Glutatyon S-transferaz: Koyun Dalak Dokusundan Saflaştırılması ve Karakterizasyonu

Abstract

Bu çalışmada, hücre içi majör antioksidan sistem olan glutatyon antioksidan sisteminin önemli enzimlerinden glutatyon S-transferaz (GST; EC 2.5.1.18) sitozolik enzimi koyun dalak dokusundan homojenat hazırlanması, salting out (amonyum sülfat tuzu ile çöktürme yöntemi) ve afinite kromatografisi (glutatyon-agaroz) olmak üzere üç adımda 3.67 EÜ mg-1 protein değeri (spesifik aktivite) ve %3.73 verim ile 122.3 kat saflaştırıldı. Koyun dalak dokusundan saflaştırılan GST enziminin saflık derecesini belirlemek ve doğal alt birim molekül kütlelerinin tespitinde sodyum dodesil sülfat poliakrilamid jel elektroforez (SDS-PAGE) metodu kullanıldı. Koyun dalak dokusu GST enziminin alt birimlerine ait molekül kütlesi yaklaşık olarak 26.36 kDa hesaplandı. Koyun dalak dokusundan saflaştırılan GST enziminin karakterizasyonu için gerçekleştirilen çalışmalarda; optimum pH, K-fosfat tamponu pH=8.0, optimum aktivite gösterdiği iyonik şiddet, K-fosfat tampon çözeltisi 1.0 M, stabil pH, K-fosfat tampon çözeltisi pH = 7.0 ve optimum sıcaklığı 60 oC olarak bulundu. Koyun dalak dokusundan saflaştırılan GST enzimine ait KM ve Vmax değerlerini belirlemek için yapılan kinetik çalışmalarda Lineweaver-Burk grafiklerinden yararlanıldı. Enzimin substratları olan indirgenmiş glutatyon (GSH) ve 1-kloro-2,4-dinitrobenzen (CDNB) için yürütülen kinetik çalışmalarda; GSH için KM değeri 0.629 mM, Vmax değeri 0.056 EÜ mL-1; CDNB için KM değeri 0.321 mM, Vmax değeri 0.129 EÜ mL-1 olarak belirlendi.

Keywords

• saflaştırma
• karakterizasyon
• koyun
• dalak
• GST

Glutathione S-transferase: Purification and Characterization from Sheep Spleen Tissue

Abstract

In this study, glutathione S-transferase (GST; EC 2.5.1.18), which is the important enzyme of the glutathione antioxidant system, which is the main intracellular antioxidant system, was purified in three steps from sheep spleen tissue; using homogenate preparation, ammonium sulfate precipitation method and affinity chromatography (glutathione-agarose) with a specific activity value of 3.67 EU mg-1, 122.3 protein fold and 3.73% yield. SDS-PAGE method was used to determine the purity level and to determine the natural molecular mass of subunits of the GST enzyme, purified from sheep spleen tissue. The molecular mass of the subunits of the sheep spleen tissue GST enzyme was calculated as approximately 26.36 kDa. In the studies carried out for the characterization of the GST enzyme purified from sheep spleen tissue; optimum pH, K-phosphate buffer pH=8.0, optimum ionic strength, K-phosphate buffer 1.0 M, stable pH, K-phosphate buffer pH = 7.0 and optimum temperature 60 oC. Lineweaver-Burk graphs were used in kinetic studies to determine the KM and Vmax values of the GST enzyme purified from sheep spleen tissue. In the kinetic studies carried out for reduced glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB), which are the substrates of the GST Enzyme; for GSH the KM value 0.629 mM, the Vmax value 0.056 EU mL-1, for CDNB the KM value 0.321 mM, and the Vmax value 0.129 EU mL-1 were determined.

Keywords

• GST
• purification
• characterization
• sheep
• spleen

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