Purifcation and Characterization of Glutathion Reductase Enzyme From Japanese Quail (Coturnix coturnix japanica) Erythrocytes

Abstract

Glutathione (γ-L-glutamyl-L-cysteinyl-glycine) is a low molecular weight thiol which protects
organism from harmful effect of oxidized molecules in cell. Glutathione reductase (GR) is a key enzyme involved
in the glutathione metabolism. In the present study GR enzyme has been purifed and characterized from Japanese
quail erythrocytes. The purifcation proses consisted of two steps, which include preparation of hemolysate, and
2’,5’-ADP Sepharose 4B afnity chromatography. The japanese quail erythrocytes GR enzyme had specifc activity
of 33.75 EU m g-1 proteins, with a yield of 46.2% and 1 028 purifcation fold. Enzyme purity and molecular weight
of subunits were determined by SDS-PAGE method as 78.7 kDa. Then, characteristics and kinetic properties of the
purifed enzyme was determined.

Keywords

• Characterization,erythrocyte,glutathion reductase,purifcation,quail

Glutatyon Redüktaz (GR) Enziminin Japon Bıldırcın (Coturnix coturnix japanica) Eritrositlerinden Saflaştırılması ve Karakterizasyonu

Abstract

Glutatyon (γ-L-glutamil-L-cysteinyl-glycine) organizmanın hücre içi okside moleküllerin zararlı
etkisinden korunmasında görevli düşük molekül ağırlıklı bir tiyoldür. Glutatyon redüktaz (GR) enzimi glutatyon
metabolizmasında yer alan temel enzimdir. Bu çalışma kapsamında GR enzimi japon bıldırcın eritrositlerinden
saflaştırılarak karakterize edilmiştir. Saflaştırma işlemi hemolizatın hazırlanması ve 2’,5’-ADP Sepharose 4B
afnite kromatografsi olmak üzere 2 basamakta gerçekleştirilmiştir. GR enzimi bıldırcın eritrositlerinden 33.75
EÜ m g-1 protein spesifk aktivite ile %46.2 verimle, 1 028 kat saflaştırılmıştır. Enzim saflığı SDS-PAGE ile
kontrol edilmiştir alt birimlerinin molekül ağırlığı aynı yöntemle 78.7 kDa olarak bulunmuştur. Daha sonra çalışma
kapsamında saflaştırılan enzimin karakteristik ve kinetik özellikleri tesbit edilmiştir.

Keywords

• Bıldırcın,eritrosit,glutatyon redüktaz,karakterizasyon,saflaştırma

References

1. Acan N.L., Tezcan E.F., 1989. Sheep brain glutathione reductase: purification and general properties. FEBS Letter, 250: (1) 72-74.
2. Açan L. 1990. Koyun beyni glutatyon redüktazının saflaştırılması ve bazı özelliklerinin incelenmesi. Doktora Tezi, Hacettepe Universitesi, Sağlık Bilimleri Enstitüsü, 100s
3. Adem S., Ciftci M. 2016. Purification and Biochemical Characterization of Glucose 6-Phosphate Dehydrogenase, 6-Phosphogluconate Dehydrogenase and Glutathione Reductase from Rat Lung and Inhibition Effects of Some Antibiotics. J Enzyme Inhib Med Chem. 31:(6) 1342-8.
4. Beütler E., 1969. Effect of flavin compound on Glutathione Reductase Activity; İn vivo and in vitro studies. J Clin Invest. 48: 1957–1966.
5. Boggaram V., Brobjer K.L., Mannervik B., 1979. Purufication of glutathione reductase from porcine erytrocytes by the use of affinity chromatography on 2’,5’-ADP-Sepharose 4B and cristalization of the enzyme. Anal. Biochem., 98: 335-340.
6. Bohme C.C., Arscott D.L, Becker K., Schirmer H.R., Williams C.H., 2000. Kinetic Characterization of Glutathione Reductase from the Malarial Parasite Plasmodium Falciparum. J. Biol. Chem. 275 (48): 37317–23.
7. Bradford M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72(1–2):248–254.
8. Carlberg I., Mannervik B., 1981. Purification and characterization of glutathione reductase from calf liver. Glutathione reductase assays. Methods in Enzymology, 113: 484-495.

9. Connell J. P., Mullet J. E., 1986. Pea Chloroplast Glutathione Reductase: Purification and Characterization. Plant Physiology. 82 :(2) 351–56.
10. Deepali P., Supriya K., Neeta B., Meena K., Aditi M., Yashwant I., Varsha D., 2013. Antioxidant Defence System, Or. Maxillofacial Pathol. J. 4: (1), 309-315.
11. Douglas K.T., 1987. Mechanizm of glutathione-dependent enzymes., Meister, A.,(ed).Avdan. Enzymol., John Wiley and Sons ınc., New York. 59: 103-167.
12. Erat M., 2002. İnsan ve sığır eritrosit glutatyon redüktaz enziminin saflaştırılması, bazı ilaç ve kimyasal maddelerin inhibisyon veya aktivasyon etkilerinin araştırılması. Doktora tezi, Atatürk Üniversitesi Fen Bilimleri Enstitüsü, 154s
13. Erat M., Sakiroglu H., Çiftci M., 2003. Purification and Characterization of Glutathione Reductase from Bovine Erythrocytes. Prep. Biochem. and Biotech. 33 (May 2014): 283–300.
14. Genestra M., 2007. Oxyl Radicals, Redox-Sensitive Signalling Cascades and Antioxidants. Cellular Signalling 19 : (9) 1807–19.
15. Harding J.J., 1973. Affinity Chromatography in the Purification of Glutathione Reductase. Journal of Chromatography 77: 191-99.
16. Hunaiti A.A., Soud M., 2000. Effect of lead concentration on the level of glutathione, glutathione S-transferase, reductase and peroxidase in human blood. Sci Total Environ. 29: 45-50.
17. Jiang F., Hellmans U., Stroga E., Bergman B., Mannervik B., 1995. Cloning, sequencing and regulation of the glutathione reductase gene from the cyanobacterium Anabaena PCC 7120. J. Biol. Chem., 270: (39), 22882-22889.
18. Kanzok S.M., Fechner A., Bauer H., Ulschmid J.K., Müller H.M., Botella-Munoz J., Schneuwly S., Schirmer R., Becker K., 2001. Substitution of the thioredoxin system for glutathione reductase in Drosophila melanogaster. Science. 26: 291(5504), 643-646.
19. Keha E., Küfrevioğlu Ö.İ., 2010. Biyokimya, Aktif Yayınevi, Erzurum, 653s
20. Knapen M.F., Zusterzeel, P.L., Peters, W.H., Steegers, E.A., 1999. Glutathione and glutathione-related enzymes in reproduction, Eur. J. Obstet. Gynecol. Reprod. Biol., 82: 171-184.
21. Laemmli U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259):680–685.
22. Lamotte F., Vianey-Liuaud N., Duviau M.P., Kobrehel K., 2000. Glutathione reductase in wheat grain. 1. Isolation and characterization. J. Agric. Food Chem. 48: 4978-4983.
23. Lineweaver H., Burk D., 1934. The determination of enzyme dissocation constants, J. Am. Chem. Soc. 57: 685.
24. Mannervik B., Jacobbson K., Boggaram V., 1976. Purification of Glutathione Reductase from Erythrocytes by the Use of Affinity Chromatography on 2’, 5’-ADP-Sepharose 4-B. FEBS Letters 66 (2): 221–24.
25. McCallum M. J., Barrett, J., 1995. Purification and properties of glutathione reductase from the cestode Moniezia expanse. Int. J. Biochem. Cell. Biol., 27: 393-401.
26. Mullineaux P., Enard C., Hellens R., Creissen G., 1996. Characterization of glutathione reductase gene and ist genetic locus from pea (Pisum sativum L.). Planta, 200: 186-194.
27. Patel M.P., Marcinkeviciene J., Blanchard J.S., 1998. Enterococus faecalis glutathione reductase: Purification, characterization and expression under normal and hyperbaric O2 conditions. FEMS Microbiology Letters, 166: 155-163.
28. Taşer P., Çiftci M., 2012. Purification and characterization of glutathione reductase from turkey liver. Turk. J. Vet. Anim. Sci, 36: 546-553.
29. Taher S.S.M., 2017. Purification And Characterization of Glutathione Reductase From Japanese Quail (Coturnix coturnix japonica) Liver, Master Thesis, Bingöl Universty institute of science, 78p.
30. Tekman B., Ozdemir H., Senturk M., Ciftci M., 2008. Purification and characterization of glutathione reductase from rainbow trout (Oncorhynchus mykiss) liver and inhibition effects of metal ions on enzyme activity.. Comp Biochem. Physiol. C Toxicol. Pharmacol. 148: 117-121.
31. Temel Y., Kufrevioglu O.I., Ciftci M., 2017. Investigation of the effects of purification and characterization of turkey (Meleagris gallopavo) liver mitochondrial thioredoxin reductase enzyme and some metal ions on enzyme activity. Turk. J. Chem 41: 48-60.
32. Toribio F., Martinet L.E., Pascual P., Lopez B.J., 1996. .Methods for purification of glutathione peroxidase and related enzymes, J. Chromatog. B. 684: 77-97.
33. Ulusu G., Erat M., Çiftci M., Şakiroğlu H., Bakan E., 2005. Purification and characterization of glutathione reductase from sheep liver. Turk J. Vet. Anim Sci. 29: 1109-1117.
34. Ulusu N.N., Tandoğan B., 2007. Purification and kinetic properties of glutathione reductase from bovin liver. Mol Cell Biochem, 303: 45-51.
35. Valko M.D., Leibfritz J., Moncol M.T., Cronin M., Mazur M., Telser J., 2007. Free Radicals and Antioxidants in Normal Physiological Functions and Human Disease. Int.J Biochem Cell Biol. 39 (1): 44–84.
36. Worthington D.J., Rosemeyer M.A., 1976. Glutathione Reductase from Human Erythrocytes. Catalytic Properties and Aggregation. Eur. J. Biochem. / FEBS, 67 (1): 231–38.