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| | Esterase Activity Assay |
| Description: | Esterase activity assay was performed based on the method of by Verpoorte et al. [Verpoorte et al., J. Biol. Chem., 242:4221-4229] as described in previous studies [Innocenti et al., Bioorg. Med. Chem., 18:2159-2164; Akıncıoğlu et al., Arch. Pharm., 347:68-76; Akıncıoğlu et al., Bioorg. Med. Chem., 21:1379-1385]. The differences in absorbances due to the hCA I and II isoenzyme activities converting 4-nitrophenylacetate (NPA) to 4-nitrophenylate ion at 348 nm were observed in 3 min duration at room temperature using a spectrophotometer (Shimadzu, UVmini-1240 UV-VIS) [Çetinkaya et al., Arch. Pharm., 347:354-359; Aksu et al., Bioorg. Med. Chem., 21:2925-2931]. The reactants were 1.4 mL 0.05 M Tris-SO4 buffer (pH 7.4), 1 mL 3 mM NPA, 0.5 mL H2O and 0.1 mL enzyme solution making a total volume of 3 mL. A cuvette containing all reactants without the enzyme solution was used as blank. For the determination of Ki values three different bromophenols concentrations with NPA as the substrate at five different concentrations were used for making of Lineweaver-Burk curves [Lineweaver et al., J. Am. Chem. Soc., 56:656-666] as previously described [Atasever et al., Food Chem., 136:864-870; Aydin et al., Int. J. Food Propert., 18:2735-2745]. |
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