Chelon auratus (Risso, 1810)’un Karaciğerinden Glukoz-6-Fosfat Dehidrojenaz’ın Saflaştırılması ve Kinetik Karakterizasyonu

Yıl 2021, Cilt: 11 Sayı: 2, 138 - 144, 15.12.2021

Salih Görgün , Emre Koç

Öz

Bu çalışmada, önemli bir kefal balığı türü olan Chelon auratus (Risso, 1810)’un karaciğerinden glukoz-6-fosfat dehidrojenaz (EC
1.1.1.49), 2′5′ ADP-Sepharose 4B afinite kromatografisi ile 252 kat saflık, 27.04 EU/mg.protein spesifik aktivite ve % 27.42 verim
ile saflaştırılmıştır. Elde edilen enzim preparatı için optimum sıcaklık, pH ve iyonik güç sırasıyla 40 ºC, pH 8, ve 0.1 M olarak
bulunmuştur. Enzimin kinetik davranışı gerek glukoz-6-fosfat (G6P) gerekse NADP+ için değerlendirilmiştir. Elde edilen verilere
göre, Km ve Vmax değerleri NADP+ için sırasıyla 0.14 mM ve 3.40 EU/mL olarak belirlenirken, G6P için sırasıyla 0.23 mM ve 2.62
EU/mL olduğu bulunmuştur. Bu veriler, substrat olarak G6P ile karşılaştırıldığında, enzimin NADP+ için daha yüksek bir afinite
sergilediğini önermektedir. Saflaştırılan enzim preparatında SDS-PAGE analizi sonucunda, molekül kütlelerinin yaklaşık olarak 108
kDa ve 60 kDa olan iki protein bandının olduğu görülmüştür. Elde edilen sonuçlar, kefal türlerinden glukoz-6-fosfat dehidrojenazın
saflaştırılmasında ek kromatografik adımların gerekli olduğunu önermektedir.

Anahtar Kelimeler

Chelon auratus (Risso, 1810), glukoz-6-fosfat dehidrojenaz, saflaştırma, kinetik karakterizasyon

Destekleyen Kurum

Sivas Cumhuriyet Üniversitesi

Proje Numarası

F-625

Teşekkür

Bu çalışma Sivas Cumhuriyet Üniversitesi Bilimsel Araştırma Projeleri (CÜBAP) Birimi tarafından F-625 No’lu proje olarak desteklenmiştir. Bu nedenle, teşekkürlerimizi sunuyoruz.

Purification and Characterization of Glucose-6-Phosphate Dehydrogenase from the Liver of Chelon Auratus (Risso, 1810)

Öz

In this study, glucose-6-phospahte dehydrogenase (EC. 1.1.1.49) from the liver of Chelon auratus (Risso, 1810), an economically
important mullet species, were purified with purification parameters of 252-fold purity, a specific activity of 27.04 EU/mg.protein
and a yield of 27.42%. The optimum temperature, pH and ionic strength were found to be 40 ºC, pH 8 and 0.1 M, respectively, for
the enzyme preparation obtained. The kinetic behavior of the enzyme was evaluated for both G6P and NADP+. According to the
data obtained, Km and Vmax values were determined as 0.14 mM and 3.40 EU/mL for NADP+, respectively, while it was found to be
0.23 mM and 2.62 EU/mL for G6P, respectively. These data have been suggested that the enzyme has higher affinity for the NADP+,
when compared with G6P as a substrate. According to the SDS-PAGE analyses of the purified enzyme, it was observed that there
were two protein bands having molecular weight of 108 kDa and 60 kDa. The results obtained have been suggested that additional
chromatographic steps might be required for the purification of glucose-6-phospahte dehydrogenase from the mullet species.

Anahtar Kelimeler

Chelon auratus, Glucose-6-phosphate dehydrogenase, Kinetic characterization, Purification

Proje Numarası

F-625

Kaynakça

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