6-Fosfoglukonat Dehidrogenaz Enziminin Japon Bıldırcın (Coturnix Coturnix Japonica) Eritrositlerinden Saflaştırılması ve Karakterizasyonu

Abstract

Bu çalışmada Japon bıldırcın eritrosit dokularından 6-fosfoglukonat dehidrogenaz enzimi (6PGD) 52,84 EU.mg-1 spesifik aktiviteyle ve % 69 verimle 2ˈ, 5ˈ-ADP Sepharose 4B afinite kromatografisi kullanılarak saflaştırıldı. Japon bıldırcın eritrositlerinden saflaştırılan 6PGD enzimi için saflaştırma katsayısı 4984 olarak bulundu. Enzim saflığının kontrolü için SDS-poliakrilamid jel elektroforezi (SDS-PAGE) yapıldı ve tek bant gözlendi. SDS-PAGE yöntemi kullanılarak enzimin alt birimlerinin molekül kutlesi 81 kDa olarak hesaplandı. Optimum iyonik şiddet 0,5 M Tris-HCl, optimum pH ve stabil pH 0,5 M Tris-HCl tamponu pH 8,0 olarak bulundu. Optimum sıcaklık 60 ºC olarak bulundu. Ayrica Japon bıldırcın 6PGD enziminin KM ve Vmax değerleri Lineweaver-Burk grafiklerinden sırasıyla 6PGA substratı için KM değeri 0,120 mM, Vmax değeri 0,191 EU/mL, NADP+ substratı için KM değeri 0,017 mM, Vmax değerise 0,228 EU/mL olarak hesaplandı.

Keywords

• Bıldırcın,6PGD,eritrosit,saflaştırma,karakterizasyon

Purification and Characterization of 6-Phosphogluconate Dehydrogenase from Japanese Quail (Coturnix, coturnix japonica) Erythrocytes

Abstract

In this study, the 6PGD enzyme from Japanese quail erythrocytes was purified with specific activity of 52.84 EU/mg and 69% yield of purification by 2ˈ, 5ˈ- ADP Sepharose 4B affinity gel in a single chromatographic method. The purification folds of the enzyme were 4984 folds. The purified enzyme was checked using SDS polyacrylamide gel electrophoresis (SDS-PAGE) method; the result of gel showed a single band. The subunit molecular weight of the enzyme was calculated as 81 kDa by the SDS-PAGE method. The characterization studies of the 6PGD enzyme from erythrocytes of Japanese quail showed: the optimum ionic strength to be at 0.5 M Tris-HCl, optimum and stable pH values to be at 0.5 M Tris-HCl buffers pH 8.0. The optimal temperature for the enzyme activity was found at 60 ˚C. Finally, the KM and Vmax values for the 6PGD enzyme from Japanese quail’s erythrocytes were calculated respectively for the 6PGA the KM value found as 0.120 mM, Vmax value as 0.191 EU/mL and for NADP+ the KM value as 0.017 mM and Vmax value as 0.228 EU/mL.

Keywords

• Quail,6PGD,erythrocytes,purification,characterization

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