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An applied photophysics stopped-flow instrument was used for assaying the carbonic anhydrase (CA) catalyzed CO2 hydration activity. Phenol red (at a concentration of 0.2 mM) was used as indicator, working at the absorbance maximum of 557 nm, with 10 mM Hepes (pH 7.5) as buffer, 0.1 M Na2SO4 (for maintaining constant ionic strength), following the CA-catalyzed CO2 hydration reaction for a period of 10 s at 25 C. The CO2 concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and activation constants. For each activator at least six traces of the initial 5–10% of the reaction have been used for determining the initial velocity. The uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of activators (Histidine methyl ester, 10 mM) were prepared in distilled–deionized water and dilutions up to 0.1 nM were done thereafter with distilled–deionized water. Activator and enzyme solutions were preincubated together for 15 min (standard assay at room temperature, or for prolonged periods of 24–72 h, at 4 C) prior to assay, in order to allow for the formation of the E–A complex.