The Effect of Safranin on Glutathione Reductase and Glucose 6-Phosphate Dehydrogenase Enzymes

Year 2011, Volume: 39 Issue: 2, 189 - 194, 01.04.2011

Müslüm Kuzu Murat Şentürk Mehmet Çiftci

Abstract

The aim of this study was to assess inhibitory effect of safranin on human erythrocyte glutathione reductase GR and glucose 6-phosphate dehydrogenase G6PD . For this purpose, human erythrocyte glutathione reductase was purified 2175-fold 31.35% yield using 2’,5’-ADP Sepharose 4B affinity gel and Sephadex G-200 gel filtration chromatography and erythrocyte glucose 6-phosphate dehydrogenase was purified 8681.3-fold 24.77% yield using 2’,5’-ADP Sepharose 4B affinity gel. Safranin exhibited inhibitory effects on GR and G6PD enzymes in vitro. The IC50 values of safranin were 1.15 mM for GR and 3.22 mM for G6PD, respectively, and the Ki constants were 2.63 ± 0.13 and 4.53 ± 0.17 mM, respectively. Safranin displayed noncompetitive inhibition for GR and G6PD.

Keywords

Safranin, glutathione reductase, glucose 6-phosphate dehydrogenase

Glutatyon Redüktaz ve Glukoz 6-Fosfat Dehidrogenaz Enzimleri Üzerine Safraninin Etkisi

Abstract

Bu çalışmanın amacı safraninin insan eritrosit glutayon redüktaz GR ve glukoz 6-fosfat dehidrogenaz G6PD üzerindeki inhibitör etkilerini değerlendirmektir. Bu amaçla, 2’,5’-ADP Sepharose 4B afinite jeli ve Sephadex G-200 jel filtrasyon kromatografisi kullanılarak insan eritrosit glutayon redüktaz 2175-kat %31.35 verim ve 2’,5’-ADP Sepharose 4B afinite jeli kullanılarak eritrosit glukoz 6-fosfat dehidrogenaz 8681.3-kat %24.77 verim ile saflaştırıldı Safranin in vitro GR ve G6PD inhibitör etkisi gösterdi. Safraninde IC50 değerleri, sırası ile GR için 1.15 mM ve G6PD için 3.22 mM, ve Ki sabitleri sırası ile 2.63 ± 0.13 ve 4.53 ± 0.17 mM oldu. Safranin GR ve G6PD için yarışmasız inhibisyon gösterdi.

Keywords

Safranin, glutatyon redüktaz, glukoz 6-fosfat dehidrogenaz

References

• 1. B. Tekman, H. Ozdemir, M. Senturk, M. Ciftci, Purification and Characterization of Glutathione Reductase from Rainbow Trout (Oncorhynchus mykiss) Liver and Inhibition Effects of Some Metal Ions on Enzyme Activity. Comp. Biochem. Physiol. C, 148 (2008) 117.
• 2. M. Poot, H. Teubert, P.S. Rabinovitch, T.J. Kavanagh, De novo synthesis of glutathione is required for both entry into and progression through the cell cycle. J. Cell. Physiol, 163 (1995) 555.
• 3. S.M. Deneke, B.L. Fanburg, Regulation of cellular glutathione. Am. J. Physiol, 257 (1989) 163.
• 4. R.H. Schirmer, R.L. Krauth-Siegel, G.E. Schulz, Glutathione reducase, New York, John Wiley and Sons Press, (1989) p 553.
• 5. W. Kuo, J. Lin, T.K. Tang, Human glucose-6-phosphate dehydrogenase (G6PD) gene transforms nih 3t3 cells and induces tumors in nude mice. Int. J. Cancer, 322 (2000) 857.
• 6. J.M. Mates, C. Perez-Gomez, I. Nunez De Castro, Antioxidant enzymes and human diseases. Clin. Biochem, 32 (1999) 595.
• 7. I. Ozmen, M. Ciftci, O.I. Kufrevioglu, M.A. Curuk, Investigation of the mutation points and effects of some drugs on glucose-6-phosphate dehydrogenasedeficient people in the Erzurum region. J. Enzyme Inhibit. Med. Chem, 19 (2004) 45.
• 8. B.N. Ames, M.K. Shigenaga, T.M. Hagen, Oxidants, antioxidants, and the degenerative diseases of aging. Proc. Natl. Acad. Sci. USA. 90 (1993) 7915.
• 9. J.M. McCord, Human disease, free radicas and the oxidant/antioxdant balance. Clin. Biochem, 26 (1993) 351.
• 10. R.R. Jenkins, A. Goldfarb, Introduction Oxidant Stress, Aging and Exercise. Med. Sci. Sports Exerc, 25 (1993) 210.
• 11. S.C. Bondy, J. Orozco, Effects of ethanol treatment upon sources of reactive oxygen species in brain and liver. Alcohol, 29 (1994) 375.
• 12. L. Rosenberg, Chemical basis for the histological use of safranin O in the study of articular cartilage. J. Bone Joint. Surg. Am, 53 (1971) 69.
• 13. I. Ozmen, O.I. Kufrevioglu, Effects of antiemetic drugs on glucose 6-phosphate dehydrogenase and some antioxidant enzymes. Pharmacol. Res, 50 (2004) 499.
• 14. A. Sahin, M. Senturk, M. Ciftci, E. Varoglu, O.I. Kufrevioglu, Effects of thallium-201 and radioactivity on human erythrocyte glucose 6-phosphate dehydrogenase activity. Nucl. Med. Biol, 37 (2010) 389.
• 15. M. Senturk, O.I. Kufrevioglu, M. Ciftci, Effects of some antibiotics on human erythrocyte glutathione reductase: An in vitro study. J. Enzyme Inhib. Med. Chem, 23 (2008) 144.
• 16. E. Beutler, G6PD deficiency. Blood, 81 (1994) 3613.
• 17. M.M. Bradford, A rapid and sensitive method for the quantition of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem, 72 (1976) 248.
• 18. D.K. Laemmli, Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature, 227 (1970) 680.
• 19. M. Senturk, O.I. Kufrevioglu, M. Ciftci, Effects of some analgesic and anestethic drugs on human erythrocyte glutathione reductase: an in vitro study. J. Enzyme Inhibit. Med. Chem, 24 (2009) 420.
• 20. H. Lineweaver, D. Burk, The determination of enzyme dissocation constants. J. Am. Chem. Soc, 57 (1934) 685.
• 21. M. Ciftci, O.I. Kufrevioglu, M. Gundogdu, I. Ozmen, Effects of some antibiotics on enzyme activity of glucose-6-phosphate dehydrogenase from human erythrocytes. Pharmacol. Res, 41 (2000) 109.
• 22. S.N. Savvides, P.A. Karplus, Kinetics and crystallographic analysis of human glutathione reductase in complex with a xanthene inhibitor. J. Biol. Chem, 271 (1996) 8101.
• 23. N. Cenas, H. Nivinskas, Z. Anusevicius, J. Sarlauskas, F. Lederer, E.S. Arner, Interactions of quinones with thioredoxin reductase: a challenge to the antioxidant role of the mammalian selenoprotein. J. Biol. Chem, 279 (2004) 2583.
• 24. W. Friebolin, B. Jannack, N. Wenzel, J. Furrer, T. Oeser, C.P. Sanchez, M. Lanzer, V. Yardley, K. Becker, E. Davioud-Charvet, Antimalarial dual drugs based on potent inhibitors of glutathione reductase from Plasmodium falciparum. J. Med. Chem, 51 (2008) 1260.
• 25. S. Bharath, M. Hsu, D. Kaur, S. Rajagopalan, J.K. Andersen, Glutathione, iron and Parkinson’s disease. Biochem. Pharmacol, 64 (2002) 1037.
• 26. U. Wullner, P.A. Loschmann, J.B. Schulz, A. Schmid, R. Dringen, F. Eblen, L. Turski, T. Klockgether, Glutathione depletion potentiates MPTP and MPP1 toxicity in nigral dopaminergic neurones. Neuroreport. 7 (1996) 921.
• 27. J.B. Schulz, J. Lindenau, J. Seyfried, J. Dichgans, Glutathione, oxidative stress and neurodegeneration. Eur. J. Biochem. 267 (2000) 4904.