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| | CA Inhibition Assay |
| Description: | 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 Tris-HCl (pH 7.5) as buffer, 0.1 M Na2SO4 (for maintaining constant the ionic strength), at 25°C, following the CA-catalyzed CO2-hydration reaction for a period of 10-80 s (the uncatalyzedreaction needs around 60-100 s in the assay conditions, whereas the catalyzed ones are of around 6-10 s). The CO2 concentrations ranged from 1.7 to 17 mM for thedetermination of kinetic parameters. For each inhibitor, tested in the concentration range between 0.01 and 100 μM, 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) dimethyl sulfoxide (which is not inhibitory at these concentrations), and dilutions up to 0.001 μM 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. |
| Affinity data for this assay | |
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