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Luteolin (LUT) is a polyphenolic compound, found in a variety of fruits, vegetables, and seeds, which has a variety of pharmacological properties. In the present contribution, binding of LUT to human serum albumin (HSA), the most abundant carrier protein in the blood, was investigated with the aim of describing the binding mode and parameters of the interaction. The application of circular dichroism, UV-Vis absorption, fluorescence, Raman and surface-enhanced Raman scattering spectroscopy combined with molecular modeling afforded a clear picture of the association mode of LUT to HSA. Specific interactions with protein amino acids were evidenced. LUT was found to be associated in subdomain IIA where an interaction with Trp-214 is established. Hydrophobic and electrostatic interactions are the major acting forces in the binding of LUT to HSA. The HSA conformations were slightly altered by the drug complexation with reduction of alpha-helix and increase of beta-turns structures, suggesting a partial protein unfolding. Also the configuration of at least two disulfide bridges were altered. Furthermore, the study of molecular modeling afforded the binding geometry.

Zn- and Cd-complexes of Quercus suber metallothionein (QsMT) were obtained by in vivo-synthesis, in order to obtain physiologically representative aggregates, and characterized by spectrometric and spectroscopic methods. The secondary structure elements and the coordination environments of the metal binding sites of the two aggregates were determined, as well as the main metal-containing species formed. The results obtained from the analysis of the Raman and IR spectra reveal that these metal-MT complexes predominantly contain beta-sheet elements (about 60%), whereas they lack alpha-helices. These structural features slightly depend on the divalent metal bound. In particular, Cd(II) binding to QsMT induces a slight increase of the beta-sheet percentage, as well as a decrease in beta-turn elements with respect to Zn(II) binding. Conversely, the in vivo capability of QsMT to inglobe metal and sulfide ions is metal-depending. Spectroscopic vibrational data also confirm the presence of sulfide ligands in the metal clusters of both Zn- and Cd-QsMT, while the participation of the spacer His residue in metal coordination was only found in Cd-QsMT, in agreement with the CD results. Overall data suggest different coordination environments for Zn(II) and Cd(II) ions in QsMT.

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The occurrence of tandem damage, due to reductive radical stress involving proteins and lipids, is shown by using a biomimetic model. It is made of unsaturated lipid vesicle suspensions in phosphate buffer in the presence of methionine, either as a single amino acid or as part of a protein such as RNase A, which contains four methionine residues. The radical process starts with the formation of H(.) atoms by reaction of solvated electrons with dihydrogen phosphate anions, which selectively attack the thioether function of methionine. The modification of methionine to alpha-aminobutyric acid is accompanied by the formation of thiyl radicals, which in turn cause the isomerization of the cis fatty acid residues to the trans isomers. The relationship between methionine modification and lipid damage and some details of the reductive radical stress obtained by proteomic analysis of irradiated RNase A are presented.

Lipidomics research, which focuses on the global changes in lipid metabolites, has recently been concerned with the type and roles of unsaturated lipids in the biological environment. The structural change induced by their conversion from the naturally occurring cis fatty acid geometry to the more thermodynamically stable trans configuration can affect membrane arrangement as well as lipid metabolism.[1] In the biomimetic model of thiyl radical-catalyzed isomerization of cis phospholipids, it was shown that when thiyl radicals are generated in the aqueous compartment and are able to diffuse in the lipid bilayer, then the interaction with unsaturated fatty acyl chains efficiently produces trans double bonds.[2] These findings suggested that radical-based degradation of sulfur-containing amino acid residues that are known to release diffusible thiol molecules could be the primer for tandem radical damage involving protein and lipid domains. We modeled such damage using g irradiation of lipid vesicle suspensions containing bovine pancreatic ribonuclease A (RNase A). The reaction of this protein with HC atoms was studied, and the inactivation was connected to the specific damage of sulfur moieties with release of low-molecular-weight thiols. [3] Liposomes were prepared by using dioleoyl phosphatidyl choline (DOPC) in the form of large unilamellar vesicles (LUVET) of 100 nm diameter.[4] The protein was added to the LUVET suspension and saturated with N 2 O prior to irradiation at a dose rate of 14.5 Gy min À1. 100 mL aliquots of the suspension were withdrawn at different irradiation times, over the interval of 2-70 min, for lipid isolation and derivatization to the corresponding fatty acid methyl esters.[5] This was followed by GC analysis to determine the cis/trans ratio. The solid circles in Figure 1 show the percentage of trans isomers (elaidate residues) formed as a function of irradiation dose. Control experiments in the absence of RNase A or by replacing RNase A with a protein without sulfur-containing amino acids, such as histone H1 type IIA from calf thymus, did not show any isomerization.In parallel, we followed changes in the enzyme activity, [6] as well as the transformation of the sulfur moieties by Raman spectroscopy, using lyophylized samples of aqueous solutions of native and irradiated RNase A. As expected from the radiation-induced inactivation, [3] a residual enzymatic activity of 67 % was found after exposure to only 33.3 Gy, followed by a slower decrease of the activity, which reached 50 % after 500 Gy. In the Raman spectra, the SÀS and CÀS stretching bands are visible in the 420-780 cm À1 spectral range. Native RNase A has four disulfide bridges that give rise to two different disulfide bands (n S-S ) at 514 and 535 cm À1

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