Mariusz Gagoś

Dimers of polyene antibiotic amphotericin B detected by means of fluorescence spectroscopy: molecular organization in solution and in lipid membranes

Fluorescence emission from amphotericin B dissolved in 2-propanol-water was recorded in the spectral region 500-650 nm. The fluorescence excitation spectrum corresponds to the absorption spectrum of the monomeric drug. The large energy shift between the excitation and emission bands indicates that emission takes place from an energy level different than that responsible for absorption. These levels were attributed to the 2(1)A(g) and 1(1)B(u) states, respectively. Excitation of the same sample with short wavelength radiation (below 350 nm) yields light emission between 400 and 550 nm. The fluorescence excitation spectrum corresponding to this emission band displays distinct maxima at 350, 334 and 318 nm. This band was analyzed in terms of the exciton splitting theory and assigned to amphotericin B in a dimeric form, in which chromophores are spaced by 4.9 A. The binding energy of the dimers, determined to be 4.9 kJ/mol, indicates that the structures are stabilized by van der Waals interactions. The same type of molecular structures was also detected in the lipid membranes formed with dipalmitoylphosphatidylcholine. Linear dichroism of amphotericin B embedded in lipid multibilayers indicates that molecules are distributed between two fractions: parallel (38%) and perpendicular (62%) with respect to the membrane. The biological importance of such membrane organization is discussed.

Spectroscopic studies of molecular organization of antibiotic amphotericin B in monolayers and dipalmitoylphosphatidylcholine lipid multibilayers

Amphotericin B (AmB) is considered the gold-standard in the treatment of serious systemic mycoses despite its numerous adverse effects. Both the mechanism of antifungal action and the toxicity of this drug are dependent on its molecular organization. The effect of AmB on the organization of lipid membranes formed with dipalmitoylphosphatidylcholine (DPPC) was studied with application of the Langmuir-Blodgett technique and ATR-FTIR spectroscopy. The aim of this research was to analyze the physical interactions leading to the formation of aggregated forms of AmB molecules in one-component monolayers and lipid multibilayers. Analysis of FTIR spectra of two-component multibilayers suggests the possibility the mutual reorientation of the amino-sugar moiety (mycosamine) and macrolide ring. This effect may be significant in the explanation of the aggregation processes of AmB in biological systems.

Molecular organization of antifungal antibiotic amphotericin B in lipid monolayers studied by means of Fluorescence Lifetime Imaging Microscopy

Amphotericin B (AmB) is a life-saving polyene antibiotic used to treat deep-seated mycotic infections. Both the mode of therapeutic action as well as toxic side effects are directly dependent on molecular organization of the drug. Binding of AmB to lipid monolayers formed with dipalmitoylphosphatidylcholine, pure and containing 40 mol% cholesterol or ergosterol, the sterols of human and fungi respectively, has been examined by means of Fluorescence Lifetime Imaging Microscopy. AmB emits fluorescence with the characteristic lifetimes dependent on actual molecular organization: tau(M2) < or = 10 ps and tau(M1) = 0.35 ns in the monomeric state, the emission from the S(2) and the S(1) states respectively and tau(D) = 14 ns and tau(A) = 3.5 ns in the form of a dimer and associated dimers respectively. Analysis of the Langmuir-Blodgett films reveals that AmB binds to the lipid membranes and to the cholesterol-containing lipid membranes preferentially in the form of associated dimers. The same form of AmB appears in the membranes containing ergosterol but additionally the monomers and dimers of the drug can be observed, which can severely affect molecular organization of the lipid membrane. The results are discussed in terms of selectivity of AmB towards the ergosterol-containing biomembranes of fungi.

Secondary structure and orientation of the pore-forming toxin lysenin in a sphingomyelin-containing membrane.

Lysenin is a sphingomyelin-recognizing toxin which forms stable oligomers upon membrane binding and causes cell lysis. To get insight into the mechanism of the transition of lysenin from a soluble to a membrane-bound form, surface activity of the protein and its binding to lipid membranes were studied using tensiometric measurements, Fourier-transform infrared spectroscopy (FTIR) and FTIR-linear dichroism. The results showed cooperative adsorption of recombinant lysenin-His at the argon-water interface from the water subphase which suggested self-association of lysenin-His in solution. An assembly of premature oligomers by lysenin-His in solution was confirmed by blue native gel electrophoresis. When a monolayer composed of sphingomyelin and cholesterol was present at the interface, the rate of insertion of lysenin-His into the monolayer was considerably enhanced. Analysis of FTIR spectra of soluble lysenin-His demonstrated that the protein contained 27% beta-sheet, 28% aggregated beta-strands, 10% alpha-helix, 23% turns and loops and 12% different kinds of aggregated forms. In membrane-bound lysenin-His the total content of alpha-helices, turns and loops, and beta-structures did not change, however, the 1636cm(-1) beta-sheet band increased from 18% to 31% at the expense of the 1680cm(-1) beta-sheet structure. Spectral analysis of the amide I band showed that the alpha-helical component was oriented with at 41 degrees to the normal to the membrane, indicating that this protein segment could be anchored in the hydrophobic core of the membrane.

The molecular organization of prenylated flavonoid xanthohumol in DPPC multibilayers: X-ray diffraction and FTIR spectroscopic studies

Xanthohumol (XN) is the major prenylated flavonoid found in hop resin. It has attracted considerable attention in recent years due to its wide spectrum of biological activities and the beneficial effect on human health. Since lipid membrane is first target for biologically active compounds, we decided to investigate the influence of XN on the dipalmitoylphosphatidylcholine (DPPC) multibilayers. Interactions of XN with DPPC were investigated as a function of temperature and its concentration by using X-ray diffraction and the ATR-FTIR spectroscopy techniques. The aim of understanding the mechanisms of molecular interactions between XN and DPPC was to indicate the localization of the XN with respect to the membrane and the type of interaction with phospholipids. The results revealed that XN changes the physical properties of the DPPC multibilayers in the form of dry film. A new complex formation between XN and DPPC is reported. The detailed analysis of refraction effect indicates the changes in electron density ratio between hydrophobic and hydrophilic zones of lipid at phase transition. This is in compliance with reported changes in FTIR spectra where at pretransition XN moves from interface region between polar heads to the neighborhood of phosphate groups.

Raman Spectroscopic Study of Aggregation Process of Antibiotic Amphotericin B Induced by H+, Na+, and K+ Ions

The normal and the preresonance Raman effects (NR and PRR) of spectroscopy have been used to monitor and explain the aggregation processes of amphotericin B (AmB) in aqueous solution at different pH values and containing the K(+) and Na(+) ions. The resonance-enhanced and normal vibrational Raman spectra were recorded with a semiconductor laser (ex 785 nm) and an argon laser (ex 514.5 nm) for investigation of interactions between AmB chromophores. The essential difference between the samples stimulated by resonance-enhanced and by near-infrared was in the C=C stretching mode region of polyene chain. The processes connected with the aggregation of AmB led to changes in the chromophore, which were only visible as a remarkable broadening of the band centered at 1558 cm(-1). The understanding of possible physical mechanisms responsible for the molecular aggregation of the drug is important from the pharmaceutical applicability standpoint.

Influence of K+ and Na+ ions on the aggregation processes of antibiotic amphotericin B: electronic absorption and FTIR spectroscopic studies.

The ionophore properties of amphotericin B (AmB) are related to the transport of Na(+) and K(+) ions across the molecular pores formed by this antibiotic in lipid membranes. In this paper, we present a new, complementary mechanism in which the -COO(-) group of the antibiotic is involved in the binding process of Na(+) and K(+) ions. Spectroscopic studies indicate that K(+) and Na(+) ions play an important role in the AmB aggregation process. Evidence in several spectral regions shows that K(+) ions exhibit a stronger ionic binding affinity to the -COO(-) group relative to Na(+). Overall, our findings indicate that monovalent ions can affect the molecular organization of AmB in substantially different ways not previously considered to be significant for their biological action.

Spectroscopic studies of amphotericin B–Cu2+ complexes

The aim of this research is to investigate amphotericin B (AmB)–Cu2+ complexes in aqueous solution at different pH values. Electronic absorption, circular dichroism (CD), Raman and FTIR spectroscopies were used in this study. We found that different concentrations of AmB and Cu2+ ions in solution leads to formation of complexes with stoichiometry of 2:1 and 1:1. The formation of AmB–Cu2+ complexes at physiological pH values is accompanied by changes of the molecular organization of AmB especially disaggregation. These observed effects might be significant from a medical point of view.

Effect of Solvent Polarizability on the Keto/Enol Equilibrium of Selected Bioactive Molecules from the 1,3,4-Thiadiazole Group with a 2,4-Hydroxyphenyl Function

Three novel 1,3,4-tiadiazole-derived compounds with biological-activity, i.e., 4-(5-(methylamino)-1,3,4-thiadiazol-2-yl)benzene-1,3-diol (MDFT), 4-(5-(phenylamino)-1,3,4-thiadiazol-2-yl)benzene-1,3-diol (PhATB), and 4-(5-(4-chlorophenylamino)-1,3,4-thiadiazol-2-yl)benzene-1,3-diol (4-CIPhATB) were characterized with the use of several spectroscopic methods. Detailed UV-vis studies revealed keto/enol tautomerism of the examined compounds. The absorption spectra recorded in nonpolar solvents exhibited bands that were characteristic of keto tautomers, while in polar solvents the enol form is predominant. A number of spectra revealed the presence of both tautomeric forms in the solution. The keto/enol equilibria observed were both solvent- and temperature-dependent. The keto/enol equilibrium was also observed using FTIR spectroscopy. A detailed analysis of the spectroscopic data leads to a conclusion that the solvent-induced tautomerism of the selected compounds from the 1,3,4-thiadiazole group does not depend on the electric dipole moment of the solvent but more likely on its average electric polarizability. Additionally, a clear effect of the substituent present in the molecule on the tautomeric equilibrium in the selected 1,3,4-thiadiazole analogues was noted.

Solvent Effects on Molecular Aggregation in 4-(5-Heptyl-1,3,4-thiadiazol-2-yl)benzene-1,3-diol and 4-(5-Methyl-1,3,4-thiadiazol-2-yl)benzene-1,3-diol.

The article presents the results of spectroscopic studies of 4-(5-methyl-1,3,4-thiadiazol-2-yl)benzene-1,3-diol (C1) and 4-(5-heptyl-1,3,4-thiadiazol-2-yl)benzene-1,3-diol (C7) in organic solvent solutions. Depending on the concentration of the compound used, three bands were observed in the fluorescence emission spectra of the compounds in DMSO solutions. A single band was observed in methanol, propan-2-ol, or ethanol. The significantly shortened fluorescence lifetimes and the different shapes of circular dichroism (CD) spectra clearly indicate association of the fluorescence effects with the aggregation processes in the analyzed compounds. The differences in the course of the CD spectra also imply an effect of the substituent group structure on the molecule aggregation interactions. Therefore, it has been postulated that the occurrence of the different spectral forms induced by changes in the compound concentration may be related to the aggregation effects of C1 and C7 molecules, which are also induced by differences in the alkyl substituent structure.