Glutatyon Redüktaz Enziminin Koyun Kalp Dokusundan Saflaştırılması ve Karakterizasyonu

Abstract

Glutatyon redüktaz (GR; E.C.1.8.1.7), GSSG’nin GSH’a indirgenmesini katalizleyen bir enzim olup oksidoredüktaz sınıfının bir çeşididir. Bu çalışmada glutatyon redüktaz enzimi koyun kalp dokusundan saflaştırıldı. Saflaştırma işleminde %30-70 arası tuz çöktürmesi ve afinite kromatografisi metodları kullanıldı. Saflaştırma sonucunda spesifik aktivitesi 5,35 EÜ/mg.protein olan enzim %72,82 oranında 191 kat şeklinde saflaştırıldı. Daha sonra enzimin saflaştırılma durumu elektroforez ile kontrol edildi ve denatüre edici şartlarda molekül kütlesi yaklaşık olarak 70 kDa olarak bulundu. Karakterizasyon çalışmalarında ise enzimin optimum pH’sı 6,0 (0,1 M KH2PO4 tamponunda), stabil pH’sı 8,0 (0,1 M KH2PO4 tamponunda), optimum iyonik şiddetti 240 mM (0,1 M KH2PO4) ve optimum sıcaklık değeri 70°C olarak bulundu. Ayrıca NADPH için KM sabiti 7,776 mM, Vmax değeri 0,1678 EÜ/mL; GSSG için KM sabiti 7,776 mM, Vmax değeri 0,1678 EÜ/mL olarak tespit edildi.

Keywords

• Glutatyon Redüktaz
• Saflaştırma
• Karakterizasyon

Supporting Institution

Bingöl Üniversitesi - BİLİMSEL ARAŞTIRMA PROJELERİ BİRİMİ (BÜBAP)

Project Number

BAP-VF.2024.002

Ethical Statement

Etik Kurul Kararına gerek yoktur

Thanks

Çalışmanın finansmanından dolayı BAP-VF.2024.002 nolu tez projemizi destekleyen Bingöl Üniversitesi BÜBAP birimine ve elemanlarına teşekkürlerimi sunarım

Purification and Characterization of Glutathione Reductase Enzyme from Sheep Heart

Abstract

Glutathione reductase (GR; E.C.1.8.1.7) is an enzyme that catalyzes the reduction of GSSG to GSH and is a type of oxidoreductase. In this study, glutathione reductase was purified from sheep heart tissue. Purification was carried out using 30-70% salt precipitation and affinity chromatography. The enzyme, with a specific activity of 5.35 EU/mg protein, was purified 191-fold at 72.82%. The purification status of the enzyme was then checked by electrophoresis, and its molecular mass was found to be approximately 70 kDa under denaturing conditions. In characterization studies, the enzyme's optimum pH was found to be 6.0 (in 0.1 M KH2PO4 buffer), its stable pH was found to be 8.0 (in 0.1 M KH2PO4 buffer), its optimum ionic strength was found to be 240 mM (0.1 M KH2PO4), and its optimum temperature was found to be 70°C. In addition, the KM constant for NADPH was found to be 7.776 mM, and the Vmax value was found to be 0.1678 EU/mL; and the KM constant for GSSG was found to be 7.776 mM, and the Vmax value was found to be 0.1678 EU/mL.

Keywords

• Glutathione Reductase
• Purification
• Characterisation

Supporting Institution

Bingöl University - Scientific Research Projects Unit (BÜBAP)

Project Number

BAP-VF.2024.002

Ethical Statement

No Ethics Committee decision is required.

Thanks

I would like to express my gratitude to the Bingöl University BÜBAP unit and its staff for supporting our thesis project, BAP-VF.2024.002, with funding for this study

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