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Assay Method Information

Assay Name:  CA 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. Phenol Red (0.2 mM) has been used as indicator, working at the absorbance maximum of 557 nm, following the CA-catalyzed CO2 hydration reaction for a period of 5-10 s. Saturated CO2 solutions in water at 25 deg C were used as substrate. Stock solutions of inhibitors were prepared at a concentration of 10 mM (in DMSO-water 1:1, v/v) and dilutions up to 0.01 nM. At least seven 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. Triplicate experiments were done for each inhibitor concentration, and the values reported throughout the paper are the mean of such results. The inhibition constants were obtained by non-linear least-squares methods using PRISM 3, and represent the mean from at least three different determinations.
Affinity data for this assay
 

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Last update November 1, 2007
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