Direct measurement of ferrous ion transport across membranes using a sensitive fluorometric assay

The fluorophore, Phen Green SK (PGSK), was assessed for its suitability to be used in an assay for ferrous ion transport into membrane vesicles. The l ong wavelengths of excitation and emission (506 and 520 nm, respectively) e nable PGSK fluorescence to be detected in membranes, such as the chloroplas t inner envelope, that contain high levels of carotenoids which absorb ligh t at lower wavelengths. At low concentrations of Fe2+, less than 3 muM, the interaction between PGSK and Fe2+ appears to result in both static and dyn amic quenching of the PGSK fluorescence. The characteristics of this quench ing were used to develop a calibration curve to determine the concentration of free Fe2+ at these low concentrations. Pronounced quenching of PGSK flu orescence entrapped within chloroplast inner envelope membrane vesicles was observed when Fe2+ was added. The extent of quenching of PGSK fluorescence trapped inside asolectin vesicles on Fe2+ addition was much less. The kine tics of the quenching of PGSK fluorescence by Fe2+ in vesicles was quite di fferent from that for PGSK and Fe2+ in solution. Using the calibration curv e developed for interaction of PGSK and low Fe2+ concentrations the initial rates of iron transport could be determined for the chloroplast inner enve lope membranes. (C) 2001 Academic Press.