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

Year 2017, , 385 - 394, 01.12.2017

Muammer Kırıcı , Mahinur Kırıcı , Muhammed Atamanalp

https://doi.org/10.17693/yunusae.v17i31121.337640

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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