[Abstract] [Materials and Methods] [Title page]

The CO2-carrying capacity of blood is greatly enhanced by a process that involves chloride-bicarbonate exchange across the red cell membrane. In vivo, CO2, produced by metabolic processes in tissue, diffuses rapidly into the red cell, where intracellular carbonic anhydrase catalyzes the conversion of CO2 into bicarbonate. Intracellular bicarbonate is then exchanged for extracellular chloride as bicarbonate flows passively down its concentration gradient. In the lungs, the process is reversed, and CO2 diffuses into the atmosphere. The anion exchange occurs by a ping-pong mechanism (1) and is mediated by band 3, an integral transmembrane protein (2).

The rate-limiting step in CO2 transport by blood is the anion exchange across the membrane. The exchange is rapid and comparable to the residence time of the red cell in capillaries (0.3-0.7 s) (3). Previous methods to characterize this rapid exchange have included radioactive tracer methods (4), pH stopped-flow methods (5, 6), and flow-tube methods (7). We report here a method to measure chloride-bicarbonate exchange in red cell ghost membranes that is based on the quenching of an intracellular fluorescent probe by halides (8). The method consists of creating gradients of chloride across the red cell membrane (with equal but oppositely-directed gradients of bicarbonate) in a stopped-flow apparatus. As chloride and bicarbonate flow down their concentration gradients, the fluorescence signal from the intracellular fluorescent probe changes in a manner that can be analyzed quantitatively to give the chloride-bicarbonate exchange time. The method is rapid and straightforward, and can be applied to any exchange system in which intracellular halide concentration changes with time.

[Abstract] [Materials and Methods] [Title page]