Glutatyon S-Transferaz Enziminin Tavuk Karaciğerinden Saflaştırılması ve Karakterizasyonu

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.

Purification and Charcterızatıon Glutathione S-Transferase Enzyme From Chicken Liver

Abstract

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.

Keywords

• Glutathione S-transferase
• chicken liver
• purification.

Kaynakça

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