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ABSTRACT: An ESR investigation of the interaction of spin-labelled penetratin with heparin, heparansulfates and several phospholipid vesicle formulations is reported. Penetratin is a 16-aa peptide corresponding to the third helix of the Antennapedia homeodomain and belonging to the cell-penetrating peptide family. The present study shows that ESR spectroscopy can provide specific and reliable information about the mechanism of interaction of penetratin with polysaccharides and lipids, at a molecular level. The study showed that: (i) heparin and heparansulfates specifically interact with spin-labelled penetratin and promote peptide aggregation and concentration on their molecular surface; (ii) penetratin does not interact with neutral lipids, whereas it enters negatively charged lipid bilayers; (iii) cholesterol plays a negative effect on the insertion of penetratin into the lipid membrane; (iv) the interaction of penetratin with lipid vesicles is strongly dependent on lipid concentration. In a low lipid regime, penetratin associates with the polar heads of phospholipids and aggregates on the membrane surface; once the lipid concentration attains a threshold, the peptide enters the lipid bilayer. This step is characterized by reduced peptide mobility and partial disaggregation.It has been shown that ESR spectroscopy is a valuable investigation tool in studies related to the still unclear mechanism of the internalization process.
Journal of Peptide Science 08/2005; 11(7):401-9. · 1.80 Impact Factor
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ABSTRACT: In this paper we report the scale-up of the purification of poly(ethylene glycol) (PEG) derivatives of the growth hormone-releasing factor 1-29, from laboratory scale (100 mg of bulk starting material) to larger scale (3 g of bulk), through the use of a cation-exchange TSK-SP-5PW chromatographic column. A one-step purification process capable of purifying large amounts of mono-PEGylated GRF species from the crude reaction mixture was developed. A simple, straightforward stepwise gradient elution separation was developed at laboratory scale and then scaled up with a larger column packed with a chromatographic resin with the same chemistry which maintained the laboratory-scale separation profile. Active material recovery and material purity remained constant through the scale-up from the 13-microm stationary phase to the 25-microm larger column. Overall, the gram GRF equivalent/batch process scale showed to be quite reproducible, and could be considered as a good platform for scale up to production scale.
Journal of Chromatography 02/2002; 944(1-2):141-8. · 4.53 Impact Factor
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ABSTRACT: The extent and distribution of N-glycosylation and the nature of most of the disulfide bond linkages were determined for bovine lactoperoxidase through proteolytic and glycolytic digestions combined with matrix-assisted laser desorption/ionization mass spectrometric analysis. In addition, 98% of the primary sequence of the protein was confirmed. All five of the asparagines present in sequons were found to be glycosylated, predominantly by high mannose and complex structures. Six disulfide bonds were assigned, including Cys 32-Cys 45, Cys 146-Cys 156, Cys 150-Cys 174, Cys 254-Cys 265, Cys 473-Cys 530 and Cys 571-Cys 596.
Journal of Mass Spectrometry 03/2000; 35(2):210-7. · 3.27 Impact Factor
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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.
JBIC Journal of Biological Inorganic Chemistry 03/1999; 4(1):12-20. · 3.29 Impact Factor
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ABSTRACT: 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.
Journal of Inorganic Biochemistry 11/1997; 68(1):17-26. · 3.35 Impact Factor
