| Assay Method Information | |
| | Enzyme Inhibition Assay |
| Description: | An SX.18MV-R Applied Photophysics (Oxford, UK) stopped-flow instrument has been used to assay the catalytic/inhibition of various CA isozymes as reported by Khalifah [Khalifah et al., J. Biol. Chem., 1:156-161]. 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 (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; pH 7.4) as buffer, 0.1 M Na2SO4 or NaClO4 (for maintaining constant the ionic strength; these anions are not inhibitory in the used concentration), following the CA-catalyzed CO2 hydration reaction for a period of 5-10 s. Saturated CO2 solutions in water at 25°C were used as substrate. Stock solutions of inhibitors were prepared at a concentration of 10 mM (in dimethyl sulphoxidewater 1:1, v/v) and dilutions up to 1 mM done with the assay buffer mentioned above. At least four different inhibitor concentrations have been used for measuring the inhibition constant. Inhibitor and enzyme solutions were preincubated together for 10 min at room temperature prior to assay, in order to allow for the formation of the E-I complex. |
| Affinity data for this assay | |
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