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Applicazioni della Spettroscopia NMR nell' analisi varietale di uve dell' Oltrepo' pavese
No full textLow-temperature intermediates to oxygen reduction reaction catalysts based on amine-modified metal-loaded carbons : an XPS and ss-NMR investigation
No full textCarbon functionalization is a major subject of interest in a number of project applications. Herein we report results on the characterization of nitrogen- and metal-loaded (Me = Fe, Co) carbon derivatives from low-T reaction steps before they are converted to catalysts for electrochemical oxygen reduction by later high-T treatments. The aim is to shed light on the state of carbon and carbon-bonded moieties before thermal modifications take place during any chosen high-T treatment. Though necessary for end catalyst activation, such thermal treatments make difficult to establish a relation between the starting reactants and finally obtained catalysts. Of interest to the paper are 13C, 15N solid-state NMR (ss-NMR) and high-resolution X-ray Photoelectron Spectroscopy (XPS) results on a commercial carbon that was reacted first with aliphatic di- and tri-amines and then with Fe, Co ions in room-T water. Data from natural abundance ss-15N NMR in combination with XPS analysis were found especially relevant to assess that, in the adopted conditions, amines preferentially bind to carbon by creating alkylimino functional groups, which spontaneously form hydrous surface metal complexes with soluble Fe and Co ions. A chemical model is thus proposed for metal coordination in such C-N species
Direct metal ion substitution at the [M(SCys)(4)](2-) site of rubredoxin
No full textThe single Fe(II) in reduced rubredoxin from Clostridium pasteurianum was found to be quantitatively displaced by either Cd2+ or Zn2+ when a modest molar excess of the substituting metal salt was anaerobically incubated with the reduced rubredoxin under mild conditions, namely, room temperature, pH 5.4-8.4, and no protein denaturants. Under the same conditions, cadmium-for-zinc substitution was also achieved upon aerobic incubation of the zinc-substituted rubredoxin with a modest molar excess of Cd2+. Displacements of Fe(II) from the reduced rubredoxin were not observed upon anaerobic incubation with Ni2+, Co2+, or VO2+ salts, and no reaction with any of the divalent metal ions was observed for the oxidized [Fe(III)] rubredoxin. Fe(II) could not be re-inserted into the Zn- or Cd-substituted rubredoxins without resorting to protein denaturation. H-1 and Cd-113 NMR experiments showed that the cadmium-substituted rubredoxin prepared by the non-denaturing substitution method retained the pseudotetrahedral M(SCys)(4) coordination geometry and secondary structural elements characteristic of the native rubredoxin, and that "unzipping" of the beta-sheet did not occur during metal substitution. Rates of Fe(II) displacement by M2+ (M = Cd or Zn) increased with increasing M2+/rubredoxin ratio, decreasing pH, and lower ionic strength. The substitution rates were faster for M = Cd than for M = Zn. Rates of Cd2+ substitution into a V8A-mutated rubredoxin were significantly faster than for the wild-type protein. The side-chain of Vg is on the protein surface and close to the metal-ligating Cys42S gamma at the M(SCys)(4) site. Therefore, the rate-limiting step in the substitution process is suggested to involve direct attack of the [M(SCys)(4)](2-) site by the incoming M2+, without global unfolding of the protein. Implications of these results for metal ion incorporation into rubredoxins in vivo are discussed
PHOTONUCLEASE ACTIVITY OF FAGOPYRIN, A PERYLENEQUINONE ISOLATED FROM Fagopyrum esculentum FLOWERS
No full textSteroid hydroxylations with Botryodiplodia malorum and Colletotrichum lini
No full textAn improved procedure for the microbial hydroxylations of dehydroepiandrosterone (DHEA, 1) and 15β,16β-methylene-dehydroepiandrosterone (2) was studied using whole cells of Botryodiplodia malorum and Colletotrichum lini. C. lini catalyzed 7α- and 15α-hydroxylation of 1 and 7α-hydroxylation of 2, while B. malorum gave 7β-hydroxylation of both the substrates. The stability of the enzymatic activity was higher in the presence of co-substrates (i.e., glucose or mannitol) allowing for repeated batches of the biotransformations. The yields of 7α,15α-dihydroxy-1 production were improved obtaining 5.8 g l−1 (recovered product) from 7.0 g l−1 of substrate. The structures of the hydroxylated products were assigned by a combination of two-dimensional NMR proton–proton and proton–carbon correlation techniques
Addressing the role of structural features of proteins in cereal processing
No full textSeveral novel methodologies have been recently proposed to investigate the significance of structural features of proteins in the processing of cereals and pseudo-cereals. Some of them address features of the proteins in the starting materials, whereas others were designed to analyze structural changes occurring upon processing, and their relevance to the textural and sensory properties of the product. Proteins in cereal-based products are unfolded and insoluble, but their structure is sensitive to the addition of water. Solvation of cereal proteins occurs with stoichiometries and time courses typical for individual grains and flours, and can be monitored by spectroscopic techniques such as fluorescence and NMR. In later stages of processing, protein networks may be formed through covalent (disulfide) and non covalent interactions, among which hydrophobic bonds are the most common.
Cysteine thiols on proteins are relevant to the disulfide exchange events involved in network formation, and their role and occurrence has been studied on the original materials by developing a so-called "thiolomics" approach, that also offers information on the accessibility of thiols. Evaluation of thiol accessibility in the presence/absence of denaturants has been proposed as a way of discriminating the compactness of the protein network in various products and the nature of the forces that ensure their stability. As for the role of hydrophobic bonds in these protein networks, basic information has been derived from solubility studies in the absence/presence of denaturants and disulfide reductants. Fluorescent hydrophobic probes have been used to monitor either the number of hydrophobic regions exposed on the protein surface in the starting material(s), as well as process-induced changes in accessibility of these same areas. These approaches may represent a useful tool also for investigating the relationship between molecular and textural/sensory properties in cereal-based materials and products even when proteins other than gluten ensure the solidity of the macromolecular network
Metal Ion Binding to Parvalbumin : A Proton NMR Study
No full textThe 1H NMR spectra of carp parvalbumin saturated with Ca2+, Cd2+, La3+ and Lu3+ were compared, using 2D 1H NMR techniques as well as conventional 1H NMR spectra. The Ca2+ and Cd2+ saturated parvalbumin (with both high affinity Ca2+-binding sites occupied) gave rise to very similar spectra. This shows that these two species have almost identical protein conformations. The 1H NMR spectrum from the Ln3+ saturated parvalbumins deviated from the other two and it was therefore concluded that Cd2+ is a better probe for Ca2+ than Ln3+ in parvalbumin and probably also for related calcium binding proteins. The addition of excess of divalent metal ions, such as Mg2+ or Ca2+, causes small changes in the chemical shift of some methyl resonances. This is presumably caused by binding of these metal ions to a third site close to the CD site which is made up of the carboxylic groups from Glu 60 and Asp 61
Polyols stabilize specific regions in the structure of betalactoglobulin
No full textPolyols have long been known to improve the stability of protein folding, but the exact mechanism of their stabilizing action remains debated. In this work, beta-lactoglobulin (BLG) was used to study the mechanism of stabilization for four common polyols: sucrose, sorbitol, glycerol, and trehalose. BLG is an excellent model protein given its well know (and complex) denaturation behavior: unfolding of BLG occur through a number of steps, each affecting separate regions of the protein.
Stabilizing effects of various concentrations of different polyols towards physical and chemical denaturation were assessed by near-UV circular dichroism, and by evaluating the reactivity of Cys121, whose thiol is hidden in native BLG. Surface hydrophobic interactions were characterized by using suitable non-covalent fluorescent probes. Effects of polyols on the dissociation equilibrium of the BLG native dimer were assessed by estimating a diffusion coefficient through NMR spectroscopy. The stabilizing effects are discussed in terms of the preferential exclusion theory, and of the effects of individual polyols on solvent properties, in order to point out structure-specific aspects of the overall effect of individual polyols.
Polyols in solution shift the Native/Denatured equilibrium towards the native state. Sucrose, sorbitol, and trehalose share many similarities in the mechanism of stabilization: they have the strongest stabilizing properties, that appear related to their molar concentration. On the contrary, glycerol presents peculiar features and the lowest stabilizing effects. The effect of polyols on the overall denaturation of BLG (as detected by thermal analysis) relates to ability of each polyol to improve surface hydrophobic interactions (i.e., within the solvent-excluded region). This interpretation is also supported by our CD data. However, individual polyols have region-specific stabilizing effects, suggesting that each of them has a somewhat specific mechanism of action.
In spite of the huge number of literature reports, properties of polyols in solution are not completely understood yet, and polyols are still used on quite empirical basis. This study indicates that stabilizing effects of individual polyols target polyol-specific structural features of the protein, and are not only a function of the polyol(s) physico-chemical features, paving the road to improving their rational application in protein stabilization
Caratterizzazione strutturale e suo ruolo nella definizione della qualità della pasta
No full textPer correlare gli indici di qualità della pasta di natura sensoriale e reologica con la struttura macro- e micro-molecolare servono approcci adatti a descrivere le caratteristiche strutturali del reticolo macromolecolare nel prodotto: la microscopia elettronica può essere utile a evidenziare la struttura complessiva; informazioni sulla mobilità dell’acqua prima e dopo cottura possono essere ottenute con approcci basati sull’imaging in Risonanza Magnetica Nucleare; l’organizzazione della frazione proteica può essere evidenziata mediante misure di fluorescenza allo stato solido o di solubilità condizionale. Le informazioni così ottenute possono essere combinate per descrivere dal punto di vista strutturale campioni di pasta diversi, evidenziando ad esempio come le modalità di essiccamento comportano una peculiare strutturazione macromolecolare che condiziona le proprietà complessive del prodotto
Structure–quality relationship in commercial pasta: a molecular glimpse
No full textPresence and stability of a protein network was evaluated by fluorescence spectroscopy, by protein solubility studies, and by assessing the accessibility of protein thiols in samples of commercial Italian semolina pasta made in industrial plants using different processes. The pasting properties of starch in each sample were evaluated by means of a viscoamylograph. Magnetic resonance imaging (MRI) was used to evaluate water distribution and water mobility in dry pasta, and at various cooking times. The molecular information derived from these studies was related to sensory indices, indicating that protein reticulation was dependent on the process conditions, which affected water penetration, distribution, and mobility during cooking. Products with a crosswise gradient of water mobility once cooked had the best sensory scores at optimal cooking time, whereas products with a less compact protein network performed better when slightly overcooked
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