Carbonic anhydrase based environmental bioanalysis

Carbonic anhydrase (CA) is a metalloenzyme catalizing the reversible idratation of CO2 in H+ and HCO3-. It is an ubiquitous enzyme in bacteria, plant and animal kingdoms, playing a fundamental role in a number of physiolgical processes. Previous studies demonstrated the sensitivity of carbonic anhydrase activity to dichlorodiphenyl-dichloro-ethane (DDT) exposure in birds [1] and to cadmium exposure in teleost [2]. The aim of the present work was to investigate the in vitro sensitivity of carbonic anhydrase activity to several organic and inorganic chemical compounds, in order to standardize a carbonic anhydrase based bioanalytical method available for monitoring environmental samples. Commercial available CA isozyme II from bovine erythrocytes was utilized for the in vitro bioassay. CA activity was determined by a modification of electrometric method previously described by Wilbur and Anderson (J. Biol. Chem., 257: 12056-12059, 1948): briefly CA activity units were calculated from the rate of H+ production in the reaction mixture (where CO2 as substrate was present) against a blank containing the specific CA inhibitor acetazolamide. [H+] variation in the reaction mixture was followed at 0°C using a Mettler Delta 350 pH-meter. In our experimental set up bovin CA activity was significantly inhibited by nanomolar concentration of heavy metals (Cd, Cu and Hg), organochlorate compounds and carbammate pesticides, showing a dose-response behaviour. Carbonic anhydrase in vitro bioassay can represent a novel tool for rapid and low cost understanding of the toxicity of environmental samples, of bioavailability of pollutants in evironmental matrices, and of their additive or synergistic biological effects when present in mixtures.