Further photophysical and photochemical characterization of flavins associated with single-shelled vesicles

Abstract

Summary. This paper is a continuation of a series of physicochemical studies of vesicle-bound flavins. In this study a detailed analysis of the Sepharose 4B elution profile of various sonicated phospholipids is given which demonstrates the presence of three distinct particle populations. Electron microscopy reveals heterogeneous, multilamellar lipoid aggregates of a diameter of more than 500 ~ in fraction I, unilamellar, closed vesicles of 200 to 250 A diameter in fraction II, and ~previously unreported micellar structures of approximately 50 A diameter in fraction III. Fraction II represents vesicles very similar to those obtained by the "deoxycholate procedure." Fine structure analysis of corrected fluorescence and fluorescence polarization spectra of amphiphilic flavin (AFI 3) bound to vesicles prepared from various phospholipids demonstrates the specificity of the flavin/membrane interaction. When bound to fractions I, II and III of sonicated egg lecithin, AFI 3 exhibits different temperature (i.e. phase-) dependencies. This demonstrates that the flavin/membrane interaction depends strongly upon the particular lipid structure as well. Evidence is presented which defines the type of excited state of flavin involved in photochemical and photophysical reactions (triplet or singlet). Finally, the dependencies of the photoreactions of vesicle-associated flavins on parameters such as pH, temperature and ionic strength are discussed. Comparison of photoreactions of isotropic and anisotropic flavins also reveal clues for the various mechanisms involved.

α-Branched 1,2-Diacyl Phosphatidylcholines as Effectors of Activity of Cytochrome P450SCC (CYP11A1)

“…Preparation, Purification, and Characterization of P450SCC-containing Phospholipid Vesicles-Small unilamellar vesicles (SUV) were prepared by ultrasonication as described previously with small modifications (12,13). 12 mg of the phospholipid(s) in chloroform/methanol (2:1, v/v) were mixed with usually 6 mol% (of total lipid) cholesterol including a small amount of [ 14 C]cholesterol in absolute ethanol in a test tube, and solvents were evaporated under N 2 and kept under vacuum for 4 h. After complete removal of the solvent 3 ml of standard buffer (20 mM Hepes, pH 7.3, 50 mM KCl, 0.1 mM dithiothreitol) were added and vortexed for 2 min.…”

“…The suspension was sonicated to clarity by using an USD 20 Desintegrator (Akademie-Werkstaetten fü r Forschungsbedarf, Berlin, Germany) at 150-watt power setting (30 min with 1:1 cooling intervals of 1 min, under a gentle stream of N 2 ) with the tubes immersed in a water bath at 25°C (DMPC) or at 0°C (DOPC), respectively. The resulting liposome suspension was chromatographed on Sepharose 4B to separate the multilamellar vesicles and micellar lipid aggregates from the SUVs (13). The size of the SUV was determined by electron microscopy to be about 42 nm in diameter.…”

Bluelight-induced, flavin-mediated transport of redox equivalents across artificial bilayer membranes

“…The present paper continues our series on vesicleassociated flavins [43,48,51], dealing with the bluelight-induced, flavin-mediated transport of redox equivalents across vesicle membranes. The basic goal of our studies is the elucidation of the.…”

“…As a minimum model system for the bluelight receptor three different amphiphilic flavins bearing C18-hydrocarbon chains at positions 3, 7 and 10 have been synthesized (AF1 3, 7, 10) [31] and anchored within artificial, single-shelled vesicles made from natural and synthetic phospholipids [48]. On this basis we hope to mimic the steric and mechanistic restrictions imposed by the apoprotein and/or the membrane in the natural photoreceptor.…”

Effect of the Incorporation Into Unilaminar Vesicle on the Photodegradation of Indoles Sensitized by Flavins

“…Little is known about the photochemical behaviour of RF and FE in membrane like systems, although there has been described that the photophysical properties of RF are affected upon compartmentalization (14,15). In this work, we have studied the incorporation of flavinic and indolic derivatives in small unilaminar vesicle (SUV) of dipalmitoylphosphatidylcholine (DPPC), and the effect of their incorporaction in the efficiency of the degradation of three indolic derivatives photosensitized by flavins.…”