Detection of superoxide anion radical in phospholipid liposomal membrane by fluorescence quenching method using 1,3-diphenylisobenzofuran

Abstract

Utilization of a fluorescence dye, 1,3-diphenylisobenzofuran (DPBF) as a detector of superoxide anion radical (O ⋅− 2) was examined. The fluorescence intensity of DPBF incorporated in phospholipid liposomes consisting of phosphatidylcholine (PC) and phosphatidylserine (PS) is effectively quenched by incubation with xanthine/xanthine oxidase system. On the other hand, xanthine or xanthine oxidase alone did not induce quenching of the DPBF fluorescence in the liposomes. Xanthine/xanthine oxidase-induced fluorescence quenching of DPBF-labeled liposomes was almost completely protected by the addition of superoxide dismutase (SOD, 1 U/ml), but not by heat-denatured SOD (10 min boiling) at the same concentration. On the other hand, catalase (1 U/ml), and hydroxyl radical and singlet oxygen scavengers (10 mM sodium benzoate, 300 mM mannitol, 1 mM tryptophan and 1 mM sodium azide) did not protect xanthine/xanthine oxidase-induced fluorescence quenching of DPBF-labeled liposomes. The concentration dependence profiles of xanthine oxidase on the DPBF fluorescence quenching and O ⋅− 2 generation showed that there is a good correlation between these parameters. Under the present experimental conditions, approximately 7 μM H 2O 2/30 min were produced, but the addition of H 2O 2 (1 mM) to DPBF-labeled liposomes did not quench the dye fluorescence in the liposomes. Temperature dependence profiles of the DPBF fluorescence quenching induced by xanthine/xanthine oxidase treatment and the excimer fluorescence formation of pyrene molecules embedded in the liposomal membrane suggested that the quenching efficiency of the DPBF fluorescence is largely dependent on their lipid dynamics. Based on these results, we proposed the possibility that DPBF fluorescence quenching method is able to be used as a simple method for detecting O ⋅− 2 inside the membrane lipid layer and that DPBF fluorescence quenching by O ⋅− 2 is controlled by the physical state of membrane lipids.