Rapid separation of carbonic anhydrase isozymes using cellulose acetate membrane electrophoresis

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Abstract
A method is described for the rapid separation of carbonic anhydrase (CA) isozymes by cellulose acetate membrane electrophoresis in which CA activity is detected using the pH-indicating dye, bromcresol purple. This method can detect bovine erythrocyte CA in a 0.3 mm3 sample applied at a concentration of 100 ng cm−3 (total of 30 pg applied) while at higher concentrations three isozymes were observed. It was found, using a potentiometric technique, that intact cells of Anabaena flos-aquae (Cyanophyceae) and Chlorella ellipsoidea had no detectable activity while C. saccharophila and Chlamydomonas reinhardtii (Chlorophyceae) had external CA activity. CA activity of the extracts suggested the presence of internal CA in all species. After electrophoresis it was found that C. saccharophila and C. reinhardtii had two isozymes while A. flos-aquae and C. ellipsoidea had only a single detectable band. Spinach had up to five detectable isozymes that were difficult to resolve. Incubation of spinach extract with the CA inhibitor ClO4− (500 mol m−3) inhibited CA activity by 90% using the potentiometric technique, but after electrophoresis had no detectable effect. This technique is useful in identifying isozymes that are substantially different in electrical charge and in monitoring CA isozyme activity during enzyme purification.

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