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| | CO2 Hydration 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., 246:2561-2573]. Phenol red (at a concentration of 0.2 mM) has been used as 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 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, in triplicate measurements. The uncatalyzedrates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (0.1 mM) were prepared in distilled-deionizedwater and dilutions up to 0.01 nM were done thereafter with distilled-deionized water. 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 [Maresca et al., Bioorg. Med. Chem. Lett., 21:1334-1337; Khalifah et al., J. Biol. Chem., 246:2561-2573; Park et al., Tetrahedron, 59:7651-7676]. |
| Affinity data for this assay | |
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