GENERATION AND REACTIONS OF SINGLET OXYGEN PHOTOSENSITIZED BY HYPERICIN IN DIPALMITOYLPHOSPHATIDYLCHOLINE VESICLES - EXPERIMENTAL EVIDENCE OF A DELAYED PHOTOOXIDATION

Hypericin ( 2,2'-dimethyl-4.4',5,5',7,7'-hexahydroxymesonaphtodianthrone) incorporated into unilamelar liposomes of dipalmitoylphosphatidylcholine (DPPC) generates O2 (1-SIGMA+g and 1-DELTA(g)) with a quantum yield of ca 0.35 upon photoexcitation at 559 nm or 308 nm as determined by photooxidation experiments using 1,3-diphenylisobenzofuran (DF) as substrate. The quantum yield of photooxidation increases when the concentration of hypericin decreases, due to the self quenching of the first excited singlet state. The rate parameters for singlet oxygen were monitored by observing the time profile of the bleaching of the reactive substrate. The lifetime of O2(1-DELTA(g)) in D2O-based suspensions was shown to be 40-mu-s and 4-mu-s in H2O. Our experiments unequivocally shows that O2(1-DELTA-(g)) can be formed in the lipid bilayer of one vesicle and react with DF in the lipid bilayer of another. From the data presented it may be concluded that liposomes of DPPC have the tendency to aggregate. the mean distance between two liposomes would be ca 100 nm (in aggregates of the vesicles). Under our conditions DF undergoes photooxidation by two mechanisms - a fast oxidation with O2(1-DELTA(g)), the bimolecular rate constant being (0,9 +/- 0,2).10(7) M-1 . s-1. - a delayed oxidation with a bimolecular rate constant of (3,3 +/- 0,6). 10(5) M-1 . s-1. It cannot be excluded that this delayed oxidation is due to a secondary reaction of peroxide primarily formed with the phospholipid and O2(1-SIGMA+(g)).