MUAMMER KIRICI, Mahinur KIRICI, ŞÜKRÜ BEYDEMİR, MUHAMMED ATAMANALP

Purification of Carbonic Anhydrase from Capoeta umbla (Heckel, 1843) Gills and Toxicological Effects of Some Metals on Enzyme Activity

Bu çalışmada, Capoeta umbla solungaç sitoplazmik karbonik anhidraz enzimi üzerine bazı metallerin (Fe+3, Cd+2, Pb+2 ve Ni+2) in vitro etkileri incelendi. Karbonik anhidraz enzimi C. umbla solungaç dokusundan ilk kez saflaştırıldı. Enzim, Sepharose 4B-L-tirozin-sulfanilamid afinite kromatografisi yöntemi ile saflaştırıldı. Bu yöntem sonucunda enzim, spesifik aktivitesi 326,73 EÜ/mg protein ve %53,3 verimle yaklaşık 31,69 kat olarak saflaştırıldı. Sodyum dodesil sülfat-poliakrilamid jel elektroforezinde (SDS-PAGE) yaklaşık 29 kDa'da tek bant olarak görüldü. Metal iyonları için enzim inhibitör kompleks sabitleri (Ki) ve %50 inhibitör değerleri (IC50), sırasıyla Lineweaver-Burk grafikleri ve % aktivite-[I] grafikleri çizilerek hesaplandı. Ki sabitleri ve IC50 değerleri, Fe+3 için 0,012±0,0135 ve 0,136 mM, Cd+2 için 0,019±0,0113 ve 0,191 mM, Pb+2için 0,041±0,0075 ve 0,289 mM ve Ni+2 için 0,120±0,034 ve 0,924 mM olarak belirlendi. Fe+3, Cd+2 ve Pb+2 iyonları enzimi, yarışmalı olarak inhibe ederken, Ni+2 iyonu yarışmasız olarak inhibe etmiştir. Sonuç olarak, Fe+3 iyonunun C. umbla solungaç karbonik anhidraz enzimi için güçlü bir inhibitör olduğu belirlendi.

Capoeta umbla (Heckel, 1843) Solungaç Dokusundan Karbonik Anhidraz Enziminin Saflaştırılması ve Bazı Metallerin Enzim Aktivitesi Üzerine Toksikolojik Etkilerinin İncelenmesi

In this study, in vitroeffects of some metal ions(Fe3+, Cd2+, Pb2+ and Ni2+)on cytoplasmic carbonic anhydrase(CA, EC 4.2.1.1) from Capoeta umbla gill was investigated. CA was purified from the gills of C. umbla for the first time. It was purified with the Sepharose-4B-L-Tyrosine Sulphanilamide affinity chromatography method. The overall purification was approx. 31.69-fold with a yield of 53.33%, and a specific activity of 326.73 EU/mg proteins. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a single band corresponding to a molecular weight of approx. 29 kDa. The constants of the enzyme inhibitor complex (Ki) and 50% inhibitory values (IC50) for metal ions were determined by Lineweaver-Burk graphs and plotting activity % vs. [I], respectively. The Kiconstants and IC50 values were 0.012±0.0135 and 0.136 mM for Fe3+, 0.019±0.0113 and 0.191 mM for Cd2+, 0.041±0.0075 and 0.289 mM for Pb2+, and 0,120±0.034 and 0.924 mM for Ni2+. It was determined that Fe3+, Cd2+ and Pb2+ inhibited the enzyme competitively while Ni2+ inhibited the enzyme noncompetitively. The potential inhibitor for C. umbla gill CA was found as Fe3+ from these results

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