Glutatyon S-Transferaz Enziminin Tavuk Karaciğerinden Saflaştırılması ve Karakterizasyonu

Yıl 2023, Cilt: 12 Sayı: 1, 117 - 124, 27.03.2023

Hakan Yılmaz , Mehmet Çiftci , Yusuf Temel

https://doi.org/10.46810/tdfd.1242764

Öz

Bu çalışmada glutatyon S-transferaz enzimi (GST; EC 2.5.1.18) tavuk karaciğerinden amonyum sülfat çöktürmesi ve glutatyon-agaroz afinite kromatografisinden yararlanılarak 8,35 EÜ/mL spesifik aktivitesine sahip olan enzim, %8 verimle 24,56 kat saflaştırıldı. Saflaştırılan enzimin saflığının kontrol edilmesi maksadıyla SDS-PAGE işlemi yapıldı ve tek bant elde edildi. Alt birimin molekül kütlesi yaklaşık olarak 30,9 kDa olarak hesaplandı. Ayrıca enzimin optimum pH değeri (Tris-HCl içinde 8,5); optimum iyonik şiddeti (Tris-HCl ile 150 mM); optimum sıcaklığı (70 oC); stabil pH değeri (Tris-HCl ile 8,5) tespit edildi. Enzimin GSH substratı için KM değeri 0,802 mM, Vmax değeri 1,833 EÜ/mL; CDNB için de KM değeri 3,6 mM ve Vmax değeri 2,829 EÜ/mL olarak hesaplandı.

Anahtar Kelimeler

Glutatyon S-transferaz, tavuk karaciğeri, saflaştırma., Glutathione S-transferase, chicken liver, purification.

Destekleyen Kurum

Bingöl Üniversitesi Araştırma Projeleri Koordinasyon Birimi

Proje Numarası

BAP-FEF-2022-001

Teşekkür

Desteklerinden dolayı Bingöl Üniversitesi Araştırma Projeleri Koordinasyon Birimine (BÜBAP) teşekkür ederiz.

Purification and Charcterızatıon Glutathione S-Transferase Enzyme From Chicken Liver

Öz

In this study, the glutathione S-transferase enzyme (GST; EC 2.5.1.18) was purified with 8.35 EU/mL specific activity, 24.56 times 8% yield, from chicken liver, using ammonium sulfate precipitation and glutathione-agarose affinity chromatography. In order to control the purity of the enzyme, SDS-PAGE was performed and a single band was obtained. The molecular mass of the subunit was calculated as approximately 30.9 kDa. In addition, the optimum pH value of the enzyme (8.5 in Tris-HCl); optimum ionic strength (150 mM with Tris-HCl); optimum temperature (70 oC); stable pH value (8.5 with Tris-HCl) was determined. The KM value for the GSH substrate of the enzyme was 0.802 mM, the Vmax value was 1.833 EU/mL; For CDNB, the KM value was calculated as 3.6 mM and the Vmax value was calculated as 2.829 EU/mL.

Anahtar Kelimeler

Glutathione S-transferase, chicken liver, purification.

Proje Numarası

BAP-FEF-2022-001

Kaynakça

• Temel Y, Ayna A, Hamdi Shafeeq I, Ciftci M. In vitro effects of some antibiotics on glucose-6-phosphate dehydrogenase from rat (Rattus norvegicus) erythrocyte. Drug Chem. Toxicol. 2020; 43(2): 219-223.
• Bayindir S, Ayna A, Temel Y, Ciftci M. The synthesis of new oxindoles as analogs of natural product 3, 3′-bis (indolyl) oxindole and in vitro evaluation of the enzyme activity of G6PD and 6PGD. Turk. J. Chem. 2018;42(2): 332-345.
• Bayindir S, Temel Y, Ayna A, Ciftci M. The synthesis of N‐benzoylindoles as inhibitors of rat erythrocyte glucose‐6‐phosphate dehydrogenase and 6‐phosphogluconate dehydrogenase. J. Biochem. Mol. Toxicol. 2018;32(9): e22193.
• Autrup H. Genetic polymorphism in human xenobiotica matebolizing enzymes as susceptibility factors in toxic response. Mutat. Res. 2000;464: 65-76.
• Lee WM. Drug-induced hepatotoxicity. N Engl J Med. 2003;349:474-485.
• Sheehan D, Meade G, Foley VM, Dowd CA. Structure, function and evolution of glutathione transferases: implications for classification of nonmammalianmembers of an ancient enzyme superfamily. Biochem J. 2001;360:1-16.
• Özaslan MS, Demir Y, Küfrevioğlu O I, Çiftci M. Some metals inhibit the glutathione S-transferase from Va Lake fish gills. J Biochem Mol Toxicol. 2017;73:e21967.
• Espinoza HM, CR Williams and EP Gallagher. Effect of cadmium on glutathione S-transferase and metallothionein gene expression in Coho Salmon liver, gill and olfactory tissues. Aquat Toxicol. 2012;110: 37-44.
• Aksoy AS. Bazı flavonoidlerin sığır karaciğer glutatyon stransferaz enzimi üzerine etkilerinin araştırılması. (Yüksek Lisans Tezi), Balıkesir Üniversitesi Fen Bilimleri Enstitüsü Kimya Anabilim Dalı, Balıkesir; 2018.
• Hayes JD, Flanagan JU, Jowsey IR. Glutathione transferases. Annu. Rev. Pharmacol Toxicol. 2005;45: 51-88.
• Cnubben NHP, Rietjens IMCM, Wortelboer H, van Zanden J, van Bladeren J. The interplay of glutathione-related processes in antioxidant defense. Environ Toxicol Pharmacol. 2001;10:141-152.
• Armstrong RN. Structure, catalytic mechanism, and evolution of the glutathione transferases. Chem Res Toxicol. 1997;10(1):2-18.
• Baş O. Dinitrocresol’ün (Rat rattus norvegicus) Sıçan glutatyon S-transferaz enzim aktivitesine etkisi (Yüksek Lisans Tezi) Uludağ Üniversitesi Fen Bilimleri Enstitüsü, Bursa; 2006.
• Fleischner GK, Kamisaka IM, Arias IM. Immunoloanalysis of rats and human ligandins. Glutathione: Metabolism and Function, 1976; 229-235.
• Toribio F, Martinez-Lara E, Pascual P, Lopez-Barea J. Methods for purification of glutathione peroxidase and related enzymes. J Chromatogr B. 1996;684:77-97.
• Güvercin S, Erat M, Şakiroğlu H. Determination of some kinetic and characteristic properties of glutathione s-transferase from bovine erythrocytes. Protein Pept. Lett. 2008;15(1): 6-12.
• Laemmli DK, Lajmanovich RC, Andre´s M, Attademo AM, Peltzer PM, Junges CM. Cleavage of structural proteins during in assembly of the head of bacteriophage T4. Nature, 1970; 277, 680-685.
• Ahmed B. M, Temel Y, Çiftçi M. Purification and characterization glutathione S transferase enzyme from quail (Coturnix, coturnix japonica) heart and investigation the effect of some metal ions on enzyme activity. C S J. 2019;40(4):802-812.
• Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976;72:248-251.
• Habig WH, Pabst MJ, Jakoby WB. Glutathione S- transferases. The first enzymmatic step in mercapturic acid formation. J Biol Chem.1974;246:7130-7139.
• Lineweaver H, and Burk D. The determination of enzyme dissociation constants. J Am Chem Soc. 1934;56 (3):658-666.
• Awasthi YC, Dao DD, Saneto RP. Interrelationship between anionic and cationic forms of glutathione S-transferases ofhuman liver. Biochem J. 1980;191:1-10.
• Nelson DL and Cox MM. Lehninger Principles of Biochemistry, Fourth edition. W.H. Freeman and Company, New York; 2004.
• Zariç Y. Glutatyon S-Transferaz Enziminin Van Gölü İnci Kefali Balığı (chalcalburnus Tarichi )Solungacından Saflaştırılması, Karakterizasyonu, Bazı Metal Ve Pestisitlerin Enzim Aktivitesi Üzerine Etkilerinin İncelenmesi. (Yüksek Lisans Tezi), Ağrı İbrahim Çeçen Üniversitesi Fen Bilimleri Enstitüsü, Ağrı; 2018.
• Taysi MŞ, Temel Y. Glutathione S-transferase: Purification and characterization from quail (Coturnix coturnix japonica) liver and the impact of some metal ions on enzyme activity. Bionanoscience. 2021;11: 91-98.
• Gözükara ME. Biyokimya, Nobel Matbaacılık, İstanbul; 2011.
• Keha E, Küfrevioglu Öİ. Biyokimya, Aktif Yayınevi, Erzurum; 2009.
• Dawn B, Marks Allan D, Mark, Colleen M, Smith. Basic Medical Biochemistry a clinical approach. A Wawely Company; 1996.
• Loscalzo J, Freedman J. Purification and characterization of human platelet glutathione-S transferase. Blood, 1986; 67, 1595-1599.
• Arca P, Garcida P, Hardisson C, Suarez JE. Purification and study of a bacteril glutathione S- transferase Area de Microbiologia, Fac. med.1990;1:77-79.
• Iizuka M, Inoue Y, Murata K, Kimura A Purification and some properties of glutathione S-transferase from escherichia coli B. J Bacteriol Res. 1989;6039-6042.
• Hiratsuka A, Sebata N, Kawashima K, Okuda H, Ogura K, Watabe T, Satoh K, Hatayama I, Tsuchida S, Ann Ishikawa TA. New class of rat glutathione Stransferase Yrs-Yrs inactivating reactive sulfate esters as metabolites of carcinogenic arylmethanols. J B C. 1990;265(20):11973-11981.
• Barcena JAE, Martinez-Lara SG, George J, Lopez-Barea. Purification and characterization of multiple glutathione s transferase isoenzymes from grey mullet liver. CMLS. 1997;759-768.
• Novoa-Valinas MC, Perez-Lopez M, Melgar MJ. Compparative study of the prufication and characterization of the cytosolic glutathione S-transferase from two salmonid species: Atlantic salmon (Salmo solar) and Brown trout (Salmo Trutta). C B P. part C, 2002;131:207-213.
• Young PR, and Briedis AV. Purification and kinetic mechanism of the major glutathione S-transferase from bovine brain. Biochem J. 1989;257:541-548.
• Huang Q, Liang L, Wei T, Zhang D, Zeng QY. Purification and partial characterization of glutathione transferase from the teleost monopterus albus. C B P Part C. 2008;147:96-100.
• Türkanoğlu A. Human serum arylesterase and glutathione S-transferase activities in patients with ischemic stroke compared to healthy controls. Approval of the Thesis Master of Science in Biochemistry Department, Middle East Technical University, Ankara; 2007.
• Tekman B, Ozdemir H, Senturk M, Ciftci M. Purification and characterization of glutathione reductase from rainbow trout (Oncorhynchus mykiss) liver and inhibition effects of metal ions on enzyme activity. C B P Part C. 2008;148:117-121.
• Çomaklı V, Çiftçi M, Küfrevioğlu Öİ. Gökkuşağı alabalık eritrositlerinden glutatyon S-transferas enziminin saflaştırılması ve bazı antibiyotiklerin enzim aktivitesi üzerine etkilerinin incelenmesi. Hacettepe J Biol Chem. 2011; 39 (4): 413-419.
• Hamed RR, Maharem TM and Guinidi RAM. Glutathione and its related enzymes in the nile fish. Fish Physiol Biochem. 2004;30 (3-4): 189-199.
• Ayna A, Khosnaw L, Temel Y, Ciftci M. Antibiotics as inhibitor of glutathione S-transferase: biological evaluation and molecular structure studies. Curr Drug Metab. 2021; 22(4):308-314.