Elena Busi

EPR and O2· — scavenger activity: Cu(II)—peptide complexes as superoxide dismutase models

Several copper(II) complexes with aminoacids and peptides are known to show superoxide dismutase (SOD)-like activity. EPR spectroscopy has proved to be a useful tool for studying the complex equilibria of the copper(II) ion and various ligands of biological importance in solution. In the present work, a variety of copper(II) complexes with di-, tri- and tetra-peptides containing only glycine residues (GG, GGG and GGGG) and others containing a histidyl residue in different positions (HGG, GHG, GGH and GGHG) have been investigated. EPR parameters obtained by extensive use of computer simulation of spectra lead to reliable spin Hamiltonian EPR parameters at both room temperature and in frozen solution. The molecular orbital coefficients computed from the anisotropic EPR data and the d-d electronic energies are used to characterize different arrangements of the complexes. Estimation of the scavenger activity of the complexes due to the particular environment created by the ligands around copper is discussed in the frame of the structure-activity relationship.

Modified cytochrome c/H2O2 system: spectroscopic EPR investigation of the biocatalytic behaviour

In recent years there has been growing interest in methods for the degradation of polycyclic aromatic hydrocarbons. Cytochrome c (Cyt c) systems in the presence of H2O2 are able to oxidize various aromatic compounds. In order to investigate ways of improving the performance of Cyt c/H2O2 oxidation systems, site-directed mutagenesis, and chemical modifications on the hemoprotein surface with poly(ethylene glycol) and methylation of the active site have been performed. The EPR technique and UV–VIS spectroscopy have been used to identify radical intermediates and heme iron spin states of the chemical modified Cyt c (PEG-Cyt-Met) and Cyt c mutants.

In situ ATR/FTIR studies of protein adsorption on polymeric materials: effectiveness of surface heparinization

The adsorption of two proteins from human plasma (human serum albumin (HSA) and human fibrinogen (HFG) onto six different polymeric surfaces (two of which are heparinized), has been studied byin situ ATR/FTIR spectroscopy. The different surface characteristics are reflected by different interfacial behaviours of the two proteins, but while both proteins unfold upon adsorption on all the different non-heparinized materials, they maintain the native conformation once adsorbed on the heparinized surfaces. These findings emphasize the effectiveness of surface heparinization.

On the mechanism of ion transport through lipid membranes mediated by PEGylated cyclic oligosaccharides (CyPLOS): An ESR study

The mechanism underlying the ionophoric activity of CyPLOS (cyclic phosphate-linked oligosaccharide, 2), a carbohydrate-based synthetic ion transporter decorated with four tetraethylene glycol (TEG) chains, has been investigated by an integrated electron spin resonance (ESR) approach. The mode of interaction of the ionophore with lipid bilayers has been studied by quantitatively analyzing the perturbations in the ESR spectrum of an ad hoc synthesized spin-labeled CyPLOS analog (6), and, in parallel, in the spectra of spin-labeled lipids mixed with 2. The results point to a positioning of the cyclic saccharide backbone close to the lipid headgroups, largely exposed to the aqueous medium. The TEG chains, carrying a terminal benzyl group, are deeply inserted among the lipid acyl chains, showing good mobility and flexibility. As a consequence, the order of the acyl chain packing is significantly reduced, and water penetration in the bilayer is enhanced. The resulting asymmetric perturbation of the bilayer leads to its local destabilization, thus facilitating, through a non-specific mechanism, the ion transport through the membrane.

Identification and structural characterization of a transient radical species in the uricase reaction mechanism

Uricase catalyzes the oxidation of urate to form allantoin, carbon dioxide and hydrogen peroxide. In this article, we demonstrate for the first time the presence of a radical intermediate involved in the reaction mechanism. Such radical species was entrapped using 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide as spin trap and the relative adduct was detected by electron paramagnetic resonance (EPR) technique. A structure of such radical (5-hydroperoxy isourate) is proposed, through chemical results and density functional theory calculations of the EPR coupling constants.

Oxygen-Ozone in orthopaedics: EPR detection of hydroxyl free radicals in ozone treated “nucleus pulposus” material

The direct and indirect application of oxygen-ozone in several orthopaedic affections was briefly reviewed, trying to understand possible mechanisms of action explaining the surprisingly good results and no toxicity. Moreover, by using EPR spin trapping technique with DEPMPO the generation of free radicals by treating human “nucleus pulposus” material with oxygen-ozone (O2-O3) was investigated. The DEPMPO spin adduct of hydroxyl radical (DEPMPO-OH) was detected. The presence of ·OH even at a very low ozone concentration (5 μg/mL) may explain why this material undergoes to a progressive breakdown with re-absorption and consequent disappearance of a hernial disk. Thus the intradiscal insufflation of a minute volume of O2-O3 may represent a new efficient, inexpensive and atoxic chemionucleolytic approach.

Redox-active polymers: synthesis and exchange reaction of amino compounds containing a cyclic disulfide

Abstract Two monoamidated amino compounds, bearing the cyclic disulfide moiety of lipoic acid, were prepared by imidazole group transfer with aliphatic diamines (1,3-diaminopropane and piperazine). The products, characterized by spectroscopic methods ( 1 H nuclear magnetic resonance and Fourier-transform infra-red), were studied in solution. Basicity constants for the protonation of the basic nitrogens were evaluated in aqueous media, and redox properties in dimethylsulfoxide, by cyclic voltammetry. Insoluble polymers were obtained by polymer-analogue exchange reaction of the monoamidated compound with a benzotriazole residue present in preformed poly( N -acryloylbenzotriazole). A copolymer of the same monomer with N -vinylpyrrolidinone was soluble in solvents, giving a response in cyclic voltammetry. Its positive anode peak was slightly higher than that of the simple monoamidated compounds.

On the microscopic and mesoscopic perturbations of lipid bilayers upon interaction with the MPER domain of the HIV glycoprotein gp41

The effect of the 665-683 fragment of the HIV fusion glycoprotein 41, corresponding to the MPER domain of the protein and named gp41MPER, on the microscopic structure and mesoscopic arrangement of palmitoyl oleoyl phosphatidylcholine (POPC) and POPC/sphingomyelin (SM)/cholesterol (CHOL) lipid bilayers is analyzed. The microscopic structuring of the bilayers has been studied by Electron Spin Resonance (ESR) spectroscopy, using glycerophosphocholines spin-labelled in different positions along the acyl chain. Transitions of the bilayer liquid crystalline state have been also monitored by Differential Scanning Calorimetry (DSC). Changes of the bilayers morphology have been studied by determining the dimension of the liposomes through Dynamic Light Scattering (DLS) measurements. The results converge in showing that the sample preparation procedure, the bilayer composition and the peptide/lipid ratio critically tune the lipid response to the peptide/membrane interaction. When gp41MPER is added to preformed liposomes, it positions at the bilayer interface and the lipid perturbation is limited to the more external segments. In contrast, if the peptide is mixed with the lipids during the liposome preparation, it assumes a trans-membrane topology. This happens at all peptide/lipid ratios for fluid POPC bilayers, while in the case of rigid POPC/SM/CHOL membranes a minimum ratio has to be reached, thus suggesting peptide self-aggregation to occur. Peptide insertion results in a dramatic increase of the lipid ordering and bilayer stiffening, which reflect in significant changes in liposome average dimension and distribution. The biological implications of these findings are discussed.

EPR Spin Trapping of a Radical Intermediate in the Urate Oxidase Reaction

Urate oxidase, or uricase (EC 1.7.3.3), is a peroxisomal enzyme that catalyses the oxidation of uric acid to allantoin. The chemical mechanism of the urate oxidase reaction has not been clearly established, but the involvement of radical intermediates was hypothesised. In this study EPR spectroscopy by spin trapping of radical intermediates has been used in order to demonstrate the eventual presence of radical transient urate species. The oxidation reaction of uric acid by several uricases (Porcine Liver, Bacillus Fastidiosus, Candida Utilitis) was performed in the presence of 5‐diethoxyphosphoryl‐5‐methyl‐pyrroline‐N‐oxide (DEPMPO) as spin trap. DEPMPO was added to reaction mixture and a radical adduct was observed in all cases. Therefore, for the first time, the presence of a radical intermediate in the uricase reaction was experimentally proved.

Complex rotational dynamics of the Cu(II)-bleomycin system in mobile and viscous media.

Metal-free bleomycin is an antitumor antibiotic. Studies on the biological activity of bleomycin indicate that the metal-free bleomycin is activated through its binding of available metal ions, such as Cu(II) and Fe(II). The Cu(II)–bleomycin [Cu(II)Blm] complex is the transport form found in blood and can be considered a “magnetopharmaceutical” compound. Its final target is cancerous tissue where it becomes localized.1,2 Living systems present a wide range of environments that can alter the dynamics of the nominally isotropically tumbling agents. Rotational correlation time, τR, is important in determining proton relaxation enhancement.3,4 A previous study in the mobilized phase showed a difference in the electron paramagnetic resonance (EPR) parameters for cupric bleomycin in the liquid as opposed to the solid state.5 Aqueous and 46% wt/vol sucrose solutions have been studied from near physiological temperature to near freezing to determine the dominant rotational correlation time in aqueous and viscous environments.