| Assay Method Information | |
| | Esterase Assay |
| Description: | CA activity was assayed by following the change in absorbance at 348 nm of 4-nitrophenylacetate (NPA) to 4-nitrophenylate ion over a period of 3 min at 25°Cusing a spectrophotometer (CHEBIOS UV-VIS) according to the method described by Verpoorte et al. [Verpoorte et al., J. Biol. Chem., 242:4221-4229] The enzymatic reaction contained 1.4 mL 0.05 M Tris-SO4 buffer (pH 7.4), 1 mL 3 mM 4-NPA, 0.5 mL H2O and 0.1 mL enzyme solution (total volume, 3.0 mL). A reference measurement was obtained by preparing the mixture without the enzyme solution. All measurements were made in triplicate. The Ki values were determined from a series of experiments using three different bromophenols concentrations and 4-NPA as the substrate at five different concentrations to construct Lineweaver-Burk curves [Lineweaver et al., J. Am. Chem. Soc., 57:685-693]. |
| Affinity data for this assay | |
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