A fluorescence quenching study on protoporphyrin IX in a model membrane system.
The interaction of protoporphyrin IX (3,7,12,15-tetramethyl-8, 13-divinyl-2,18-porphyrine-dipropionic acid) (PPIX) with unilamellar dimyristoyl-L-alpha-phosphatidylcholine (DMPC) phospholipid vesicles has been studied by means of steady-state fluorescence quenching spectroscopy. The method of fluorescence-quenching-resolved spectroscopy has been applied in order to resolve the complex emission spectrum of a membrane-bound PPIX into two component spectra, attributed to distinct fluorophore species with different accessibilities to the iodide quencher. It is shown that PPIX associated with liposomes exists in two different microenvironments. One part of the fluorophore is embedded inside the lipid bilayer and is inaccessible to iodide. Its fluorescence spectrum exhibits the maximum characteristic of protoporphyrin found in the apolar medium. The other fraction of PPIX is located near the membrane surface, close to the polar phospholipid heads. Its emission is blue-shifted, resembling that of PPIX in a polar environment. It is quenched by iodide, although it reveals significant shielding from the quencher as compared to a buffer PPIX solution. Fluorescence quenching using 1-oxyl-4-oxo-2,2,6,6-tetramethyl-piperidine (TEMPONE) does not discriminate between the two protoporphyrin species. However, the accessibility of protoporphyrin IX to this quencher is much lower in a liposome system than in water.
Kuszaj
S
S
Department of Physical Biochemistry, Jagiellonian University, Kraków, Poland.
Kaszycki
P
P
Wasylewski
Z
Z
D004134:Dimyristoylphosphatidylcholine / Q000737:chemistry; D066298:In Vitro Techniques; D008051:Lipid Bilayers / Q000737:chemistry; D008081:Liposomes / Q000737:chemistry; D008567:Membranes, Artificial; D008956:Models, Chemical; D011524:Protoporphyrins / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D014216:Triacetoneamine-N-Oxyl
D008051:Lipid Bilayers; D008081:Liposomes; D008567:Membranes, Artificial; D011524:Protoporphyrins; D014216:Triacetoneamine-N-Oxyl; C028025:protoporphyrin IX; D004134:Dimyristoylphosphatidylcholine