Characterization and anions inhibition studies of an a-carbonic anhydrase from the teleost fish Dicentrarchus labrax Deniz Ekinci a,⇑ , Saltuk Bug ˘rahan Ceyhun b,c , Murat S ßentürk d,e , Deryanur Erdem d , Ömer _ Irfan Küfreviog ˘lu d , Claudiu T. Supuran f,⇑ a Ondokuz Mayıs University, Agricultural Faculty, Department of Agricultural Biotechnology, Enzyme and Microbial Biotechnology Division, 55139 Samsun, Turkey b Atatürk University, Hınıs Vocational Training School, Aquaculture Department, Erzurum, Turkey c Atatürk University, Biotechnology Research Center, Erzurum, Turkey d Atatürk University, Science Faculty, Chemistry Department, Erzurum, Turkey e Ag ˘rı _ Ibrahim Çeçen University, Art and Science Faculty, Chemistry Department, Ag ˘rı, Turkey f Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino, Florence, Italy article info Article history: Received 10 September 2010 Accepted 9 December 2010 Available online 16 December 2010 Keywords: Carbonic anhydrase Alpha-class Teleost fish Esterase Anion inhibitor Sulfamic acid Thiocyanate abstract Carbonic anhydrase (CA; EC 4.2.1.1) was purified from the gill of the teleost fish Dicentrarchus labrax (European seabass). The purification procedure consisted of a single step affinity chromatography on Sepharose 4B-tyrosine-sulfanilamide. The enzyme was purified 84.9-fold with a yield of 58%, and a spe- cific activity of 838.9 U/mg proteins. It has an optimum pH at 8.0; an optimum temperature at 10 °C. The kinetic parameters of this enzyme were determined for its esterase activity, with 4-nitrophenyl acetate (NPA) as substrate. The following anions, H 2 NSO 3 À ,I À , SCN À , NO 3 À , NO 2 À ,N 3 À , Br À , Cl À , SO 4 2À , and F À showed inhibitory effects on the enzyme. Sulfamic acid, iodide, and thiocyanate exhibited the strongest inhibitory action, in the micromolar range (K i s of 87–187 lM). NO 3 À , NO 2 À and N 3 À were moderate inhib- itors, whereas other anions showed only weak actions. All tested anions inhibited the enzyme in a com- petitive manner. Our findings indicate that these anions inhibit the fish enzyme in a similar manner to other a-CAs from mammals investigated earlier, but the susceptibility to various anions differs signifi- cantly between the fish and mammalian CAs. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction Carbonic anhydrases (CAs; EC 4.2.1.1) are metalloenzymes which catalyze the interconversion between CO 2 and HCO 3 À . They play key roles in diverse physiological processes, such as pH control, gas balance, calcification, ion transport, secretion of electrolytes, tumorigenesis, etc. 1 Sixteen CA isozymes have been described in mammals, that differ in their subcellular localization, catalytic activity and susceptibility to different classes of inhibi- tors. Some of these isozymes are cytosolic (CA I, CA II, CA III, CA VII, and CA XIII), others are membrane bound (CA IV, CA IX, CA XII, and CA XIV), two are mitochondrial (CAVA and CA VB), and one is secreted in saliva (CA VI). 1 Up to now, CA has been purified from many different vertebrate tissues including human erythrocytes, 2–4 rainbow trout brain and liver, 5 fish gills. 6 Considering the role of gill CA in osmoregulatory processes, the relation between branchial CA activity and/or distri- bution and ambient salinity has been also investigated in several species. 7–14 The teleost fish Dicentrarchus labrax (European seabass), is a primarily ocean-living fish that sometimes enters brackish and fresh water. Its habitats include estuaries, lagoons, coastal waters and rivers. It is found in the waters in and around Europe, including the eastern Atlantic Ocean (from Norway to Senegal), the Mediterranean Sea and the Black Sea. 15 It has economically great importance due to its meat quality in addition to being one of the most cultured animals among other seawater fish. Although there are studies regarding purification of CA from var- ious tissues, no reports have been found on purification and charac- terization of the enzyme from European seabass (Dicentrarchus labrax). In the present study, we purified and characterized an a-CA from Dicentrarchus labrax gill for the first time, and investi- gated its kinetic properties and inhibitory effects of anions including H 2 NSO 3 À ,I À , SCN À , NO 3 À , NO 2 À ,N 3 À , Br À , Cl À , SO 4 2À , and F À on enzyme activity. 2. Results and discussion 2.1. Purification and characterization of carbonic anhydrase from European seabass gill Carbon dioxide, produced in fish tissues, is hydrated rapidly by CA, being converted into bicarbonate, and transported in the 0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmc.2010.12.033 ⇑ Corresponding authors. Tel.: +90 362 3121919; fax: +90 362 4576034 (D.E.); tel.: +39 055 4573005; fax: +39 055 4573385 (C.T.S.). E-mail addresses: dekinci80@gmail.com (D. Ekinci), claudiu.supuran@unifi.it (C.T. Supuran). Bioorganic & Medicinal Chemistry 19 (2011) 744–748 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc