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| | Inhibition Assay |
| Description: | The inhibition constants (K) the compounds for four CA isozymes, CA I, II, IX and XII were determined. An Applied Photophysics (Oxford, UK) stopped-flow instrument has been used for assaying the CA-catalyzed CO2 hydration activity (Khalifah, 1971). Phenol red (at a concentration of 0.2 mM) has been used as indicator, working at the absorbance maximum of 557 nm, with 10 mM Hepes (pH 7.5) as buffer, 0.1 M Na2SO (for maintaining constant the ionic strength), following 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. 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 (1 mM) were prepared in distilled-deionized water with 10-20% (v/v) DMSO. |
| Affinity data for this assay | |
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