İskorpit Balığı Solungaç Dokusundan Glutatyon Redüktaz Enziminin Saflaştırılması ve Metal İnhibisyonunun İncelenmesi

Yıl 2023, Cilt: 38 Sayı: 1, 221 - 233, 28.02.2023

Kübra Işık , Ercan Soydan

https://doi.org/10.7161/omuanajas.1226399

Öz

Bu çalışmada iskorpit balığı solungaç dokusundan glutatyon redüktaz enzimi literatürde ilk kez kısmi olarak saflaştırılmış ve ağır metal iyonlarının enzim aktivitesi üzerindeki etkileri belirlenmiştir. Saflaştırma işlemi homojenat hazırlanması, amonyum sülfat çöktürmesi ve diyaliz olarak üç aşamada gerçekleştirilmiştir. Çalışma sonucunda optimum pH 6.5, optimum substrat konsantrasyonu 2 mM NADPH ve optimum tampon 400 mM KH₂PO₄ olarak bulunmuştur. Kısmi saflaştırma sonrasında ağır metal iyonları olarak Ni⁺², Zn⁺² ve Cd⁺²’nin inhibisyon etkileri araştırılmış ve ağır metallerin IC50 değerleri sırasıyla 31 µM, 56 µM ve 74 µM olarak hesaplanmıştır. En güçlü inhibitörün çinko oldu tespit edilmiştir.

Anahtar Kelimeler

Enzim saflaştırılması, glutatyon redüktaz, iskorpit balığı, metal iyonları

Purification and Metal Inhibition of Glutathione Reductase Enzyme from Gill Tissue of Scorpionfish

Öz

In this study, glutathione reductase enzyme was partially purified from gill tissue of scorpion fish and the effects of heavy metal ions on enzyme activity were determined. The purification process was achieved in three steps as preparation of homogenate, ammonium sulfate precipitation and dialysis. As a result of the study, optimum pH 6.5, optimum substrate concentration 2 mM NADPH and optimum buffer 400 mM KH₂PO₄ were determined. After partial purification, the inhibition effects of Cd⁺², Ni⁺², Zn⁺² as heavy metal ions were investigated. The IC50 values of heavy metals were calculated as 74 µM, 31 µM, and 56 µM, respectively. The most potent inhibitor was determined to be zinc.

Anahtar Kelimeler

Enzyme purification, glutathione reductase, metal ion, scorpion fish

Kaynakça

• Açan, L., 1990. Koyun beyni glutatyon redüktazının saflaştırılması ve bazı özelliklerinin incelenmesi. Doktora Tezi. Ankara Üniversitesi Fen Bilimleri Enstitüsü, Ankara.
• Aksakal, E., Ekinci, D., Supuran, C.T., 2021. Dietary inclusion of royal jelly modulates gene expression and activity of oxidative stress enzymes in zebrafish. J Enzyme Inhib Med Chem, 36(1): 885-894. doi: 10.1080/14756366.2021.1900167.
• Akşiray, F., 1987. Türkiye deniz balıkları ve tayin anahtarı. Kardeşler basımevi, No:2, 811 s, İstanbul. Anderson, M.E., 1998. Glutatyon: biyosentez ve modülasyona genel bir bakış, Kimyasal-biyolojik etkileşimler, 111: 1-14.
• Bayir, A., Sirkecioglu, A.N., Haliloglu, H.I., Aksakal, E., Gunes, M., Aras N.M., 2011. Influence of season on antioxidant defense systems of Silurus glanis Linnaeus (Siluridae) and Barbus capito capito Güldenstädt (Cyprinidae), Fresen Environ Bull, 20(1): 3-11.
• Beutler, E., 1963. Effect of flavin compound on Glutathione Reductase Activity, In vivo and in vitro studies. Bewley, R.J.F., 1980. Effect of heavy metal pollution on oak leaf microorganism, App. Enviro. Microbiol, 40: 1053-1059.
• Carlberg, I., Mannervik, B., 1975. Purification and characterization of the flavoenzyme glutathione reductase from rat liver, J Biol Chem, 250:5475–5480.
• Carlberg, I., Mannervik, B., 1985. Glutathione reductase. In Methods in Enzymology, Vol. 113, pp. 484-490.
• Chang, S. S., Peterson, R. J., and Ho, C. T. (1978). Chemical reactions involved in the deep‐fat frying of foods1. Journal of the American Oil Chemists' Society, 55(10), 718-727.
• Çakmak, R., Durdagi, S., Ekinci, D., Şentürk, M., Topal, G., 2011. Design, synthesis and biological evaluation of novel nitroaromatic compounds as potent glutathione reductase inhibitors, Bioorganic and medicinal chemistry letters, 21(18): 5398- 5402.
• Demirdağ, R., Yerlikaya, E., Aksakal, E., Küfrevioğlu, Ö.I., Ekinci, D., 2012. Influence of pesticides on the pH regulatory enzyme, carbonic anhydrase, from European Seabass liver and bovine erythrocytes, Environ Toxicol Pharmacol, 34(2): 218-222. doi: 10.1016/j.etap.2012.04.007.
• Durdagi, S., Senturk, M., Guney, M., Ekinci, D., Aksoydan, B., Erol, I., Kantarcioglu, I., Cavdar, H., 2016. Design of novel uracil derivatives as inhibitors of carbonic anhydrase I and II, acetylcholinesterase, butyrylcholinesterase, and glutathione reductase using in silico, synthesis and in vitro studies, FEBS Journal, 283:106.
• Ekinci, D., Beydemir, S., 2009. Effect of some analgesics on paraoxonase-1 purified from human serum, J Enzyme Inhib Med Chem, 24(4): 1034-1039.
• Ekinci, D., Ceyhun, S. B., Şentürk, M., Erdem, D., Küfrevioğlu, Ö. İ., and Supuran, C. T. (2011). Characterization and anions inhibition studies of an α-carbonic anhydrase from the teleost fish Dicentrarchus labrax. Bioorganic & medicinal chemistry, 19(2), 744-748.
• Ekinci, D., Şentürk, M., 2013. Assesment of metal inhibition of antioxidant enzyme glutathione reductase from rainbow trout liver, Journal of Enzyme Inhibition and Medicinal Chemistry, 28 (1): 11-15.
• 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, Erzurum.
• Fidan, I., Salmas, R.E., Arslan, M., Senturk, M., Durdagi, S., Ekinci, D., Senturk, E., Cosgun, S., Supuran, C.T, 2015. Carbonic anhydrase inhibitors: design, synthesis, kinetic, docking and molecular Dynamics analysis of novel glycine and phenylalanine sulphonamide derivatives, Bioorg Med Chem, 23:7353.
• Garcia-Alfonso, C., Martinez-Galisteo, E., Llobell, A., Barcena, J.A., Lopez-Barea, J., 1993. Horse-liver glutathione reductase: purification and characterization, Int J Biochem, 25(1): 61-68.
• Hisar, O., Erdogan, O., Aksakal, E., Hisar, S.A., 2006. Authentication of fish species using a simple PCR-RFLP method, Isr J Aquac – Bamidgeh, 58 (1): 62-65.
• Isık, S., Vullo, D., Durdagi, S., Ekinci, D., Senturk, M., Cetin, A., Senturk, E., Supuran, C.T., 2015. Interaction of carbonic anhydrase isozymes I, II and IX with some pyridine and phenol hydrazinecarbothioamide derivatives, Bioorg Med Chem Lett, 25: 5636.
• Karagözoğlu, Y., Çiftçi, M., 2017. Bazı ağır metal iyonlarının tavuk böbreğinden saflaştırılan glutatyon redüktaz enzimi üzerine in vitro etkilerinin araştırılması, Türk Doğa ve Fen Dergisi, Vol. 6, No. 1
• Keha, E.E., Küfrevioğlu, Ö.İ., 2012. Biyokimya, Aktif yayınları, Erzurum.
• Keleştemur, T.G., 2012. Phisiological effects created on fish of hypoxic waters (in Turkish with English abstract), Turkish Journal of Scientific Reviews, 5(1): 87-90.
• Kocaoglu, E., Talaz, O., Cavdar, H., Senturk, M., Supuran, C.T., Ekinci, D., 2019. Determination of the inhibitory effects of N-methylpyrrole derivatives on glutathione reductase enzyme, J Enzyme Inhib Med Chem, 34:51.
• Levy, H.R., 1979. Glucose-6-phosphate dehydrogenase, Adv.Enzymol, 48, 97-192.
• Liebman, J.F., Greenberg, A., VCH Publishers, 1988. New York.
• Mate, S., J.M., 2000. Effects of antioxidant enzymes in the molecular control of reactive oxygen species toxicology, Toxicology, 153, 83–104.
• Meister, A., Anderson, M.E., 1983. Glutathione, Annu. Rev. Biochem, 52, 711–760.
• Mitchell, J.B., Russo, A., 1987. Br. J. Cancer, 8:96
• Mullineaux, P.M., 1997. Glutathione reductase: regulation and role in oxidative stress, Oxidative stress and the molecular biology of antioxidant defence, 667-713.
• O 'Hara, J., 1973. Cadmium uptake by fiddler crabs exposed to temperature and salinitystress, J. Fish. Res. Board Can, 30: 846–848.
• Ogus, I.H., Ozer, N., 1998. Purification of NADPH-free glutathione disulfide reductase from human erythrocytes, Protein Expr Purif, 13(1): 41-44. doi:10.1006/prep.1997.0865.
• Öğüs, H., Özer, N., 1991. Human jejunal glutathione reductase: purification and evaluation of the NADPH-and glutathione-induced changes in redox state, Biochemical medicine and metabolic biology, 45(1): 65-73
• Sen, S., Chakraborty, R., Sridhar, C., Reddy, Y.S.R., De, B., 2010. Free radicals, antioxidants, diseases and phytomedicines, Current status and future prospect, International Journal of Pharmaceutical Sciences and Research, 3(1): 91-100.
• Senturk, M., Talaz, O., Ekinci, D., Cavdar, H., Kufrevioglu, O.I., 2009. In vitro inhibition of human erythrocyte glutathione reductase by some new organic nitrates, Bioorganic and Medicinal Chemistry Letters, 19:3661.
• Siktar, E., Ekinci, D., Siktar, E., Beydemir, S., Gulcin, I., Gunay, M., 2011. Protective role of L-carnitine supplementation against exhaustive exercise induced oxidative stress in rats, Eur J Pharmacol, 668(3): 407-413. doi: 10.1016/j.ejphar.
• Tandogan, B., Ulusu, N.N., 2010. Purification and kinetics of bovine kidney cortex glutathione reductase, Protein Pept Lett, 17(5): 667-674.
• Tas, M., Senturk, E., Ekinci, D., Demirdag, R., Comakli, V., Bayram, M., Akyuz, M., Senturk, M., Supuran, C.T., 2019. Comparison of blood carbonic anhydrase activity of athletes performing interval and continuous running exercise at high altitude, J Enzyme Inhib Med Chem, 34:219.
• 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, Comparative Biochemistry and Physiology C-Toxicology & Pharmacology, 148(2): 117-121. doi: 10.1016/j.cbpc.2008.04.005.
• Toribio, F., Martinet, L.E., Pascual, P., Lopez, B.J., 1996. Methods for purification of glutathione peroxidase and related enzymes, Journal of Chromatography B, 684: 77-97.
• Townsend, D.M., Tew, K.D., Tapiero, H., 2003. The importance of glutathione in human disease, Biomed Pharmacother, 57(3-4): 145- 155.
• Ulusu, G., Erat, M., Ciftci, M., Sakiroglu, H.,Bakan, E., 2005. Purification and characterization of glutathione reductase from sheep liver, Turkish Journal of Veterinary and Animal Sciences, 29(5): 1109-1117.
• Valavanidis, A., Vlahogianni, T., Dassenakis, M., Scoullos, M., 2006. Molecular biomarkers of oxidative stress in aquatic organisms in relation to toxic environmental Pollutants, Ecotoxicol. Environ, 64: 178–189. doi: 10.1016/j.ecoenv.2005.03.013.
• Willmore, W.G., Storey, K.B., 2007. Purification and properties of glutathione reductase from liver of the anoxia-tolerant turtle, Trachemys scripta elegans. Molecular and Cellular Biochemistry, 297(1-2): 139-149.
• Wu, G., Fang, Y.Z., Yang, S., Lupton, J.R., Turner, N.D., 2004. Glutathione metabolism and its implications for health, The Journal of nutrition,134(3): 489-492.