Krzysztof Polewski

Antioxidant activity of daidzein, a natural antioxidant, and its spectroscopic properties in organic solvents and phosphatidylcholine liposomes

Daidzein, one of major isoflavones found in soybeans, has a wide spectrum of physiological and pharmacological functions. The observed biological effects involve its interactions with lipid bilayers, usually detected by indirect methods. In this study we use the native fluorescence of daidzein to report changes observed during its interactions with organic solvents and in a phosphatidylcholine membrane. We have investigated interactions of daidzein with lipid bilayers of egg phosphatidylcholine (PC) by absorption and fluorescence methods. The data obtained indicate emission arises from the conjugate anion in excited singlet state. The fluorescence is found to increase with the basicity of the solution and the polarity of the solvent. An increase in fluorescence anisotropy in the presence of membranes suggests partial incorporation of daidzein molecules into the bilayer. Two fluorescence lifetime components, 1.5 ns and 3.5 ns, reflects the partition of daidzein between aqueous and membrane environments, respectively. On the basis of the obtained spectroscopic data we conclude that up to 15% of daidzein is located in hydrophilic region of the membrane whereas the rest is distributed in aqueous bulk and aqueous/membrane interface. For studying the antioxidant activity of daidzein against lipid peroxidation initiated by AAPH the molecule of C11-BODIPY581/591 has been used as a fluorescent oxidation indicator. The results show that the presence of daidzein anions in the membrane interface increases the inhibitory effect on lipid peroxidation compared to the neutral form of daidzein.

Simultaneous resolution of spectral and temporal properties of UV and visible fluorescence using single-photon counting with a position-sensitive detector

A new fluorescence spectrometer has been assembled at the U9B beamline of the National Synchrotron Light Source to allow simultaneous multiwavelength and time‐resolved fluorescence detection, as well as spatial imaging of the sample fluorescence. The spectrometer employs monochromatized, tunable UV and visible excitation light from a synchrotron bending magnet and an imaging spectrograph equipped with a single‐photon sensitive emission detector. The detector is comprised of microchannel plates in series, with a resistive anode for encoding the position of the photon‐derived current. The centroid position of the photon‐induced electron cascade is derived in a position analyzer from the four signals measured at the corners of the resistive anode. Spectral information is obtained by dispersing the fluorescence spectrum across one dimension of the detector photocathode. Timing information is obtained by monitoring the voltage divider circuit at the last MCP detector. The signal from the MCP is used as a ‘‘sta...

Fluorescence of matrix isolated guanine and 7-methylguanine.

We have prepared argon and nitrogen matrices containing guanine and 7-methylguanine, and measured their absorption, fluorescence excitation and fluorescence emission spectra. The fluorescence excitation spectrum of guanine shows four well-resolved bands in the range from 170 to 290 nm; excitation at the wavelengths of each of these bands results in a fluorescence emission with maximum intensity near 350 nm and a single-exponential decay with a lifetime of about 10 ns. There are significant differences between the fluorescent excitation and emission spectra of guanine and of 7-methylguanine, suggesting that the fluorescence observed from the guanine sample does not arise from a minority tautomer.

Time-resolved fluorescence using synchrotron radiation excitation: A powered fourth-harmonic cavity improves pulse stability

The pulsed nature or ‘‘time structure’’ of synchrotron radiation from electron storage rings is used to measure the kinetics of the decay of electronically excited states and is particularly useful because the wavelength of excitation can be chosen at will. However, changes in the length of the pulses of radiation from a storage ring resulting from the gradual decrease of current circulating in the ring during the course of a ‘‘fill’’ limit the duration of data collection, and hence photometric sensitivity. A fourth‐harmonic cavity that was recently added to the vacuum ultraviolet (VUV) storage ring at the National Synchrotron Light Source slows the loss of current during a fill, and thus increases the total number of photons produced. When operated in a passive (unpowered) mode, however, the fourth‐harmonic cavity increases both the average width of the photon pulses and the changes in width that occur during a fill, thus reducing the usefulness of the VUV ring in timing experiments. We demonstrate that ...

Photostability of alpha-tocopherol ester derivatives in solutions and liposomes. Spectroscopic and LC-MS studies.

α-Tocopherol (Toc) is known to degrade to the tocopheroxyl radicals (Toc) by exposure to UV light irradiation. In the present study, the stability of Toc ester derivatives exposed to UV light was investigated and compared with Toc in organic solution and in phospholipid vesicles. To follow the depletion of Toc and its esters the absorbance and fluorescence methods were applied whereas degradation products were detected using LC-MS method. The irradiation with UVB light of air-equilibrated solutions of di-α-Tocopheryl malonate (DTMO), α-Tocopheryl malonate (TMO) and α-Tocopheryl succinate (TS) strongly modifies their absorption and fluorescence spectra. Upon UVB irradiation, absorption band at 279/285nm becomes less pronounced indicating the photodegradation of esters. During irradiation, the fluorescence maximum of esters at 305nm shifts to 326nm, a maximum characteristic for Toc. Photorecovery of Toc from its esters derivatives was finally confirmed by LC-MS method. Among studied esters, only α-tocopheryl nicotinate (TN) did not undergo depletion and appeared resistant to UVB radiation. Kinetic studies indicated that photoinduced transformation occurs through the first order consecutive reaction chain mechanism. The photodissociation of Toc esters in the liposomes occurred with one order of magnitude slower than in organic solvents. Using MS/MS method it was found that final stable product of irradiation was α-tocopheryl quinone (TQ), an animal and plant metabolite of Toc.

Disruptive effect of tocopherol oxalate on DPPC liposome structure: DSC, SAXS, and fluorescence anisotropy studies

α-Tocopherol oxalate (TO), a tocopherol ester derivative, was investigated for its effect on the structural changes of fully hydrated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes, as a function of concentration and temperature, by applying differential scanning calorimetry (DSC), small angle X-ray scattering (SAXS), and DPH fluorescence anisotropy methods. The DSC and DPH anisotropy data indicated that TO embedded into DPPC membrane lowered the enthalpy (ΔHm) and temperature (Tm) of the main phase transition as well its cooperativity. Fluidization of the membrane at a lowered temperature was accompanied by formation of mixed structures of tocopherol-enriched domains. SAXS studies showed the formation of various ordered structures in DPPC gel-phase during incorporation of TO into the bilayer, as evidenced by the existence of lamellar phases with repeat distances (d) of 6.13 and 6.87 nm, assigned to TO-enriched domains and a lamellar, liquid-ordered DPPC phase with d = 8.45 nm at increasing TO concentrations with lowering and broadening of the Bragg peaks, and diffuse scattering, characteristic of a fluid Lα phase, were observed. In DPPC fluid-phase, the increasing presence of TO at low concentrations resulted in the appearance of a liquid-ordered phase with repeat d = 6.9 nm coexistent with a lamellar structure with d = 9.2 nm, assigned to liquid-disordered structures. An increasing repeat distance observed with raising the TO amount in the DPPC bilayer evolved from an increasing interlamellar water layer of increasing thickness. Presence of TO facilitated penetration of water molecules into the acyl chain region which decreased van der Waals interactions in the bilayer. The DSC, SAXS, and fluorescence anisotropy data established that TO exhibited pronounced disruptive activity in DPPC membranes compared to α-tocopherol. The driving force of the observed action was attributed to electrostatic and dipole interactions of the acidic moiety with the polar head group of phospholipids in the interface region of the bilayer.

Incorporation of Ag Nanoparticles Into Micelles. Stability Studies of Self-Organized Nanoparticlesmicelles Structures

Abstract In this paper we present the results of measured physical parameters of self-organized structures consisting of hydrophobic functionalized silver nanoparticles and amphiphilic molecules capable of micelles formation. Those systems may be considered as simple models for transfer of nanoparticles through the biological membrane. Three different surfactants were used: negatively charged sodium dodecyl sulphite, SDS, neutral Triton X-100 and positively charged tetredodecyltrimethyl ammonium bromide, TTABr. We have found that hydrophobic functionalized Ag nanoparticles are encapsulated in neutral Triton X-100 micelles with a diameter of 10 nm without significant change in the size of the micelles. The efficiency of encapsulation of Ag by SDS micelles is lower compared to Triron X-100 and no incorporation of Ag nanoparticles into TTABr occurs. Obtained results indicate that in aqueous environment ionic properties of molecules creating micelles and concentration ratios between components determine the efficiency and kinetics of two competitive processes association or aggregation of nanoparticles and encapsulation of Ag nanoparticles within micelles.

Soft x-ray circular dichroism and scattering using a modulated elliptically polarizing wiggler and double synchronous detection

We have constructed an experimental station (beamline) at the National Synchrotron Light Source to measure circular dichroism (CD) using soft x-rays (250 less than or equal to hv less than or equal to 900 eV) from a time modulated elliptically polarizing wiggler. The polarization of the soft x-ray beam switches periodically between two opposite polarizations, hence permitting the use of phase-sensitive (lock-in) detection. While the wiggler can be modulated at frequencies up to 100 Hz, switching transients limit the actual practical frequency to approximately equals 25 Hz. With analog detection, switching transients are blocked by a chopper synchronized to the frequency and phase of the wiggler. The CD is obtained from the ratio of the signal recovered at the frequency of polarization modulation, f, to the average beam intensity, which is recovered by synchronous detection at frequency 2f.