In an experimental system where both Fe2+ autoxidation and generation of reactive oxygen species is negligible, the effect of FeCl2 and FeCl3 on the peroxidation of phosphatidylcholine (PC) liposomes containing different amounts of lipid hydroperoxides (LOOH) was studied; Fe2+ oxidation, oxygen consumption and oxidation index of the liposomes were measured. No peroxidation was observed at variable FeCl2/FeCl3 ratio when PC liposomes deprived of LOOH by triphenylphosphine treatment were utilized. By contrast, LOOH containing liposomes were peroxidized by FeCl2. The FeCl2 concentration at which Fe2+ oxidation was maximal, defined as critical Fe2+ concentration [Fe2+]*, depended on the LOOH concentration and not on the amount of PC liposomes in the assay. The LOOH-dependent lipid peroxidation was stimulated by FeCl3 addition; the oxidized form of the metal increased the average length of radical chains, shifted to higher values the [Fe2+]* and shortened the latent period. The iron chelator KSCN exerted effects opposite to those exerted by FeCl3 addition. The experimental data obtained indicate the kinetics of LOOH-dependent lipid peroxidation depends on the Fe2+/Fe3+ ratio at each moment during the time course of lipid peroxidation. The results confirm that exogenously added FeCl3 does not affect the LOOH-independent but the LOOH-dependent lipid peroxidation; and suggest that the Fe3+ endogenously generated exerts a major role in the control of the LOOH-dependent lipid peroxidation.

p13suc1 acts in the fission yeast cell division cycle as a component of p34 cdc2 . In the present work, structural information contained in the intrinsic fluorescence of p13 suc1 has been extracted by steady-state and time-resolved fluorescence techniques. In its native form, the steady-state emission spectrum of p13 suc1 is centered at 336 nm. Upon denaturation by guanidine HCl (4.0 M), the emission spectrum is shifted to 355-360 nm and the fluorescence intensity decreases 70%. The same changes are not obtained with p13 suc1 at 56°C or after incubation at 100°C, and the protein appears to be substantially temperature-stable. The fluorescence decay of p13 suc1 is best described by three discrete lifetimes of 0.6 ns ( 1 ), 2.9 ns ( 2 ), and 6.1 ns (

The fluorescence properties of the phospholipid derivative,N-[1-(2-naphthol)]-phosphatidylethanolamine (NAPH-PE), have been studied by steady-state and time-resolved fluorescence techniques. The new probe is a naphthol adduct of phosphatidylethanolamine. The emission spectrum of the fluorescent phospholipid depends on the pH and on the proton acceptor concentration as expected for a typical two-state excited-state proton transfer reaction. In ethanol solutions at an apparent pH of 6.7 and in the presence of acetate anion (0.14M), a biexponential decay is obtained from global analysis of the data. The lifetimes,τ 1=3.9 ns andτ 2=6.2 ns. are constant across the spectral region 350-460 nm. The decay-associated spectra and the species-associated spectra reproduce well the profiles reported for a two-state excited-state proton transfer reaction. The fluorescent phospholipid has been incorporated into dimyristoyllecithin and dipalmitoyllecithin vesicles. Although lower proton transfer is found, the reaction appears to be dependent on the gel-to-liquid-crystalline phase transition of the lipid membrane. In addition, the steady-state anisotropy of NAPH-PE measured as a function of temperature trace the phase transition of the two vesicle systems. Thus, it is shown that the physical state of the bilayer affects a reaction which takes place at the membrane surface. In the presence of acetate ions (0.3M), global analysis, performed in terms of fluorescence decay parameters, recovers preexponential coefficients that are consistent with an excited-state proton transfer reaction. The short lifetime drops from 3.9 to 0.44 ns without significant changes of the longer-lifetime component.

This report describes a method to conjugate lucifer yellow to the external surface of liposomes. The heterobifunctional cross-linking reagentN-succinimidyl 3-(2-pyridyldithio)propionate has been used to activate DMPE molecules. The DMPE-dithiopyridine product has been mixed with DMPC to prepare liposome vesicles. These have been reduced by DTT and finally reacted with lucifer yellow-iodoacetamide to produce the fluorescence-labeled vesicles. The quenching of their fluorescence intensity by Kl is consistent with fully exposed fluorophores. The decay of the fluorescence intensity of the lipid-bound lucifer yellow is biexponential (τ1=7.9 ns; τ2=1.1 ns), with a relative yield of 0.16. When the fluorescent liposomes are mixed with cells, the lucifer yellow-DMPE derivative is transferred. Boar spermatozoa and peripheral human blood lymphocytes have been used as cellular models. The extent of incorporation is dependent on the incubation time and temperature. At 36°C, lucifer yellow fluorescence appears in the spermatozoa cells after 10 min of incubation and reaches its maximum at about 60 min. The fluorescent phospholipid derivative seems to incorporate specifically into membrane structures. The highest labeling ratio is observed with integer, scarcely motile, spermatozoa. A poorer labeling yield (≈15%) is found with lymphocytes. Interestingly, photobleaching due to epiillumination of the labeled cells is apparently negligible and cells are clearly visible after irradiation times ranging from several minutes to few hours.