27.5 and 28.5 KDa while germinated GST3 was expressed as a homodimer with molecular weight of 27 KDa. Characterization of both dormant GST1 & GST2 and germinated GST2 and GST3 including optimum pH, kinetic parameters [kmGSH of 1.17±0.39, 1.22±0.13, 0.84±0.19, 0.96±0.24mM, respectively and kmCDNB of 0.65±0.095, 0.57±0.11, 0.59±0.14, 1.02±0.18 mM, respectively]. The substrate selectivity using different electrophilic compounds and inhibitor effects were carried out. In opposite to other isoenzymes, germinated GST3 exhibited enzymatic activity towards ethacrynic acid with specific activity of 0.073±0.005 µmol/min/mg protein while cibacron blue was the most potent inhibitor for these isoenzymes. VL - 3 IS - 4 ER -
Email: