Purification, Characterization of NADPH-Cytochrome P450 Reductase from Turkey Liver and Effects of Some Metal Ions on Enzyme Activity

Öz

NADPH-Cytochrome P450 reductase (CPR) is a key enzyme catalyzing electron transfer in

detoxification metabolism. In this study, two methods were employed to purify CPR enzyme from turkey liver

microsomes. In the first method, NADPH-Cytochrome P450 reductase was purified using 2ʹ, 5ʹ-ADP Sepharose 4B

affinity column with ~114 purification fold and ~23% yield. In the second method, CPR was purified using DE-52

Cellulose anion exchange column and 2ʹ, 5ʹ-ADP Sepharose 4B affinity column with 124 purification fold and 8%

yield. Enzyme purity was checked in both methods with SDS-PAGE. Characteristics kinetic features of the purified

enzyme were identified. The effects of some metal ions on purified PCR enzyme activity have been investigated

in vitro conditions. It has been found that Ag+, Hg2+ and Cu2+ metal ions have an inhibitory effect on CPR enzyme

activity.

Anahtar Kelimeler

• Characterization,cytochrome P450 reductase,inhibition,metal ions,turkey liver

Hindi Karaciğerinden NADPH-Sitokrom P450 Redüktaz’ın Saflaştırılması, Karakterizasyonu ve Bazı Metal İyonlarının Enzim Aktivitesi Üzerindeki Etkileri

Öz

NADPH-Sitokrom P450 redüktaz (CPR) enzimi, detoksifikasyon metabolizmasında elektron transferini

katalizleyen anahtar bir enzimdir. Bu çalışmada, hindi karaciğer mikrozomlarından CPR enziminin saflaştırılması

için iki yöntem kullanılmıştır. Birinci yöntemde, CPR enzimi ~114 saflaştırma katsayısıyla ve ~%23 verimle

2’, 5’-ADP Sefaroz 4B afinite kolonu kullanılarak saflaştırılmıştır. İkinci yöntemde, CPR, DE-52 selüloz anyon

değişim kolonu ve 2’, 5’-ADP Sefaroz 4B afinite kolonu kullanılarak, ~124’lük saflaştırma katsayısı ve %8

verimle saflaştırılmıştır. Enzim saflığı her iki yöntemde de SDS-PAGE ile kontrol edilmiştir. Saflaştırılmış enzimin

karakteristik kinetik özellikleri belirlenmiştir. Bazı metal iyonlarının saflaştırılmış CPR enzim aktivitesi üzerindeki

etkileri in vitro şartlarda araştırılmıştır. Ag+, Hg2+ ve Cu2+ metal iyonlarının CPR enzim aktivitesi üzerinde inhibisyon

etkisi gösterdiği tespit edilmiştir.

Anahtar Kelimeler

• Hindi karaciğeri,inhibisyon,karakterizasyon,metal iyonları,sitokrom P450 redüktaz

Kaynakça

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