Shingles R, Moroney J V
Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218-2685, USA.
Anal Biochem. 1997 Oct 1;252(1):190-7. doi: 10.1006/abio.1997.2305.
The dehydration reaction of bicarbonate was measured using the fluorescent pH indicator, 8-hydroxypyrene-1,3,6-trisulfonate (pyranine), in combination with stopped-flow spectrofluorometry. The initial rate of bicarbonate dehydration was measured after mixing a pH 6.0 solution with a pH 8.0 solution containing bicarbonate. Addition of carbonic anhydrase to the pH 6.0 solution enabled the measurement of the initial rate of activity at physiological temperatures with resolution times of 2 ms. This assay was used to resolve differences in activity and sensitivity to sulfonamides by comparing mammalian carbonic anhydrase isoforms. The fluorescent technique used in this study is very sensitive, allowing the determination of initial rates with a protein concentration as little as 65 ng/ml. Pyranine can also be loaded into membrane vesicles to follow carbonic anhydrase activity within vesicles. The change in pH within vesicles is dependent on the concentration of externally added bicarbonate and the presence of carbonic anhydrase on either side of the membrane. Therefore, this assay can be used to measure carbon dioxide flux across membranes and to assess the contribution of carbonic anhydrase to this flux.
使用荧光pH指示剂8-羟基芘-1,3,6-三磺酸盐（吡喃荧光素）结合停流荧光光谱法测定碳酸氢盐的脱水反应。将pH 6.0的溶液与含碳酸氢盐的pH 8.0溶液混合后，测定碳酸氢盐脱水的初始速率。向pH 6.0的溶液中添加碳酸酐酶，能够在生理温度下以2毫秒的分辨时间测定活性的初始速率。通过比较哺乳动物碳酸酐酶同工型，该测定法用于分辨活性差异和对磺胺类药物的敏感性。本研究中使用的荧光技术非常灵敏，能够测定蛋白质浓度低至65纳克/毫升时的初始速率。吡喃荧光素也可以加载到膜囊泡中，以追踪囊泡内的碳酸酐酶活性。囊泡内pH的变化取决于外部添加的碳酸氢盐的浓度以及膜两侧碳酸酐酶的存在情况。因此，该测定法可用于测量跨膜的二氧化碳通量，并评估碳酸酐酶对该通量的贡献。
