A micromethod for measuring carbonic anhydrase activity using 18O exchange between CO2 and H2O.
We have developed a method of measuring the activity and characteristics of carbonic anhydrase (CA) using the disappearance of 18O from CO2 in 1 ml of gas contained in a glass chamber as it exchanges with H2O in 0.01 ml 0.25 M NaHCO3 solution in a thin (25 micron) porous membrane. Serial gas samples (approximately 0.02 ml) are analyzed in a mass spectrometer to obtain the rate of disappearance of the label. The enzyme activity can be measured inside intact cell or particle membranes. As little as 10(-15) mol of high-activity type CA can be detected at 25 degrees C, and the activity of 200 times this amount can be measured. The uncatalyzed hydration reaction velocity constant was 0.056 +/- 0.004 s-1, in agreement with published data.
Bitterman
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Department of Physiology, School of Medicine, University of Pennsylvania, Philadelphia 19104-6085.
Lin
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Forster
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D001639:Bicarbonates; D002245:Carbon Dioxide; D002256:Carbonic Anhydrases / Q000378:metabolism*; D055598:Chemical Phenomena; D002621:Chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013058:Mass Spectrometry; D008832:Microchemistry; D008851:Micropore Filters; D010103:Oxygen Isotopes; D012964:Sodium; D017693:Sodium Bicarbonate; D014867:Water
D001639:Bicarbonates; D010103:Oxygen Isotopes; D014867:Water; D002245:Carbon Dioxide; D017693:Sodium Bicarbonate; D012964:Sodium; D002256:Carbonic Anhydrases