Capoeta trutta Böbrek Dokusundan Saflaştırılan Glutatyon Redüktaz Enzim Aktivitesine Bazı Metallerin in vitro İnhibisyon Etkileri

Abstract

Bu çalışmada, glutatyon redüktaz (EC: 1.8.1.7; GR) iç su balıklarından Capoeta trutta böbrek dokusundan saflaştırılmıştır. Saflaştırma işlemi, homojenatın hazırlanması, amonyum sülfat çöktürmesi ve 2’,5’-ADP Sepharose 4B afinite kromatografisi olmak üzere 3 basamakta gerçekleştirilmiştir. Bu yöntemlerle, enzim 11.91 EÜ/mg protein spesifik aktivite ve %35.4 verimle 794 kat saflaştırılmıştır. Saflaştırma sonucu elde edilen saf enzimin saflık kontrolü sodyum dodesilsülfat poliakrilamid jel elektroforezi (SDS-PAGE) ile yapıldı ve enzimlerin tek band olduğu görülmüştür. Enzimin molekül kütlesi yaklaşık olarak 55 kDa olarak hesaplanmıştır. Ayrıca, balık böbrek dokusundan saflaştırılan glutatyon redüktaz enzimi üzerine bazı metallerin (Ag⁺, Co⁺², Ni⁺², Cu⁺² ve Zn⁺²) in vitro inhibisyon etkileri incelenmiştir. Metallerin K_i sabiti ve IC₅₀ değerleri sırasıyla Lineweaver-Burk ve Yüzde (%) Aktivite-[Metal] grafikleri ile hesaplanmıştır. Bu grafiklerden, Ag⁺, Co⁺², Ni⁺², Cu⁺² ve Zn⁺² metalleri için IC₅₀ değerleri sırasıyla 0.00078, 0.622, 0.722, 0.073 ve 0.519 mM, ve K_i sabitleri sırasıyla 0.000394 ± 0.0002, 0.235 ± 0.027, 0.279 ± 0.048, 0.026 ± 0.008 ve 0.382 ± 0.024 mM olarak hesaplanmıştır. Sonuç olarak, Ag⁺ metalinin diğer metallerden daha fazla C. trutta böbrek GR enzimini inhibe ettiği tespit edilmiştir.

Keywords

• Glutatyon redüktaz,Capoeta trutta,böbrek,inhibitör,saflaştırma

In vitro Inhibition Effects of Some Metal Ions on Glutathione Reductase Purified from Capoeta trutta Kidney

Abstract

Glutathione reductase (EC: 1.8.1.7; GR) was purified from the kidney of the teleost fish Capoeta trutta. The purification procedure consisted of three steps: preparation of homogenate, ammonium sulfate fractionation and affinity chromatography on 2’,5’-ADP Sepharose 4B. The enzyme was purified 794-fold with a yield of 35.4% and a specific activity of 11.91 U/mg proteins. In order to control enzyme purity, SDS-PAGE was done and showed a single band for enzyme. A single band was obtained approximately at 55 kDa. In addition, inhibitory effects of metal ions (Ag⁺, Co⁺², Ni⁺², Cu⁺² and Zn⁺²) on fish kidney glutathione reductase were investigated. K_i constants and IC₅₀ values for metal ions were determined by Lineweaver–Burk graphs and plotting activity % vs. [I], respectively. IC₅₀ values were 0.00078, 0.622, 0.722, 0.073 and 0.519 mM, and K_i constants were 0.000394 ± 0.0002, 0.235 ± 0.027, 0.279 ± 0.048, 0.026 ± 0.008 and 0.382 ± 0.024 mM for Ag⁺, Co⁺², Ni⁺², Cu⁺² and Zn⁺², respectively. From these results, we showed that Ag⁺ is the most potent inhibitor of glutathione reductase enzyme.

Keywords

• Glutathione reductase,Capoeta trutta,kidney,inhibitor,purification

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