X-ray absorption spectroscopy studies of the adducts formed between cytotoxic gold compounds and two major serum proteins.

Gold metallodrugs form a class of promising antiproliferative agents showing a high propensity to react with proteins. We exploit here X-ray absorption spectroscopy (XAS) methods [both X-ray absorption near-edge spectroscopy (XANES) and extended X-ray absorption fine structure (EXAFS)] to gain insight into the nature of the adducts formed between three representative gold(I, III) metallodrugs (i.e., auranofin, [Au(2,2'-bipyridine)(OH)(2)](PF(6)), Aubipy, and dinuclear [Au(2)(6,6'-dimethyl-2,2'-bipyridine)(2)(µ-O)(2)](PF(6))(2), Auoxo6) and two major plasma proteins, namely, bovine serum albumin (BSA) and human serum apotransferrin (apoTf). The following metallodrug-protein systems were investigated in depth: auranofin/apoTf, Aubipy/BSA, and Auoxo6/apoTf. XANES spectra revealed that auranofin, upon protein binding, conserves its gold(I) oxidation state. Protein binding most probably takes place through release of the thiosugar ligand and its subsequent replacement by a thiol (or a thioether) from the protein. This hypothesis is independently supported by EXAFS results. In contrast, the reactions of Aubipy with serum albumin and of Auoxo6 with serum apoTf invariantly result in gold(III) to gold(I) reduction. Gold(III) reduction, clearly documented by XANES, is accompanied, in both cases, by release of the bipyridyl ligands; for Auoxo6 cleavage of the gold-gold dioxo bridge is also observed. Gold(III) reduction leads to formation of protein-bound gold(I) species, with deeply modified metal coordination environments, as evidenced by EXAFS. In these adducts, the gold(I) centers are probably anchored to the protein through nitrogen donors. In general, these two XAS methods, i.e., XANES and EXAFS, used here jointly, allowed us to gain independent structural information on metallodrug/protein systems; detailed insight into the gold oxidation state and the local environment of protein-bound metal atoms was achieved in the various cases.

Use of EPR and FTIR to detect biological effects of ultrasound and microbubbles on a fibroblast cell line.

Structural and functional effects of exposing murine fibroblasts (NIH 3T3) to therapeutic ultrasound at 1 MHz frequency are described. These bioeffects can be attributed to the formation of free radical species by sonolysis of water. When cavitation occurs, dissociation of water vapor into H atoms and OH radicals is observed; these H atoms and OH radicals combine to form H(2), H(2)O(2), and HO(2). The radicals can chemically modify biomolecules, for example enzymes, DNA, and lipids. Generation of free radicals during exposure to ultrasound with or without encapsulated microbubbles (contrast agents) was studied by use of electron paramagnetic resonance with DMPO spin trapping. Recently the potential for possible use of these microbubbles in gene therapy has been investigated, because of the ability of the stabilized microbubbles to release their content when exposed to ultrasound. Structural changes were studied by Fourier-transform infrared spectroscopy, and induction of possible genotoxic damage by exposure of the cells to therapeutic ultrasound at 1 MHz frequency with our experimental device was verified by use of the cytokinesis-block micronucleus assay.

Deprotonation of glutamic acid induced by weak magnetic field: an FTIR-ATR study.

It has been claimed that weak extremely low frequency electromagnetic fields (ELF-EMFs) can affect biochemical reactions and a wide-ranging body of literature is available on this topic. Nevertheless, the physical nature of these effects remains largely unknown. We investigated the influence of ELF-EMF on glutamic acid solutions using Fourier transform infrared-attenuated total reflectance (FTIR-ATR) spectra. Samples were exposed for 10, 20, or 30 min to a weak EMF generated by Helmoltz coils, and then placed in a spectrometer. After exposure, those solutions that had a pH lower than the isoelectric point tended to show a shift toward the deprotonation of the carboxylic group, while solutions having a pH greater than the isoelectric point showed the deprotonation of the residual amine group. Moreover, at low pH values, we also detected a shift of the δ(antisym) band of the amine. The effects lasted a few minutes after exposure before the native configuration was restored. The spectral modifications were observed after each independent exposure to EMFs, and the same effects were seen by varying the frequencies in the range of 0-7 kHz. Therefore, the hypothesis of the existence of a resonant frequency that has been proposed elsewhere cannot be supported by the results of this study. The most surprising characteristic of this effect is the long-lasting nature of the perturbation, which is hard to be explained in terms of short-living excitations in biological matter.

UVB radiation induced effects on cells studied by FTIR spectroscopy.

We have made a preliminary analysis of the results about the effects on tumoral cell line (lymphoid T cell line Jurkat) induced by UVB radiation (dose of 310 mJ/cm(2)) with and without a vegetable mixture. In the present study, we have used two techniques: Fourier transform infrared spectroscopy (FTIR) and flow cytometry. FTIR spectroscopy has the potential to provide the identification of the vibrational modes of some of the major compounds (lipid, proteins and nucleic acids) without being invasive in the biomaterials. The second technique has allowed us to perform measurements of cytotoxicity and to assess the percentage of apoptosis. We already studied the induction of apoptotic process in the same cell line by UVB radiation; in particular, we looked for correspondences and correlations between FTIR spectroscopy and flow cytometry data finding three highly probable spectroscopic markers of apoptosis (Pozzi et al. in Radiat Res 168:698-705, 2007). In the present work, the results have shown significant changes in the absorbance and spectral pattern in the wavenumber protein and nucleic acids regions after the treatments.

Roughness of the plasma membrane as an independent morphological parameter to study RBCs: a quantitative atomic force microscopy investigation.

A novel approach to the study of RBCs based on the collection of three-dimensional high-resolution AFM images and on the measure of the surface roughness of their plasma membrane is presented. The dependence of the roughness from several parameters of the imaging was investigated and a general rule for a trustful analysis and comparison has been suggested. The roughness of RBCs is a morphology-related parameter which has been shown to be characteristic of the single cells composing a sample, but independent of the overall geometric shape (discocyte or spherocyte) of the erythrocytes, thus providing extra-information with respect to a conventional morphology study. The use of the average roughness value as a label of a whole sample was tested on different kinds of samples. Analyzed data revealed that the quantitative roughness value does not change after treatment of RBCs with various commonly used fixation and staining methods while a drastic decrease occurs when studying cells with membrane-skeletal alteration both naturally occurring or artificially induced by chemical treatments. The present method provides a quantitative and powerful tool for a novel approach to the study of erythrocytes structure through an ultrastructural morphological analysis with the potential to give information, in a non-invasive way, on the RBCs function.

Changes in Fe site structure from fetal to adult hemoglobin probed by XANES.

Iron X-ray absorption near edge structure (XANES) spectra of human fetal (F) and adult (A) deoxyhemoglobin (deoxyHb) measured at the Frascati synchrotron radiation facility reveal the different geometrical structure of the Fe-porphyrin complexes in the two proteins. By this method, having determined for the first time the variation of atomic positions in fetal and adult hemoglobin in solution (close to the 'in vivo' situation), we give further insight into the structure-function relationship in hemoglobins.

Influence of allosteric effectors on the heme conformation of dromedary ferrous nitrosylhemoglobin detected by XANES spectroscopy.

The changes of the Fe heme-active site conformation of dromedary (Camelus dromedarius) nitrosylhemoglobin (HbNO) induced by inositol hexakisphosphate (IHP) and chlofibric acid (CFA) have been studied by using X-ray absorption near-edge structure (XANES) spectroscopy. Structural information has been determined by multiple scattering analysis of the Fe K-edge XANES spectra. The proximal histidine is found to move away from iron centers by about 0.4 Angstrom on the average over the four hemes upon binding of CFA or stoichiometric amount of IHP. In molar excess of polyanion or in the simultaneous presence of IHP, CFA and chloride, the proximal histidine moves back to a position very close to that observed in pure buffer; yet, the structure modulation induced by the allosteric effectors is not completely reversible. Such findings parallel with the functional properties and the spectroscopic (e.g., EPR and absorbance) characteristics of HbNO.

Iron site structure of two irreversible hemichromes from human hemoglobin, untreated and oxidized to sulfoxide at MetD6(55)beta.

The Fe K-edge X-ray absorption near-edge structure (XANES) spectra of two irreversible human hemichromes, spontaneously formed from HbA and HbMetSO (a hemoglobin derivative, where MetD6(55)beta has been previously oxidized to sulfoxide by chloramine T) were determined. The results show that the hemichrome from HbMetSO is characterized by the distal histidyl imidazole moved within the bonding distance of the heme iron. Such structure is different from that of the hemichrome spontaneously produced from native human hemoglobin, which probably has a hydroxide group as sixth heme ligand.

Heterogeneity of the isolated subunits of the fetal and adult human hemoglobin in solution, detected by XANES spectroscopy.

Differences in the local structure of the heme in the isolated alpha-, beta- and gamma-chains of the adult and fetal human hemoglobin are detected by XANES (X-ray absorption near-edge structure) spectroscopy. The ligand bonding angle to the iron ion in the ligated forms and the displacement of the Fe respect to the porphyrin plane in the deoxy forms are found to be different for each chain.

XANES study of iron displacement in the haem of myoglobin.

The XANES (X-ray absorption near edge structure) spectra of deoxy human adult haemoglobin (HbA) and myoglobin (Mb) have been measured at the wiggler beam line of the Frascati synchrotron radiation facility. The XANES are interpreted by the multiple scattering cluster theory. The variations in the XANES between HbA and Mb are assigned to changes in the Fe-porphyrin geometry.

MRS study of the interaction of dihydropyridines with lipid molecules in phosphatidylcholine vesicles.

Dihydropyridines (DHPs), synthetic molecules used as antihypertensive agents, bind to plasma membrane receptors following diffusion through the hydrophobic phase. In this study, MRS technique has been used to clarify the interactions of the dihydrophyridines Nifedipine and Lacidipine within the lipid bilayer. 1D and 2D 1H MRS at high field have been employed to examine the behavior of unilamellar dimyristoyl-phosphatidylcholine liposomes when the two drugs have been inserted in the bilayer. In particular, the study represents an innovative application of 2D 1H NOESY technique to clarify different mechanisms of interactions of small molecules inside model membranes. On the other hand, 31P measurements have been performed in multilamellar dimyristoyl-phosphatidylcholine lipsomes to detect alterations of lipid polymorphic phases. The experiments show that the two dihydropyridines interact with the lipids by different modalities. Lacidipine undergoes a very strong interaction with lipids, possibly inducing a phase segregation of lipid molecules into the bilayer, while self-association seems to be the prevalent interaction of Nifedipine inside the bilayer.

Structural changes induced in proteins by therapeutic ultrasounds.

The structural effect induced by therapeutic ultrasound on proteins in aqueous solution has been investigated with FTIR spectroscopy, UV-VIS spectroscopy, circular dichroism and light scattering. Six proteins (cytochrome, lysozyme, myoglobin, bovine serum albumin, trypsinogen, and alpha-chymotrypsinogen A) with different molecular weight and secondary structure have been studied. The experiment has been performed using an ultrasound source at resonant frequency of 1 MHz and sonication times of 10, 20, 30, 40, 50, and 60 min. A different behaviour of proteins under sonication depends on the dominant secondary structure type (alpha-helix or beta-sheets) and on the grade of the ordered structure. The results suggest that the free radicals, produced by water sonolysis, have an important role in the changes of structural order.

Conformational changes of bovine beta-trypsin and trypsinogen induced by divalent ions: an energy-dispersive X-ray diffraction and functional study.

The radius of gyration (R(g)) of bovine trypsinogen and beta-trypsin was measured by an energy-dispersive X-ray technique as a function of Ca(2+) or SO(4)(2-) concentration; these results have been supplemented with measurements of association equilibrium constants of Ca(2+) to its binding site(s) on both serine proteases and some of their adducts (with an effector and/or an inhibitor). As a whole, all information reported in the present work demonstrates that: (i) the strains exerted by different ions on these proteases produce diverse structural modifications; and (ii) at least in the case of Ca(2+), the changes in R(g) can be ascribed to the direct interaction of the binding site(s) on the protein matrix with the cation.

Dynamic properties of an oriented lipid/DNA complex studied by neutron scattering.

The formation of lipid-DNA (CL-DNA) complexes called lipoplexes, proposed as DNA vectors in gene therapy, is obtained by adding DNA to a solution containing liposomes composed of cationic and neutral lipids. The structural and dynamic properties of such lipoplexes are determined by a coupling between the electrostatic interactions and the elastic parameters of the lipid mixture. An attempt to achieve a better understanding of the structure-dynamics relationship is reported herein. In particular, an elastic neutron scattering investigation of DOTAP-DOPC (dioleoyl trimethylammonium propane-dioleoyl phosphatidylcoline) complexed with DNA is described. Proton dynamics in this oriented CL-DNA lipoplex is found to be strongly dependent upon DNA concentration. Our results show that a substantial modification of the membrane dynamics is accompanied by the balancing of the total net charge inside the complex, together with the consequent displacement of interlayer water molecules.

Structural characterization of a new lipid/DNA complex showing a selective transfection efficiency in ovarian cancer cells.

We investigated, for the first time, by using Energy Dispersive X-ray Diffraction, the structure of a new ternary cationic liposome formulated with dioleoyl trimethylammonium propane (DOTAP), 1,2-dioleoyl-3-phosphatidylethanolamine (DOPE) and cholesterol (Chol) (DDC) which has been recently found to have a selective high gene transfer ability in ovarian cancer cells. Our structural results provide a further experimental support to the widely accepted statement that there is not a simple and direct correlation between structure and transfection efficiency and that the factors controlling cationic lipid/DNA (CL-DNA) complexes-mediated gene transfer depend not only on the formulations of the cationic liposomes and their thermodynamic phase, but also significantly on the cell properties.

Intermediate states in ligand photodissociation of carboxymyoglobin studies by dispersive X-ray absorption.

The ligand photodissociation of sperm whale carboxymyoglobin (MbCO) at low temperature (15K-100K) under extended illumination has been studied by X-ray Absorption Near Edge Structure (XANES) spectroscopy using the dispersive technique. XANES simulations through the multiple scattering (MS) approach allow one to interpret the spectroscopic data in structural terms, and to investigate the Fe site structure configurations of the states that follow the CO photodissociation as a function of temperature. The Fe site in the photoproduct is unbound, with an overall structure similar to the deoxy-form (Mb) of the protein. The Fe site structure changes from T < 30K(Mb*) to T > 50K (Mb**), revealing the existence of a slower unbound state Mb**. A model is proposed which includes the faster state (Mb*) as a planar porphyrin ring with a displacement of Fe from the heme plane of less than 0.3 A, and the slower state (Mb**) with a domed heme.

Room-temperature magnetic properties of oxy- and carbonmonoxyhemoglobin.

The magnetic susceptibility and the density of human oxy-(HbO(2)) and carbonmonoxyhemoglobin (HbCO) solutions of various concentrations have been measured at room temperature, with pure water used as a calibrant. Solutions of unstripped and stripped HbO(2) at pH 7.2 in unbuffered water solvent were always found to be less diamagnetic than pure water, whereas solutions of HbCO in identical conditions were always found to be more diamagnetic than pure water. After correcting for concentration-dependent density changes and assuming the HbCO samples to be fully diamagnetic, the paramagnetic reduction of the diamagnetic susceptibility of HbO(2) corresponds to a molar susceptibility per heme (chi(M) (heme)) of 2460 +/- 600 x 10(-6) cgs/mol.

Structure-function relationship in the serotransferrin: the role of the pH on the conformational change and the metal ions release.

We report experimental evidence that the pH value of the micro-environment of the protein is the key for the conformational changes of transferrin, rather than the ligated for unligated state of the binding sites. The "open" and "closed" conformation of protein, suggested by X-ray crystallography in static condition, is triggered by the pH value and the effect is independent of the metal ion being transported. We propose a simple description of the structure-function relationship that allows this protein to deliver, in particular, iron or aluminum ions in the biological cycle.