The impact of heavy metals on the activity of carbonic anhydrase from rainbow trout ( Oncorhynchus mykiss ) kidney

Abstract

Many environmental and health problems have become a consequence of contamination of soil and water by toxic heavy metals and organic pollutants in the present age of technology. Heavy metals play vital roles in enzyme activities and other metabolic events with their bioaccumulative and nonbiodegradable properties among aquatic pollutants. Metal toxicity causes irregular metallothioneins protein synthesis, renal damage, and disruption of bone structure in humans and wildlife. In this study, we investigated in vitro effects of some metals on chemical-targeted carbonic anhydrase (CA) enzyme from rainbow trout kidney. The enzyme was purified with a specific activity of 17,285 EU × mg⁻¹ and 31.7% yield and approximately 1800-fold using simple affinity purification method. Molecular weights of the subunit and native enzyme were estimated as 28.7 kDa and 26.9 kDa via sodium dodecyl sulfate polyacrylamide gel electrophoresis and Sephadex-G 200 column, respectively. Other kinetic properties of the enzyme were determined. Apparent K_m , V _max and k _cat values were 0.40 mM, 0.097 µmol min⁻¹ and 15.2 s⁻¹ for p-nitrophenylacetate substrate, respectively. Inhibitory effects of cobalt, zinc, copper, cadmium and silver on CA activity were determined using the esterase method under in vitro conditions. IC₅₀ and K_i values were calculated for metals. K_i values for Co²⁺, Zn²⁺, Cu²⁺, Cd²⁺ and Ag⁺ were 0.035, 1.2, 34.8, 103 and 257 from Lineweaver–Burk graphs, respectively. Consequently, in vitro inhibition rank order was determined as Co²⁺ > Zn²⁺ > Cu²⁺ > Cd²⁺ > Ag⁺. The potential inhibitor for CA was found as Co²⁺ from these results.