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| | Esterase Activity Assay |
| Description: | CA activity was assayed according to method of Verpoorte et al. [Verpoorte et al., J. Biol. Chem., 242:4221-4229] described previously by Innocenti et al. [Innocenti et al., Bioorg. Med. Chem., 18:2159-2164; Innocenti et al., Bioorg. Med. Chem. Lett., 20:5050-5053] CA activity was determined by following thechange in absorbance at 348 nm of 4-nitrophenylacetate (NPA) to 4-nitrophenylate ion over a period of 3 min at 25°C using a spectrophotometer (CHEBIOS UV-VIS). The enzymatic reaction, in a total volume of 3.0 mL, contained 1.4 mL 0.05 M Tris-SO4 buffer (pH 7.4), 1 mL, 3 mM 4-nitrophenylacetate, 0.5 mL H2O and 0.1 mL enzyme solution. A reference measurement was obtained by preparing the same cuvette without enzyme solution. The inhibitory effects of ampicillin sulfate, ceftriaxone, ceftizoxime, ranitidine were examined. All compounds were tested in triplicate at each concentration used. Different inhibitor concentrations were used. HCA-I enzyme activities were measured for ceftriaxone (0.003-0.033 mM), ceftizoxime (0.017-0.096 mM), ranitidine (0.026-0.053 mM) at cuvette concentrations and HCAII enzyme activities were measured for ampicillin sulfate (0.038-0.095 mM), ceftriaxone (0.003-0.021 mM), ceftizoxime (0.026-0.061 mM) and ranitidine (0.032-0.058 mM) at cuvette concentrations. |
| Affinity data for this assay | |
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