Mehmet ÇİFTCİ, Yusuf TEMEL, Barzan Mirza AHMED

Glutatyon S-transferaz enziminin Japon bıldırcın (Coturnix, coturnix japonica) yürek dokusundan saflaştırılması, karakterizasyonu ve bazı metal iyonlarının enzim aktivitesi üzerine etkilerinin araştırılması

Bu çalışmada japon bıldırcın yürek dokusundan glutatyon S-transferaz enzimi (EC: 2.5.1.18) 34.0 EU/mg spesifik aktivite ile % 10.44 verimle ve 78.29 kat saflaştırılmıştır. Saflaştırma işlemi üç aşamada gerçekleştirilmiştir. Bu aşamalar homojenatın hazırlanması, amonyum sülfat çöktürmesi ve glutatyon-agaroz jel afinite kolon kromatografisinden oluşmaktadır. Bıldırcın yürek dokusu GST enziminin saflığını test etmek için SDS-PAGE yöntemi kullanılmıştır. Daha sonra SDS-PAGE yöntemi ile enzimin alt birimlerinin molekül kütlesi yaklaşık olarak 26.3 kDa olarak hesaplanmıştır. Enzimatik aktivite 340 nm'de Beutler yöntemine göre spektrofotometrik olarak belirlenmiştir. Yapılan karakterizasyon çalışmalarında enzyme ait optimum pH Tris/HCl tamponu pH = 8.0, stabil pH Tris / HCL tamponu pH = 9.0, optimum sıcaklık 60 °C, optimum iyonik şiddeet Tris/HCl tamponu 1.2 M olarak bulunmuştur. GST enzimi substrat olarak hem glutatyon hem de 1-kloro 2,4-dinitrobenzen kullanmaktadır. Yapılan kinetik çalışmalarda GST enzimi için KM ve Vmax değerleri, GSH substratı için sırasıyla 3.880 mM ve 0.588 EU/mL, CDNB substrat için sırasıyla 1.642 mM ve 0.502 EU/mL bulunmuştur. Buna ek olarak bazı metal iyonlarının (Cu2+, Cd2+, Fe2+, Fe3+ Zn2+, Ag+, Co2+ ve Ti1+) GST enzim aktivitesi üzerine in vitro etkileri araştırılmıştır.

Purification and characterization glutathione S-transferase enzyme from quail (Coturnix, coturnix japonica) heart and investigation the effect of some metal ions on enzyme activity

In this study glutathione S-transferase enzyme (EC: 2.5.1.18) from the heart of japonica quail was purified with 34.0 EU/mg specific activity, 10.44% purification yield and 78.29 purification folds and characterized. Purification processes are consist of three steps, firstly homogenate was prepared, and then ammonium sulfate precipitation was performed and finally glutathione-agarose gel affinity column chromatography was performed. To check the purity of GST enzyme used SDS-PAGE method. Then the M.W calculated at approximately 26.3 kDa by SDS-PAGE method. Enzymatic activity was determined spectrofotometrically according to Beutler`s method at 340 nm. Also characterizations study carry out, and the results obtained are stability-pH = 9.0 in Tris/HCL buffer, optimum pH = 8.0 in Tris/HCl buffer, optimum temperature 60 °C, optimum ionic strength was 1.2 M in Tris/HCl buffer. And kinetic studies performed for GST enzyme purified from quail heart by used both glutathione and 1-chloro 2,4-dinitrobenzen as substrate. KM and Vmax values are determined as 1.642 mM and 0.502 EU/mL respectively for GSH substrate and 3.880 mM and 0.588 EU/mL respectively for CDNB substrate. In addition, the effect of some metal ions (Cu2+, Cd2+, Fe2+, Fe3+ Zn2+, Ag+, Co2+, and Ti1+) were investigated on the GST enzyme activity in vitro.

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