Modified Micromethod for Assay of Carbonic Anhydrase Activity
The introduction of an assay for microliter samples of carbonic anhydrase (CA) (EC 126.96.36.199.) by Dr. T. H. Maren provided a simple, rapid method by which CA activity could be detected in 0.05 μl of dog blood.3 Quantitation results from the change of pH caused by the hydration of CO2, continuously supplied as gas, following the addition of alkaline buffer; the time needed for the indicator phenol red to change color from red to yellow depends on CA activity. We have modified the assay first by blowing CO2 on the surface of the enzyme solution (instead of through it; this allows us to use samples that tend to foam), second by adding a magnetic stirrer, third by reducing the reaction volume 50%, thus increasing the sensitivity of the assay. Another modification is the use of barbital buffer—as an alternative to the original CO 3 2− /HCO 3 − buffer—which further increases the assay’s sensitivity, especially in the case of anion-sensitive CA isozymes such as CA I or CA III, which are inhibited by HCO 3 − but not by barbital.5 The technique cannot be used to determine Michaelis-Menten constants since the initial velocities of substrate turnover cannot be obtained.
KeywordsCarbonic Anhydrase Reaction Chamber Carbonic Anhydrase Activity Indicator Solution Substrate Turnover
Unable to display preview. Download preview PDF.