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

Abstract

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 (K-i) and 50% inhibitory values (IC50) for metal ions were determined by Lineweaver-Burk graphs and plotting activity % vs. [I], respectively. The Klconstants 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.