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Khalifah, R. G, J. Biol. Chem. 1971, 246, 2561. An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalysed CO2 hydration activity. Phenol red (at a concentration of 0.2 mM) has been used as indicator, working at the absorbance maximum of 557 nm, with 10-20 mM Hepes (pH 7.5, for α-CAs) or TRIS (pH 8.3 for β-CAs) as buffers, and 20 mM Na2SO4 (for α-CAs) or 20 mM NaCl-for β-CAs (for maintaining constant the ionic strength, following the initial rates of the CA-catalyzed CO2 hydration reaction for a period of 10-100 s. For each inhibitor 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 inhibitor (10 mM) were prepared in distilled-deionized water with 5-10, v/v) DMSO which is not inhibitory at these concentrat
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