NMR approach for monitoring the photo-degradation of riboflavin and methionine

The light exposure of white wine is responsible for several reactions leading to changes on colour, flavours and, consequently, affecting the sensory profile. These reactions can take place when the white wine is bottled in clear glass and their mechanisms are dependent on both light exposure and chemical composition of white wine. Particular attention has been given to the reaction involving riboflavin (RF), a photo-sensitizer compound, and methionine (Met), a sulfur-containing amino acid, that can cause the formation of volatile sulphur compounds (VSCs), namely methanethiol and dimethyl disulfide. These compounds are responsible for a defect known as light-struck taste. Previous studies showed that hydrolysable tannins, in particular those from nut galls, limited both the degradation of Met and the formation of VSCs. The effectiveness of hydrolysable tannins was also proved after light exposure and storage for 24 months. In order to better understand the role of tannins in the photo-degradative reactions, an NMR approach was carried out. A solution containing RF (0.2 mM) and Met (2 mM) acidified at pH 3.2 was exposed to light by using fluorescence light bulbs. The solution was exposed to light up to two hours sampling it every 15 minutes. The same experimental conditions were applied in presence of gallic acid (2 mM), a constitutive unit of nut gall tannins. The degradation of RF and Met was monitored and, as expected, their signals decreased as the light exposure increased. Results provided evidence that a new signal appeared at 2.64 ppm. This signal was assigned to the SOCH3 moiety of methionine sulfoxide through the addition of the standard solution and standard 2D-NMR assignment techniques. The formation kinetic of methionine sulfoxide was measured for increased duration of light exposure and its rate resulted two-folds lower with the addition of gallic acid. This result suggests that the limited degradation of Met in presence of tannins, also observed in previous studies, is due to their action as competitor with Met in reducing RF from its excited form. The NMR technique was suitable for monitoring the photo-degradative reaction of RF and Met. Further researches have been carried out in order to verify and prove the ability of tannins in quenching both singlet oxygen and RF

Spatial and temporal variations in Pb concentrations and isotopic composition in road dust, farmland soil and vegetation in proximity to roads since cessation of use of leaded petrol in the UK

Results are presented for a study of spatial distributions and temporal trends in concentrations of lead (Pb) from different sources in soil and vegetation of an arable farm in central Scotland in the decade since the use of leaded petrol was terminated. Isotopic analyses revealed that in all of the samples analysed, the Pb conformed to a binary mixture of petrol Pb and Pb from industrial or indigenous geological sources and that locally enhanced levels of petrol Pb were restricted to within 10 m of a motorway and 3 m of a minor road. Overall, the dominant source of Pb was historical emissions from nearby industrial areas. There was no discernible change in concentration or isotopic composition of Pb in surface soil or vegetation over the decade since the ban on the sale of leaded petrol. There was an order of magnitude decrease in Pb concentrations in road dust over the study period, but petrol Pb persisted at up to 43% of the total Pb concentration in 2010. Similar concentrations and spatial distributions of petrol Pb and non petrol Pb in vegetation in both 2001 and 2010, with enhanced concentrations near roads, suggested that redistribution of previously deposited material has operated continuously over that period, maintaining a transfer pathway of Pb into the biosphere. The results for vegetation and soil transects near minor roads provided evidence of a non petrol Pb source associated with roads/traffic, but surface soil samples from the vicinity of a motorway failed to show evidence of such a source

Low-​temperature intermediates to oxygen reduction reaction catalysts based on amine-​modified metal-​loaded carbons : an XPS and ss-​NMR investigation

Carbon 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

The 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

Investigating the role of antioxidant compounds in riboflavin-mediated photo-oxidation of methionine: a 1H-NMR approach

Riboflavin (RF) is a well-known photosensitizer, responsible for the light-induced oxidation of methionine (Met) leading to the spoilage of wine. An NMR approach was used to investigate the role of gallic acid (GA) and sulfur dioxide (SO2) in the RF-mediated photo-oxidation of Met. Water solutions of RF and Met, with and without GA or SO2, were exposed to visible light for increasing time in both air and nitrogen atmosphere. Upon light exposure, a new signal appeared at 2.64 ppm that was assigned to the S(O)CH3 moiety of methionine sulfoxide. Its formation rate was lower in nitrogen atmosphere and even lower in the presence of GA, supporting the ability of this compound in quenching the singlet oxygen. In contrast, SO2 caused relevant oxidation of Met, moderately observed even in the dark, making Met less available in donating electrons to RF. The competition of GA vs Met photo-oxidation was revealed, indicating effectiveness of this antioxidant against the light-dependent spoilage of wine. A pro-oxidant effect of SO2 toward Met was found as a possible consequence of radical pathways involving oxygen

\u201cIron Priming\u201d Guides Folding of Denatured Aporubredoxins

The relationship between iron uptake by aporubredoxins (apoRds) and formation of native holorubredoxins (holoRd), including their Fe(SCys) 4 sites, was studied. In the absence of denaturants, apoRds exhibited spectroscopic features consistent with structures very similar to those of the folded holoRds. However, additions of either ferric or ferrous salts to the apoRds in the absence of denaturants gave less than 40% recovery of the native holoRd circular dichroism and UV-vis spectroscopic features. In the presence of either 6 M urea or 6 M guanidine hydrochloride, the nativelike structural features of the apoRds were absent. Nevertheless, nearly quantitative recoveries of the native holoRd spectroscopic features were achieved by addition of either ferric or ferrous salts to the denatured apoRds without diluting the denaturant. Consistent with this observation, the native spectroscopic features were unaffected by addition of the same denaturant concentrations to the as-isolated holoRds. Denaturing concentrations of urea or guanidine hydrochloride also increased the rates of holoRd recoveries from apoRds and ferrous salts. Mass spectrometry confirmed that ferric iron binding to the denatured apoRds precedes the recoveries of protein secondary structures and Fe(SCys)4 sites. Thus, iron binding to the apoRds guides, both kinetically and thermodynamically, refolding to the native holoRd structures. Our results imply that the ferrous oxidation state would more efficiently drive formation of the native holoRd structure from the nascent apoprotein in vivo, but that the Fe(SCys)4 site must attain the ferric state in order to achieve its native structure

Steroid hydroxylations with Botryodiplodia malorum and Colletotrichum lini

An improved procedure for the microbial hydroxylations of dehydroepiandrosterone (DHEA, 1) and 15\u3b2,16\u3b2-methylene-dehydroepiandrosterone (2) was studied using whole cells of Botryodiplodia malorum and Colletotrichum lini. C. lini catalyzed 7\u3b1- and 15\u3b1-hydroxylation of 1 and 7\u3b1-hydroxylation of 2, while B. malorum gave 7\u3b2-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 7\u3b1,15\u3b1-dihydroxy-1 production were improved obtaining 5.8 g l 121 (recovered product) from 7.0 g l 121 of substrate. The structures of the hydroxylated products were assigned by a combination of two-dimensional NMR proton\u2013proton and proton\u2013carbon correlation techniques

Polyols stabilize specific regions in the structure of betalactoglobulin

Polyols 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

Per correlare gli indici di qualit\ue0 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 pu\uf2 essere utile a evidenziare la struttura complessiva; informazioni sulla mobilit\ue0 dell\u2019acqua prima e dopo cottura possono essere ottenute con approcci basati sull\u2019imaging in Risonanza Magnetica Nucleare; l\u2019organizzazione della frazione proteica pu\uf2 essere evidenziata mediante misure di fluorescenza allo stato solido o di solubilit\ue0 condizionale. Le informazioni cos\uec ottenute possono essere combinate per descrivere dal punto di vista strutturale campioni di pasta diversi, evidenziando ad esempio come le modalit\ue0 di essiccamento comportano una peculiare strutturazione macromolecolare che condiziona le propriet\ue0 complessive del prodotto