| Summary: | A simple, reliable, and universal method is demanded for routine determination of carbonic anhydrase (CA) activity, overcoming the limitations of previous assays that are inaccurate, complicated, expensive, or limited to a specific enzyme family. The most widely used Wilbur–Anderson assay was modified to improve the speed, accuracy, and precision by employing a temperature controllable UV/Vis spectrophotometer and the pH indicator phenol red. The experimental setting, measurement, and data analysis were facile and straightforward. The assay was validated using a commercially available bovine CA, showing that the obtained activity was directly proportional to the amount of enzyme. The measured activity (2540 WAU mg<sup>−1</sup>) agreed well with the previously reported data. The comparison results with esterase assay showed that the CO<sub>2</sub> hydration assay should not be substituted by the esterase assay in the measurement of CA activity. The simple and reliable colorimetric method can be widely adopted for the routine determination of CO<sub>2</sub> hydration activity, substituting for the traditional Wilbur–Anderson assay.
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