Ewa Szajdzinska-Pietek

Interaction of cationic protoberberine alkaloids with human serum albumin. No spectroscopic evidence on binding to Sudlow's site 1.

Physicochemical studies on drug interactions with human serum albumin (HSA) are relevant for elucidation, at the molecular level, of the processes occurring in vivo. In this work using optical spectroscopic methods (fluorescence, absorption, circular dichroism), we have investigated aqueous HSA solutions containing pharmaceutically important isoquinoline alkaloids, berberine and palmatine. The primary objective was to verify whether the two compounds are located in the subdomain IIA of the secondary HSA structure as reported in literature. We prove that the excited state of Trp214 residue is not quenched by the alkaloids; all observed changes in fluorescence spectra are due to inner filter effects. Furthermore, differential absorption spectra indicate that the ligands remain in a waterlike microenvironment. We infer that bound alkaloid molecules are located at the protein/water interface. Yet, such binding mode can induce some unfolding of the HSA molecule detectable in the far-UV circular dichroism (CD) spectra. We have also performed, for the first time, pulse radiolysis studies of hydrated electron scavenging in the HSA/alkaloid systems and have measured steady-state absorption spectra of irradiated samples. The results reveal that neither berberine nor palmatine is effectively protected by the protein against one-electron reduction, which is consistent with the aforementioned conclusion.

Unexpected radical generation on γ-irradiating metastable forms of water at 77 K. Invited Lecture

The effects of γ-irradiating three metastable forms of water at 77 K have been studied by electron spin resonance spectroscopy and are compared with those of hexagonal ice. Two amorphous forms of water were made by so-called hyperquenching of liquid water droplets (hyperquenched glassy water, HGW) and by deposition of water vapours (amorphous solid water, ASW) at 77 K. Metastable cubic ice was made either by heating HGW, or by hyperquenching liquid water droplets at 170 K. Whereas γ-irradiation of hexagonal ice at 77 K produces only OH radicals as primary radiation product, on γ-irradiation of HGW and ASW comparable yields of OH and HO2 radicals were observed, and their ratio did not depend on radiation dose. Mainly OH radicals were formed on γ-irradiating cubic ice made directly from liquid water. In contrast, comparable amounts of OH and HO2 radicals were formed on irradiation of cubic ice made by heating HGW, and their relative yields depend strongly on the irradiation dose. HO2 radicals are formed as primary products of radiation, as are OH radicals, and their formation is attributed to the abundant presence of defects, with two oxygens facing each other without hydrogen in between, in HGW, in ASW and in disordered regions of cubic ice. When small amounts of thymine were added to HGW, thymine radicals were formed by addition of atomic hydrogen on γ-irradiation already at 77 K at the expense of the relative contribution of HO2 radicals. This is not due to scavenging of once-formed HO2 radicals by thymine, but it occurs concurrently with HO2 radical formation. Therefore, it is rationalized by a concerted process involving the primary intermediate H2O+. The radiation effect on thymine must be indirect because of the large water-to-thymine ratio of ≈104 in homogeneous glassy solution. Possible implications of these studies are discussed for our understanding of (i) defects in metastable forms of water and their detection via HO2 radical formation, (ii) radiation damage to biomolecules in aqueous solution, and (iii) radical generation on high-energy irradiation of ASW in outer space and the secondary formation of stable molecules on decay of the radicals.

Pulse radiolytic investigation of perfluorinated surfactants in aqueous solutions

Pulse radiolysis studies were performed for aqueous solutions of ammonium perfluorooctanoate, tetraethylammonium perfluorooctyl sulfonate, and an ammonium salt of a carboxylic perfluoropolyether derivative. The protiated system of sodium octanoate was also examined for comparison. The rate constants of hydrated electron (e−aq) and ·OH radical reactions with these amphiphiles, and absorption spectra of the intermediate products are reported for the first time. It is demonstrated that the critical micellar concentrations (CMC) can be determined from the kinetic data. The observed differences in e−aq reactivity towards fluorinated amphiphiles above CMC are rationalized in terms of different charge density at the micellar interface.

QUENCHING OF PYRENE FLUORESCENCE BY AMPHIPHILIC NITROXIDE RADICALS EMBEDDED IN CATIONIC MICELLES

Abstract Steady-state and nanosecond time-resolved studies have been carried out on the fluorescence quenching of excited pyrene by n-doxylstearic methyl esters (n-DSE, n = 5, 10, 12) in an aqueous solution of cationic micelles of hexadecyltrimethylammonium chloride (HTAC, 0.1 M). The aggregation number (N = 114 ± 6) and the rate constants of intramicellar quenching (1.4 × 107, 1.3 × 107 and 1.05 × 107 s−1 for 5-, 10- and 12-DSE respectively) have been determined. The results are discussed in terms of the average location of the luminophore and the quencher molecules in the aggregates, considering previous findings of electron spin echo modulation studies of n-doxylstearic acid spin probes in micellar systems.

Electron spin resonance studies on tryptophan photolysis in frozen micellar systems of anionic surfactants

Abstract UV photolysis of tryptophan (Trp) was studied by the electron spin resonance method in frozen solutions of simple micelles (SMs) and reversed micelles (RMs) of anionic surfactants: sodium bis(2-ethylhexyl)sulphosuccinate, sodium dodecylsulphate and ammonium perfluoropolyether carbonate. The primary species stabilized at 77 K in protiated systems are Trp + , Trp and alkyl-type radicals while, in the perfluorinated RM system, only Trp + is observed. The yield of paramagnetic species is an order of magnitude higher in microheterogeneous systems than in homogeneous aqueous solutions. It is higher in RM than in SM samples, parallel to the fraction of Trp molecules bound to micellar interface. In the presence of oxygen the primary photoproducts transform into peroxy radicals on heating the sample above 100 K. In deaerated samples, Trp + survives up to temperatures of 190 K or higher (depending on the system).

Kinetic patterns of bimolecular reactions: H atom and Cl−2 radical decay in γ-irradiated zinc chloride/hydrochloric acid glasses

Abstract Isothermal recombination of H atoms and disproportionation of Cl−2 radicals in HClH2O- ZnCl2 glasses can be described with the use of time-dependent rate constants of the form k(t) = Btα−1, 0

Radiation cryochemistry of frozen dilute aqueous solutions: influence of the extent of solute segregation on the radiolysis pathway

Polycrystalline hexagonal ice containing thymine was γ-irradiated at 77 K, and X-band electron paramagnetic resonance spectra of the resulting radicals were recorded at this temperature immediately after irradiation, and after thermal annealing of the samples at temperatures up to 250 K. The examined frozen samples were obtained from hyperquenched glassy aqueous solution containing 0.005 M of thymine, by using two different procedures: (i) crystallization of the glass by heating up to 250 K, and (ii) warming the glass up to room temperature to melt it, and subsequent cooling of the solution in liquid N2. Thymine-derived radicals were detectable only in the samples obtained by crystallization of the glass. We conclude that the extent of solute segregation is less for the frozen aqueous solution made by route (i) than by route (ii). Therefore, the solute is more hydrated in the frozen sample made by route (i) and the primary products of water radiolysis can react with the solute. The present results are compared with those reported for the thymine/hyperquenched glassy water system (Bednarek et al. , J. Phys. Chem. B 103 , 6824 (1999)).

Quenching of excited states of pyrene derivatives by amphiphilic nitroxide radicals in cationic micellar solutions. Dynamics and location of the guest molecules in the aggregates

Abstract Steady-state and nanosecond time-resolved studies have been performed on fluorescence quenching of excited pyrene sulfonic acid and pyrene butyric acid (PBA and PSA, respectively, in the form of sodium salts) by n -doxylstearic methyl esters ( n -DSE, n =5, 10, 12) in aqueous solution of cationic micelles of hexadecyltrimethylammonium chloride (HTAC, 0.1 M). The results are compared with those reported earlier for pyrene (P) as a luminophore (Szajdzinska-Pietek and Wolszczak, Chem. Phys. Lett., 270 (1997) 527). It is shown that PBA/ n -DSE and PSA/ n -DSE luminophore/quencher pairs, unlike P/ n -DSE pairs, are not suitable for determination of the micellar aggregation number, neither by the ‘quasi-static’ method of Turro—Yekta nor by the dynamic method of Infelt—Tachiya. It is suggested that the fluorescence decay curves in the examined systems can be adequately described in terms of dispersive kinetics using the time-dependent rate coefficient for intramicellar quenching process. The average location of the luminophore and the quencher molecules in the aggregates is discussed, considering previous findings of electron spin echo modulation studies of n -doxylstearic acid spin probes in micellar systems.

ESR study of γ-irradiated hyperquenched glassy water doped with thymine and its nucleoside

Using X-band ESR spectroscopy we have studied radicals produced by g-irradiation at 77 K in hyperquenched glassy water (HGW) doped with thymine, 2 0 -deoxythymidyne, 2-deoxy-d-ribose, and the equimolar mixture of thymine and 2deoxy-d-ribose. In HGW matrix the solute molecules are not freeze-concentrated, so the indirect radiation effects on biomolecules can be followed. The results are discussed in terms of the mechanism of radical formation and the role of sugar in radiation damage of nucleosides. r 2003 Elsevier Science Ltd. All rights reserved.

A dispersive model of radical accumulation in irradiated solids

Abstract- A dispersive model of radical accumulation in irradiated solids is discussed by the example of hexagonal ice (Ih) and cubic ice (Ic) obtained by annealing of hyperquenched glassy water at 160 K. The model assumes that radical production upon γ-irradiation is accompanied by their second-order decay, which proceeds according to dispersive kinetics with the time-dependent specific reaction rate k(t) = Btα-1, where B = constant and α is the dispersion parameter equal to 0.40 for both the hexagonal and cubic ices. The radicals, OH in Ih, and OH plus HO2 in Ic are produced at the same rate upon γ-irradiation. The observed enhancement of radical accumulation in ice Ic in comparison with Ih is due to the marked decrease of radical recombination in ice Ic. This is rationalized by the hindrance of radical transport in the lattice of ice Ic containing substantial amounts of Bjerrum-type L-defects.