Elena Maria Ghibaudi

Multiple aspects of the interaction of biomacromolecules with inorganic surfaces

The understanding of the mechanisms involved in the interaction of biological systems with inorganic materials is of interest in both fundamental and applied disciplines. The adsorption of proteins modulates the formation of biofilms onto surfaces, a process important in infections associated to medical implants, in dental caries, in environmental technologies. The interaction with biomacromolecules is crucial to determine the beneficial/adverse response of cells to foreign inorganic materials as implants, engineered or accidentally produced inorganic nanoparticles. A detailed knowledge of the surface/biological fluids interface processes is needed for the design of new biocompatible materials. Researchers involved in the different disciplines face up with similar difficulties in describing and predicting phenomena occurring at the interface between solid phases and biological fluids. This review represents an attempt to integrate the knowledge from different research areas by focussing on the search for determinants driving the interaction of inorganic surfaces with biological matter.

A neurotoxic prion protein fragment induces rat astroglial proliferation and hypertrophy

Prion‐related encephalopathies are characterized by the accumulation of an abnormal prion protein isoform (PrPSc) and the deposition of PrP amyloid in the brain. This process is accompanied by neuronal loss and astrogliosis. We recently showed that a synthetic peptide corresponding to residues 106–126 of human PrP is amyloidogenic and causes neuronal death by apoptosis in vitro. In the present study we investigated the effects of 1‐ and 14‐day exposures of rat astroglial cultures to mtcromolar concentrations of this peptide as well as peptides homologous to other portions of PrP, a peptide corresponding to residues 25–35 of amyloid‐β protein, and a scrambled sequence of PrP 106–126. No significant changes were observed after 1‐day exposure of cultures to any peptide. Conversely, 14‐day treatment with PrP 106–126 (50 μM) resulted in a 5‐fold increase in glial fibrillary acidic protein (GFAP) expression, as evaluated by Northern and Western blot analyses, and a 1.5‐fold increment in cell number. Light and electron microscopy immunohistochemistry showed an enlargement in size and density of astroglial processes, and an increase in GFAP‐immunoreactive intermediate filaments. These changes were not observed after 14‐day treatment of cultures with the other peptides, including PrP 106–126 scrambled. The increase in GFAP expression of astroglial cultures exposed to PrP 106–126 was quantitatively similar to that found in scrapie‐infected hamster brains. These results suggest that the PrP region corresponding to residues 106–126 is biologically active, and that cerebral accumulation of peptides including this sequence might be responsible for both the neuronal degeneration and the astrogliosis that occur in prion‐related encephalopathies.

An integrated approach to the study of the interaction between proteins and nanoparticles.

The rapid development of nanotechnology has raised some concerns about the effects of engineered nanoparticles (NPs) on human health and the environment. At the same time, NPs have attracted intense interest because of their potential applications in biomedicine. Hence, the requirement of detailed knowledge of what takes place at the molecular level when NPs get inside living organisms is a necessary step in assessing and likely predicting the behavior of an NP. The elicited effects strongly depend on the early events occurring when NPs reach biological fluids, where the interaction with proteins is the primary process. Whereas the adsorption of proteins on biomaterials has been thoroughly investigated, the mechanisms underlying the interaction of proteins with NPs are still largely unexplored. Here we report a study of the behavior of four model proteins differing in their resistance to conformational changes, net charge, and surface charge distributions, adsorbed on two nanometric silica powders with distinct hydrophilicity. An integrated picture of the adsorption process has been obtained by applying a whole set of techniques: the extent of coverage of the silica surface and the reversibility of the process were evaluated by combining the adsorption isotherms with the changes in the zeta potential and the point of zero charge for NPs at different protein coverages; the occurrence of protein deformation was evaluated by Raman spectroscopy, and EPR spectroscopy of spin-labeled proteins provided insight into their orientation on the silica surface. We have found that the extent of coverage of the nanoparticle surface is strongly influenced by the protein structural stability as well as by the distribution of charges at the protein surface.

Oxidation of 2,4-dichlorophenol catalyzed by horseradish peroxidase: characterization of the reaction mechanism by UV-visible spectroscopy and mass spectrometry.

The hydrogen peroxide-oxidation of 2,4-dichlorophenol catalyzed by horseradish peroxidase has been studied by means of UV-visible spectroscopy and mass spectrometry in order to clarify the reaction mechanism. The dimerization of 2,4-dichlorophenol to 2,4-dichloro-6-(2,4-dichlorophenoxy)-phenol and its subsequent oxidation to 2-chloro-6-(2,4-dichlorophenoxy)-1,4-benzoquinone together with chloride release were observed. The reaction rate was found to be pH-dependent and to be influenced by the pK(a) value of 2,4-dichlorophenol. The dissociation constants of the 2,4-dichlorophenol/horseradish peroxidase (HRP) adduct at pH 5.5 and 8.5 were also determined: their values indicate the unusual stability of the adduct at pH 5.5 with respect to several adducts of HRP with substituted phenols.

Tyrosinase-catecholic substrates in Vitro model: kinetic studies on the o-quinone/o-semiquinone radical formation☆

Abstract The mechanism of o-semiquinone production was examined in the tyrosinase and peroxidase catalyzed oxidations of a series of catecholic compounds using the electron spin resonance (ESR) spin-stabilization approach and in the presence of 3-methyl-2-benzothiazolinone hydrazone (MBTH). In the tyrosinase process, the nonenzymatic o-semiquinone formation by inverse disproportion mechanism was clearly confirmed. Mechanistic and kinetic studies of o-semiquinone and o-quinone formation by mushroom tyrosinase were carried out by ESR spin stabilization and optical spectroscopy. Two different types of cyclizable catecholic substrates ( l -dopa and dopamine 3,4-dihydroxyphenylacetic acid and 3-(3,4-dihydroxyphenyl)propionic acid) together with an uncyclizable substrate (3,4-dihydroxybenzoic acid) were examined. The reactive quinones were monitored by measuring the apparent initial rates of the o-quinone-MBTH adducts. The transient behaviour of the o-semiquinone was studied by determining the pseudo first-order formation constants (k values in the range 0.226–0.035 s −1 ), the relative second-order decay kinetic constants (k = 3.3·10 2 M −1 s −1 for dopamine o-semiquinone) and the maximum concentrations of the o-semiquinone complexes formed in situ with Mg 2+ ions. The o-semiquinone formation constants are not directly correlated with their maximum concentrations; in fact, the o-semiquinone maximum concentration of the uncyclizable substrate is comparable with that derived from l -dopa. Furthermore, the secondary semiquinone formation is slow and not competitive with the primary semiquinone generation. Then, in our model the limiting factor for the o-semiquinone formation, is not simply the substrate ability to cyclize, and, therefore, the potential toxicity of the secondary semiquinone is questionable.

Spectroscopic and binding studies on the interaction of inorganic anions with lactoperoxidase

The interaction of several inorganic species (SCN-, I-, Br-, Cl-, F-, NO2-, N3-, CN-) with bovine lactoperoxidase was investigated through kinetic and binding studies by using UV-Vis spectroscopy. The above ligands form 1:1 complexes with the protein and can be assigned to three different groups, on the basis of the dissociation constant values (KD) of the adducts: (1) SCN-, I-, Br-, and Cl- (KD increases along the series); (2) F- (which shows a singular behavior); (3) NO2-, N3-, and CN- (that bind at the iron site). KD values for the LPO/SCN- adduct appeared to be modified in the presence of other inorganic species; a strong competition between this substrate and all other anions (with the exception of F-) was evidentiated. Binding investigations on the natural substrates SCN- and I-, at varying pH and temperature, showed that their interaction with lactoperoxidase involves the protonation of a common site in proximity of the iron (possibly distal histidine). Michaelis-Menten constants for SCN-, I-, and Br- followed roughly the same trend as KD; KM for hydrogen peroxide is strongly dependent on the cosubstrate. Computer-assisted docking simulations showed that all ligands can penetrate inside the heme pocket.

A theoretical three-dimensional model for lactoperoxidase and eosinophil peroxidase, built on the scaffold of the myeloperoxidase X-ray structure

Abstract Lactoperoxidase (LPO), eosinophil peroxidase (EPO) and myeloperoxidase (MPO) belong to the class of haloperoxidases, a group of mammalian enzymes able to catalyze the peroxidative oxidation of halides and pseudohalides, such as thiocyanate. They all play a key role in the development of antibacterial activity. The homology in their functional role is emphasized by the striking similarity of their primary structures. A theoretical model for the three-dimensional structure of LPO and EPO has been developed on the basis of the X-ray structure of MPO, a high degree of similarity having been found in their sequences. Evidence supporting the hypothesis of an ester linkage between heme and apoprotein in LPO and EPO, originally proposed by Hultquist and Morrison is discussed.

Can estrogenic radicals, generated by lactoperoxidase, be involved in the molecular mechanism of breast carcinogenesis?

Abstract Mutations of regulatory genes, which perturb the mechanism of cell replication resulting in abnormal cell proliferation, are the main cause of cancer. Many endogenous and exogenous chemicals (including estrogenic hormones) are known to represent a major carcinogenic risk for humans. 2-OH- and 4-OH-derivatives of estrogenic molecules have been shown to form stable adducts with purine DNA bases and act as ‘depurinating’ agents, thus altering gene transcription (Cavalieri EL, Stack DE, Devanesan PD et al. Proc Natl Acad Sci USA 1997; 94: 10937–10942). Lactoperoxidase (LPO), which is produced by mammary glands, is likely to be involved in breast carcinogenesis, because of its ability to interact with estrogenic hormones and oxidise them through two one-electron reaction steps. We investigated the reactivity of LPO towards five molecules: 17-β-estradiol (a natural hormone), diethylstilbestrol (a synthetic drug, supplied to pregnant women for preventing spontaneous abortion), exestrol (a synthetic antigonadotropic estrogen), 2-OH- and 4-OH-estradiol (catabolic products of estradiol). Enzymatically generated radical derivatives of such molecules were stabilized by spin-trapping or by chelation of a diamagnetic metal ion and characterized with EPR spectroscopy. A kinetic study of the oxidation process was carried out using EPR and UV-visible spectroscopy.

Intramolecular electron transfer versus substrate oxidation in lactoperoxidase: investigation of radical intermediates by stopped-flow absorption spectrophotometry and (9-285 GHz) electron paramagnetic resonance spectroscopy.

We have combined the information obtained from rapid-scan electronic absorption spectrophotometry and multifrequency (9-295 GHz) electron paramagnetic resonance (EPR) spectroscopy to unequivocally determine the electronic nature of the intermediates in milk lactoperoxidase as a function of pH and to monitor their reactivity with organic substrates selected by their different accessibilities to the heme site. The aim was to address the question of the putative catalytic role of the protein-based radicals. This experimental approach allowed us to discriminate between the protein-based radical intermediates and [Fe(IV)=O] species, as well as to directly detect the oxidation products by EPR. The advantageous resolution of the g anisotropy of the Tyr (*) EPR spectrum at high fields showed that the tyrosine of the [Fe(IV)=O Tyr (*)] intermediate has an electropositive and pH-dependent microenvironment [g(x) value of 2.0077(0) at pH >or= 8.0 and 2.0066(2) at 4.0 <or= pH <or= 7.5] possibly related to the radical stability and function. Two types of organic molecules (small aromatic vs bulkier substrates) allowed us to distinguish different mechanisms for substrate oxidation. [Fe(IV)=O Por (*+)] is the oxidizing species of benzohydroxamic acid, o-dianisidine, and o-anisidine via a heme-edge reaction and of mitoxantrone via a long-range electron transfer (favored at pH 8) not involving the tyrosyl radical, the formation of which competed with the substrate oxidation at pH 5. In contrast, the very efficient reaction with ABTS at pH 5 is consistent with [Fe(IV)=O Tyr (*)] being the oxidizing species. Accordingly, the identification of the ABTS binding site by X-ray crystallography may be a valuable tool in rational drug design.

Catechol(amine)s as probes of lactoperoxidase catalytic site structure: spectroscopic and modeling studies

Abstract Binding affinities to lactoperoxidase (LPO) of a homologous series of substituted catechol(amine)s [such as catechol, 4-methylcatechol, 3,4-dihydroxybenzoic acid, 3,4-dihydroxyphenylacetic acid, 3-(3,4-dihydroxyphenyl)propionic acid; dopamine, noradrenaline, adrenaline;l-3,4-dihydroxyphenylalanine] were studied by UV-visible spectroscopy and docking simulations. Dissociation constant (Kd) values were calculated by direct fitting of the experimental data and fall in a range of 3–95 mM. Thermodynamic parameters are comparable with those reported for the interaction of LPO with p-substituted phenols, suggesting a similar general mode of binding. Furthermore, the relative contributions to binding energy, described by the unimolecular constant Ku, show that interaction between protein and ligands originates from a relatively large number of groups. Docking and molecular dynamics simulations, in agreement with experimental evidence, predict that the substrate is localized into the access channel in the vicinity of heme distal pocket. This channel is characterized by a hydrophobic patch (six Phe residues) and by a charged contribution (two Glu and one His residues). All of the substrates, except caffeic acid, may approach the protein active site. Positively charged Arg372 acts as a gate above the heme distal pocket and seems to address substrate orientation in relation to the side-chain terminal group.