The dehydration reaction of bicarbonate was meaknown isozymes, having a turnover rate of 1 1 10 4 sured using the fluorescent pH indicator, 8-hydroxys 01 , about 100-fold less than that of human carbonic pyrene-1,3,6-trisulfonate (pyranine), in combination anhydrase II (HCA II) (1, 2). These two isoforms also with stopped-flow spectrofluorometry. The initial rate differ in their sensitivity to sulfonamides; HCA II is of bicarbonate dehydration was measured after mixvery sensitive to acetazolamide, while HCA III is pecuing a pH 6.0 solution with a pH 8.0 solution containing liarly insensitive to acetazolamide (3).
bicarbonate. Addition of carbonic anhydrase to the pH
Carbonic anhydrase activity has been measured by 6.0 solution enabled the measurement of the initial various methods (for a review, see Ref. 4). The Wilburrate of activity at physiological temperatures with res-Anderson method (5), utilizing a pH electrode, is probaolution times of 2 ms. This assay was used to resolve bly the most widely used assay. Unfortunately, this differences in activity and sensitivity to sulfonamides method does not give a linear response over a broad by comparing mammalian carbonic anhydrase isoenzyme concentration range and cannot measure small forms. The fluorescent technique used in this study is differences in activity. In addition, the slow response very sensitive, allowing the determination of initial times of pH electrodes do not give accurate determinarates with a protein concentration as little as 65 ng/ml. tions of the initial rate of the reaction. This assay also Pyranine can also be loaded into membrane vesicles to requires nonphysiological conditions as the temperafollow carbonic anhydrase activity within vesicles.
ture must be near 0ΠC to give the highest sensitivity.
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