3.7. Enzyme Inhibition Assays

An applied photophysics stopped-flow instrument has been used for assaying the fungal and human CA-catalyzed CO₂ hydration activity [48]. Phenol red (0.2 mM) has been used as the indicator, working at the absorbance maximum of 557 nm, with 20-mM HEPES (pH 7.5 for α-CAs) or TRIS (pH 8.3 for the β-CA) as buffers and 20-mM NaClO₄ (for maintaining constant the ionic strength), following the initial rates of the CA-catalyzed CO₂ hydration reaction for a period of 10–100 s. The CO₂ concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. In particular, CO₂ was bubbled in distilled deionized water for 30 min until saturation. A CO₂ kit (Sigma-Aldrich, Milan, Italy) was used to measure the concentration in serially diluted solutions from the saturated one at the same temperature. For each inhibitor, at least six traces of the initial 5–10% of the reaction have been used for determining the initial velocity. The uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of the inhibitor (1 µM) were prepared in distilled-deionized water, and dilutions up to 0.1 nM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were preincubated together for 15 min at room temperature prior to the assay in order to allow for the formation of the E-I complex or for the eventual active site-mediated hydrolysis of the inhibitor. The inhibition constants were obtained by the nonlinear least-squares methods using PRISM 3 and the Cheng-Prusoff equation and represent the average from at least three different determinations. All recombinant CA isoforms were obtained in-house, as previously reported [49,50].