Simona Baroni

Relaxometric characterization of human hemalbumin

Hemalbumin [i.e., Fe(III)-protoporphyrin IX-human serum albumin; Fe(III)heme-HSA] is an important intermediate in the recovery of heme iron following hemolysis. Relaxometric data are consistent with the occurrence of a hexacoordinated high-spin Fe(III) center with no water in the inner coordination sphere. The relatively high relaxation enhancement observed for an aqueous solution of Fe(III)heme-HSA (r1p=4.8 mM–1 s–1 at 20 MHz, pH 7, and 25 °C) is ascribed to the occurrence of a strong contribution from water molecules in the second coordination sphere. Structural analysis of the putative binding region has been performed by a Monte Carlo simulated annealing procedure, which allowed us to identify His105 and Tyr148 as axial ligands. The role of a tyrosinate as the sixth Fe(III)heme ligand is supported by the pH-dependent analysis. Interestingly, when Fe(III) is replaced by Mn(III), the occurrence of a fast exchanging water molecule at pH values close to neutrality is detected. As the pH is increased, the Mn(III) containing system behaves analogously to Fe(III)heme-HSA. At higher pH, the phenolate ligand is eventually displaced by OH– from both Fe(III) and Mn(III) centers. Support for the proposed bonding scheme has been gained also from competitive binding assays for the sixth coordination site by fluoride, azide, and imidazole ligands.

Relaxometric Investigations and MRI Evaluation of a Liposome-Loaded pH-Responsive Gadolinium(III) Complex

Accurate measurement of the tissue pH in vivo by MRI may be of clinical value for both diagnosis and selection/monitoring of therapy. To act as pH reporters, MRI contrast agents have to provide responsiveness to pH that does not require prior knowledge of the actual concentration of the contrast agent. This work deals with the use of a paramagnetic gadolinium(III) complex, loaded into liposomes, whose relaxometric properties are affected by the pH of the medium. In this system, the amphiphilic metal complex, which contains a moiety whose protonation changes the coordination properties of the metal chelate, experiences a different intraliposomial distribution depending on the pH conditions. The pH of the solution can be unambiguously identified by exploiting the peculiar characteristics of the resulting NMRD profiles, and a ratiometric pH-responsive method has been set up by comparing the relaxation enhancement at different magnetic field strengths.

Characterization of human hair melanin and its degradation products by means of magnetic resonance techniques

Melanin granules (MGs) have been extracted from human Chinese black hairs by either acid hydrolysis (CH‐type MGs) or enzymatic digestion (CP‐type MGs), and their chemical structure investigated at the solid state by means of 13C cross polarization magic angle spinning (CPMAS NMR) and EPR spectroscopy. Both types of MGs contain a large amount of protein that is tightly bound to the true melanin polymer, with CP‐type MGs having a larger protein content than CH‐type ones. Moreover, MGs may also contain variable amounts of lipid‐like material. A high amount of paramagnetic metals is detected by EPR in CP‐type MGs, in particular Fe(III). Iron can be bound in two chemical forms: as isolated high spin Fe(III) ions with rhombic symmetry and as small oxy‐hydroxy Fe(III) aggregates. Iron is poorly available to chelators. CH‐type MGs contain much fewer metals. CP‐type MGs have then been subjected to partial bleaching by hydrogen peroxide in ammonia, yielding a residual solid, called residual oxidized melanin (ROM) and a soluble but still pigmented fraction called melanin free acid (MFA). MFA can be isolated by precipitation at acidic pH. The 13C‐CPMAS NMR and EPR spectra of these derivatives indicated that ROM has a structure very similar to that of parent MGs, whereas MFA shows a decrease of the protein content with respect to the melanin and a decreased amount of bound iron. Thus, the oxidative degradation of CP‐type MGs is a process not involving the bulk of MGs, but rather it proceeds from the solvent‐exposed outer parts to the interior. Copyright

Binding and Relaxometric Properties of Heme Complexes with Cyanogen Bromide Fragments of Human Serum Albumin

The spectroscopic and reactivity properties of hemin complexes formed with cyanogen bromide fragments B (residues 1-123), C (124-298), A (299-585), and D (1-298) of human serum albumin (HSA) have been investigated. The complex hemin-D exhibits binding, spectral, circular dichroism, and reactivity characteristics very similar to those of hemin-HSA, indicating that fragment D contains the entire HSA domain involved in heme binding. The characteristics of the other hemin complexes are different, and a detailed investigation of the properties of hemin-C has been carried out because this fragment contains the HSA binding region of several important drugs. Hemin-C contains a low-spin Fe(III) center, with two imidazole ligands, but the complex undergoes a reversible structural transition at basic pH leading to a high-spin, five-coordinated Fe(III) species. This change determines a marked increase in the relaxation rate of water protons. Limited proteolysis experiments and mass spectral analysis carried out on fragment C and hemin-C show that the region encompassing residues Glu-208 to Trp-214 is protected from activity of proteases in the complex and, therefore, is involved in the interaction with hemin. A structural model of fragment C enables us to propose that His-242 and His-288 are the axial ligands for the Fe(III) center.

Relaxometric studies for food characterization: the case of balsamic and traditional balsamic vinegars

NMR spectroscopy is a powerful technique for investigating the structure and composition, as well as the physicochemical properties, of foodstuff. NMR-field cycling modality reports about the relaxation times of solvent molecules as a function of the applied magnetic field strength. In the case of aqueous solutions, this methodology is particularly valuable in assessing the interactions of water molecules with paramagnetic and large-size macromolecular systems. (1)H NMR field cycling relaxometry has been used to characterize traditional balsamic vinegars and balsamic vinegars of Modena. It has been found that the longitudinal relaxation time (T(1)) of the water proton resonance is mainly determined by the water molar fraction and the occurrence of dissolved macromolecules and paramagnetic metal ions. Actually, the observed (1)H nuclear magnetic resonance dispersion (NMRD) profiles appear markedly affected by the formation of paramagnetic macromolecular adducts. It has been shown that counterfeit specimens can be identified on the basis of the comparison of their T(1) and T(2) (transverse relaxation time) values with respect to the corresponding values of genuine samples. For the latter ones, a relationship has been found that relates the observed T(1) to the age of the vinegar.

Water exchange across the erythrocyte plasma membrane studied by HR-MAS NMR spectroscopy.

Water exchange across the plasma membrane of erythrocytes (red blood cells (RBCs)) was studied by means of high‐resolution magic angle spinning (HR‐MAS) NMR spectroscopy. Under HR‐MAS conditions, the centrifugal force causes the splitting of RBC suspensions into a two‐phase system composed of a central core of cell free water and an outer layer of tightly packed cells. Water belonging to each of these phases gives rise to two separated resonances. Chemical exchange between them is not detectable on the chemical shift or saturation transfer (ST) NMR time scale because of the physical separation between the phases. When the RBCs are dispersed and immobilized within a matrix made of cross‐linked albumin, the splitting into a two‐phase system is prevented and a single exchange‐averaged peak for water is detected in 1H HR‐MAS NMR spectra. The lineshape of this peak is dependent on transmembrane exchange kinetics, since MAS averages out all the anisotropic magnetic interactions that are responsible for additional line‐broadening under conventional liquid conditions. Line‐shape analysis according to a two‐site exchange model yielded a residence lifetime on the order of about 10 ms (at 37°C) for a water molecule within the intracellular compartment, which is not too far from the generally accepted value of 9.6–14.8 ms. Magn Reson Med, 2006.

Frequency-Encoded MRI-CEST Agents Based on Paramagnetic Liposomes/RBC Aggregates

Paramagnetic liposomes containing Dy-HPDO3A in their inner water compartment and carrying a residual positive charge on their outer surface have been electrostatically bound to the membrane of red blood cells (RBCs). These aggregates yield two chemical exchange saturation transfer (CEST) pools represented by liposomal water protons (LipoCEST) and cytoplasmatic water protons (ErythroCEST), respectively. The absorption frequencies of the two pools fall at the negative and positive side of the solvent water resonance as expected from the dipolar (LipoCEST) and BMS (bulk magnetic susceptibility) (ErythroCEST) origin of the paramagnetic induced shift of their water protons resonances, respectively. In vivo magnetic resonance imaging (MRI) shows that the liposomes/RBC aggregates report about the vascular volume whereas the residual LipoCEST effect informs about the presence of released liposomes in the region of interest (ROI). Besides being an innovative blood cell labeling for MRI, the LipoCEST/RBC aggregates provide a route to improve the circulation lifetime of the liposomes and the CEST procedure allows assessing the deassembly of the aggregates and accumulation of the liposomes in the ROI.

Modulation of the antioxidant activity of HO* scavengers by albumin binding: a 19F-NMR study.

The interaction between different HO(z.rad;) radical scavengers in a three-component antioxidant system has been investigated by means of 19F-NMR spectroscopy. This system is composed of bovine serum albumin (BSA), trolox, and N-(4-hydroxyphenyl)-trifluoroacetamide (CF(3)PAF). The antioxidant capacity of BSA and trolox has been assessed by measuring the amount of trifluoroacetamide (TFAM) arising from the radical mediated decomposition of CF(3)PAF. When assayed separately, both trolox and BSA behaved as antioxidants, as they were effective to protect CF(3)PAF from HO* radical-mediated decomposition. By contrast, trolox enhanced the production of TFAM in the presence of BSA, thus behaving as a pro-oxidant. Urate, carnosine, glucose, and propylgallate showed antioxidant properties both with or without BSA. CF(3)PAF and trolox were found to bind to BSA with association constants in the order of 5 x 10(3)M(-1) and to compete for the same binding sites. These results have been discussed in terms of BSA-catalysed cross-reactions between trolox-derived secondary radicals and CF(3)PAF.

Synthesis and characterization of an MRI Gd-based probe designed to target the translocator protein

DPA‐713 is the lead compound of a recently reported pyrazolo[1,5‐a]pyrimidineacetamide series, targeting the translocator protein (TSPO 18 kDa), and as such, this structure, as well as closely related derivatives, have been already successfully used as positron emission tomography radioligands. On the basis of the pharmacological core of this ligands series, a new magnetic resonance imaging probe, coded DPA‐C6‐(Gd)DOTAMA was designed and successfully synthesized in six steps and 13% overall yield from DPA‐713. The Gd‐DOTA monoamide cage (DOTA = 1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetic acid) represents the magnetic resonance imaging reporter, which is spaced from the phenylpyrazolo[1,5‐a]pyrimidineacetamide moiety (DPA‐713 motif) by a six carbon‐atom chain. DPA‐C6‐(Gd)DOTAMA relaxometric characterization showed the typical behavior of a small‐sized molecule (relaxivity value: 6.02 mM−1 s−1 at 20 MHz). The good hydrophilicity of the metal chelate makes DPA‐C6‐(Gd)DOTAMA soluble in water, affecting thus its biodistribution with respect to the parent lipophilic DPA‐713 molecule. For this reason, it was deemed of interest to load the probe to a large carrier in order to increase its residence lifetime in blood. Whereas DPA‐C6‐(Gd)DOTAMA binds to serum albumin with a low affinity constant, it can be entrapped into liposomes (both in the membrane and in the inner aqueous cavity). The stability of the supramolecular adduct formed by the Gd‐complex and liposomes was assessed by a competition test with albumin. Copyright

Thermodynamic analysis of hydration in human serum heme-albumin

Ferric human serum heme-albumin (heme-HSA) shows a peculiar nuclear magnetic relaxation dispersion (NMRD) behavior that allows to investigate structural and functional properties. Here, we report a thermodynamic analysis of NMRD profiles of heme-HSA between 20 and 60 degrees C to characterize its hydration. NMRD profiles, all showing two Lorentzian dispersions at 0.3 and 60 MHz, were analyzed in terms of modulation of the zero field splitting tensor for the S=5/2 manifold. Values of correlation times for tensor fluctuation (tau(v)) and chemical exchange of water molecules (tau(M)) show the expected temperature dependence, with activation enthalpies of -1.94 and -2.46+/-0.2 kJ mol(-1), respectively. The cluster of water molecules located in the close proximity of the heme is progressively reduced in size by increasing the temperature, with DeltaH=68+/-28 kJ mol(-1) and DeltaS=200+/-80 J mol(-1) K(-1). These results highlight the role of the water solvent in heme-HSA structure-function relationships.