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| | CA Inhibition Assay |
| Description: | An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalyzed CO2 hydration activity [Khalifah et al., J. Biol. Chem., 246:2561-73]. 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 7.4 for hCA I and II) or with 20 mM Tris (pH 8.4, for ScCA) 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. 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 (10 mM) were prepared in distilled-deionized water and dilutions up to 0.001 nM were done thereafter with the assay buffers. Inhibitor and enzyme solutions were preincubated together for 15 min at room temperature prior to assay, in order to allow for the formation of the E-I complex. The concentration of the enzymes in the assays was of 8.0 nM for hCA I, of 5.9nM for hCA II and of 12.5 nM for ScCA, respectively. |
| Affinity data for this assay | |
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