No abstract

The saccharide profiles of 5 different botanical species in 86 Italian honey samples were investigated by ¹H and ¹H-¹³C NMR spectroscopy. Nineteen saccharides were identified in the aqueous extracts, namely, fructose, glucose, gentiobiose, isomaltose, kojibiose, maltose, maltulose, melibiose, nigerose, palatinose, sucrose, turanose, erlose, isomaltotriose, kestose, maltotriose, melezitose, raffinose, and maltotetraose. PCA performed on NMR spectral regions, in particular between 4.400 and 5.700 ppm and the fructose signal at 4.050 ppm, revealed a partial sample grouping. The score contribution plots derived from PCA performed using the mean values for the buckets of the anomeric region for each floral source allowed the identification of saccharides characterizing different honeys. OPLS-DA models were further evaluated to confirm the previous findings. OPLS-DA models were also built to highlight differences between polyfloral and high mountain polyfloral honeys and between high mountain polyfloral and rhododendron honeys, both collected at high altitude; S-plots highlighted the characteristic saccharides.

Bile acid‐binding proteins (BABPs) are cytosolic lipid chaperones that play central roles in driving bile flow, as well as in the adaptation to various pathological conditions, contributing to the maintenance of bile acid homeostasis and functional distribution within the cell. Understanding the mode of binding of bile acids with their cytoplasmic transporters is a key issue in providing a model for the mechanism of their transfer from the cytoplasm to the nucleus, for delivery to nuclear receptors. A number of factors have been shown to modulate bile salt selectivity, stoichiometry, and affinity of binding to BABPs, e.g. chemistry of the ligand, protein plasticity and, possibly, the formation of disulfide bridges. Here, the effects of the presence of a naturally occurring disulfide bridge on liver BABP ligand‐binding properties and backbone dynamics have been investigated by NMR. Interestingly, the disulfide bridge does not modify the protein‐binding stoichiometry, but has a key role in modulating recognition at both sites, inducing site selectivity for glycocholic and glycochenodeoxycholic acid. Protein conformational changes following the introduction of a disulfide bridge are small and located around the inner binding site, whereas significant changes in backbone motions are observed for several residues distributed over the entire protein, both in the apo form and in the holo form. Site selectivity appears, therefore, to be dependent on protein mobility rather than being governed by steric factors. The detected properties further establish a parallelism with the behaviour of human ileal BABP, substantiating the proposal that BABPs have parallel functions in hepatocytes and enterocytes.

The investigation of multi-site ligand-protein binding and multi-step mechanisms is highly demanding. In this work, advanced NMR methodologies such as 2D (1)H-(15)N line-shape analysis, which allows a reliable investigation of ligand binding occurring on micro- to millisecond timescales, have been extended to model a two-step binding mechanism. The molecular recognition and complex uptake mechanism of two bile salt molecules by lipid carriers is an interesting example that shows that protein dynamics has the potential to modulate the macromolecule-ligand encounter. Kinetic analysis supports a conformational selection model as the initial recognition process in which the dynamics observed in the apo form is essential for ligand uptake, leading to conformations with improved access to the binding cavity. Subsequent multi-step events could be modelled, for several residues, with a two-step binding mechanism. The protein in the ligand-bound state still exhibits a conformational rearrangement that occurs on a very slow timescale, as observed for other proteins of the family. A global mechanism suggesting how bile acids access the macromolecular cavity is thus proposed.

Four comonomers bearing a highly efficient phenolic antioxidant unit and different methylene spacers between the aromatic ring and the double bond have been prepared and tested in copolymerization with ethylene using metallocene‐based catalysts. The possibility of obtaining a “masterbatch” suitable for melt blending with commercial polyolefins has been evaluated by modifying: (i) the structure of the functionalized comonomer, (ii) the kind of catalyst, and (iii) the polymerization conditions. Characterization of monomers and copolymers was accomplished by using 1H and 13C NMR, size exclusion chromatography (SEC), differential scanning calorimetry (DSC), and thermal gravimetric analysis (TGA). Using the comonomer with the longest methylene spacer between the aromatic ring and the double bond, and rac‐(EBTHI)ZrCl2 as catalyst, adjustable amounts of the antioxidant moiety can be incorporated into the polyethylene chains. TGA analysis carried out on some of the copolymers containing the antioxidant group showed no oxygen uptake before decomposition. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 6393–6406, 2008