A method for measuring anion transfer across red cell membranes by continuous monitoring of fluorescence

A fluorometric method for continuous monitoring of anion transport across red blood cell membranes was developed. The method was based on the use of a fluorescent substrate, N-(2-aminoethylsulfonate)-7-nitrobenz-2-oxa-3-diazole) (NBD-taurine), whose intracellular fluorescence was quenched by hemoglobin. Cells loaded with NBD-taurine, free of extracellular probe, were suspended in various media, and the efflux of probe was recorded as a time-dependent increase in fluorescence. The method was highly reproducible and was sensitive down to 10³ cells as well as to transport rates in the range 10–30 s. A single fluorescence trace allowed determination of the Michaelis-Menten parameters of NBD-taurine efflux.