In Vitro Effects of Diclofenac Sodium on Glucose-6-Phosphate Dehydrogenase and Glutathione Reductase Enzymes Purified from Sheep Spleen Tissue

Abstract

In this study, in vitro effects of diclofenac sodium, which is used to prevent inflammation and alleviate pain in arthritis and acute injuries, on glucose-6-phosphate dehydrogenase enzyme (G6PD; EC 1.1.1.49) and glutathione reductase enzyme (GR; EC 1.8.1.7) purified from sheep spleen tissue were investigated. In the first stage of the study, G6PD and GR enzymes were purified from sheep spleen tissue by ammonium sulfate (NH4)2SO4 precipitation and 2', 5' ADP-Sepharose-4B gel affinity chromatography. The purity levels of the enzymes were checked by the sodyum dodesilsülfat poliakrilamid jel elektroforezi (SDS-PAGE) method. In the second stage of the study, the effects of diclofenac sodium on the pure enzyme activities were investigated. IC50 values were calculated by plotting the % Activity-[I] graph for diclofenac sodium, which has an inhibitory effect on enzymes. In addition, Lineweaver-Burk graphs were drawn by making measurements with five different substrate concentrations and three different inhibitor concentrations. From these graphs, it was determined that G6PD enzyme inhibited with 0.51 mM IC50 value and 0.48 ± 0.01 mM Ki value non competitively, by diclofenac sodium. It was determined that the GR enzyme inhibited with an 6.55 mM IC50 value of and 15.69 ± 0.84 mM Ki value non competitively.

Keywords

• G6PD
• GR
• diclofenac sodium
• inhibition

Koyun Dalak Dokusundan Saflaştırılan Glukoz-6-Fosfat Dehidrogenaz ve Glutatyon Redüktaz Enzimi Üzerine Diklofenak Sodyumun İn Vitro Etkileri

Abstract

Bu çalışmada, artrit ve akut yaralanmalarda enflamasyonu önlemek ve ağrıyı dindirmek için kullanılan diklofenak sodyumun, koyun dalak dokusundan saflaştırılan glukoz-6-fosfat dehidrogenaz enzimi (G6PD; E.C. 1.1.1.49) ve glutatyon redüktaz enzimi (GR; E.C. 1.8.1.7) üzerine in vitro etkileri araştırıldı. Çalışmanın ilk aşamasında G6PD ve GR enzimleri koyun dalak dokusundan, amonyum sülfat (NH4)2SO4 çöktürmesi ve 2', 5' ADP-Sepharose-4B jel afinite kromatografisi ile saflaştırıldı. Enzimlerin saflık dereceleri sodyum dodesilsülfat poliakrilamid jel elektroforezi (SDS-PAGE) metodu ile kontrol edildi. Çalışmanın ikinci aşamasında saf olarak elde edilen enzim aktiviteleri üzerine diklofenak sodyumun etkileri araştırıldı. Enzimler üzerinde inhibisyon etkisi gösteren diklofenak sodyumun için % Aktivite-[I] grafiği çizilerek IC50 değerleri hesaplandı. Ayrıca beş farklı substrat konsantrasyonu ve üç farklı inhibitör konsantrasyonlarıyla ölçümler yapılarak Lineweaver-Burk grafikleri çizildi. Elde edilen bu grafiklerden diklofenak sodyumun G6PD enzimini 0.51 mM, IC50 değeri ve 0.48 ± 0.01 mM Ki değeri ile yarışmasız olarak inhibe ettiği belirlendi. GR enzimini ise 6.55 mM IC50 değeri ve 15.69 ± 0.84 mM Ki değeri ile yarışmasız olarak inhibe ettiği tespit edildi.

Keywords

• G6PD
• GR
• diklofenak sodyum
• inhibisyon

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