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| | CA Inhibition Assay |
| Description: | An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalysed CO2 hydration activity. Phenol red (at a concentration of 0.2 mM) has been used as an indicator, working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5) as buffer, and 20 mM Na2SO4 (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 thedetermination of the kinetic parameters and inhibition constants19. 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 inhibitor (0.1 mM) were prepared in distilled-deionized water and dilutionsup to 0.01 nM were done thereafter with distilled deionized water. Inhibitor and enzyme solutions were preincubated together for 15 min-72 h at room temperature(15 min) or 4 °C (all other incubation times) prior to assay, in order to allow for the formation of the E-I complex or for the eventual active site mediated hydrolysis of the inhibitor. |
| Affinity data for this assay | |
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