Efflux of dipicolinic acid from human erythrocytes, sealed membrane fragments, and band 3-liposome complexes: a fluorescence probe for the erythrocyte anion transporter

The greatly enhanced fluorescence of Tb3+ when complexed with dipicolinic acid affords a simple and highly sensitive method for monitoring continuous anion flux through the erythrocyte anion transporter, band 3. Dipicolinic acid (pyridine-2,6-dicarboxylic acid) is entrapped in human erythrocytes and other band 3-membrane preparations. Efflux of dipicolinic acid from preequilibrated systems into Tb3+-containing medium is monitored fluorometrically. Dipicolinic acid efflux is demonstrated in intact erythrocytes, sealed red cell membrane fragments, and band 3-liposome complexes. In each system, dipicolinic acid efflux is blocked by the band 3 inhibitor 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid. The technique is highly sensitive and reproducible, and is applicable to hemoglobin-containing and hemoglobin-free systems of widely varying surface area to volume ratios.