Journal of Applied Physiology, Vol 65, Issue 4 1902-1906, Copyright © 1988 by American Physiological Society
A micromethod for measuring carbonic anhydrase activity using 18O exchange between CO2 and H2O
N. Bitterman, L. Lin and R. E. Forster
Department of Physiology, School of Medicine, University of Pennsylvania, Philadelphia 19104-6085.
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.
