NMR investigation of enzymatic coupling of sulfonamide antimicrobials with humic substances

Phenoloxidases mediate the oxidative transformation of soil phenolic constituents, contributing to the formation of humic substances and the chemical incorporation of some xenobiotic organic compounds into natural organic matter. We previously demonstrated phenoloxidase-mediated covalent coupling of sulfonamide antimicrobials with model humic constituents. Here, we investigate fungal peroxidase-mediated covalent coupling of 13C-sulfamethazine and 15N-sulfapyridine to humic substances. 1H-13C heteronuclear single quantum correlation (HSQC) nuclear magnetic resonance spectroscopy provided an initial indication of peroxidase-mediated covalent binding of 13C-sulfamethazine to humic acid. To confirm the role of the sulfonamide anilinic nitrogen in coupling to humic acid and to determine the nature of the covalent linkage, we incubated 15N-sulfapyridine with humic acid and peroxidase and examined reaction products in 1H-15N heteronuclear multiple bond (HMBC) experiments. The HMBC spectra revealed the presence of Michael adducts (i.e., anilinohydroquinones, anilinoquinones) and possibly other covalent linkages. No evidence for Schiff base formation was observed. Analogous experiments with the model humic constituent catechol provided corroborating evidence for these assignments. Michael adducts are expected to exhibit greater environmental stability than imine linkages that can form between sulfonamides and 2,6-dimethoxyphenols. Because the free anilinic nitrogen is required for the bioactivity of sulfonamide antimicrobials, nucleophilic addition occurring through this moiety could result in the biochemical inactivation of these compounds.     

Sulphite adducts of cyclic monoterpene ketones

Structures of sulphite addition products of some cyclic monoterpene ketones with Na₂SO₃ and/or NaHSO₃ were studied. The conjugated enones reacted with Na₂SO₃ and gave 1,4-addition products. The non-conjugated compounds did not react with Na₂SO₃. NaHSO₃ made radical additions to non-conjugated olefinic bonds. On the other hand, ketones having no olefinic bond did not form addition products. 1,4-addition products underwent a reverse reaction when alkali was added but the radical addition product was stable towards alkali and did not undergo reverse reaction.     

Laccase-mediated michael addition of 15N-sulfapyridine to a model humic constituent

Chemical incorporation of sulfonamide antimicrobials into natural organic matter may represent an important process influencing the fate of these synthetic, primarily agents in soil and sediment environments. We previously demonstrated that a fungal peroxidase mediates covalent coupling of sulfonamide antimicrobials to model humic constituents; reactions with the 2,6-dimethoxyphenol syringic acid produced Schiff bases (Bialk et al. Environ. Sci. TechnoL 2005, 39, 4436-4473). Here, we show that fungal laccase-mediated reaction of sulfapyridine with the orthodihydroxyphenol protocatechuic acid yields a Michael adduct. We synthesized 15N-enriched sulfapyridine to facilitate determination of the covalent linkage(s) formed between sulfapyridine and protocatechuic acid by NMR spectroscopy. 1H-(15)N heteronuclear multiple bond correlation experiments and tandem mass spectrometry demonstrated that the sulfapyridine anilinic nitrogen engaged in a Michael addition reaction to oxidized protocatechuic acid to form an anilinoquinone. Michael adducts are more stable than the previously reported imine linkages between sulfonamides and 2,6-dimethoxyphenols. Michael addition to quinone-like structures in soil organic matter is expected to diminish the mobility and biological activity of sulfonamide antimicrobials.     

功能化氧化石墨烯吸附材料的研究进展

为推进氧化石墨烯材料在水处理领域的应用,综述了近几年功能化氧化石墨烯吸附材料的研究成果。首先介绍了氧化石墨烯的制备方法与结构特征,然后将功能化氧化石墨烯吸附材料分成共价键改性材料和非共价键复合材料。针对氧化石墨烯上不同官能团的化学修饰,将共价键改性材料划分成5类:羰基功能化、羧基功能化、羟基功能化、环氧基功能化和碳碳双键功能化。根据复合材料结构形态的不同,将非共价键复合材料划分成3类:氧化石墨烯基凝胶、氧化石墨烯基分离膜、氧化石墨烯基磁性吸附剂。最后讨论了功能化氧化石墨烯吸附材料在水处理中存在的问题,指出共价键和非共价键的联合功能化处理有望成为氧化石墨烯吸附材料的发展方向。     

Quinone emissions from gasoline and diesel motor vehicles

Gas- and particle-phase emissions from gasoline and diesel vehicles operated on chassis dynamometers were collected using annular denuders, quartz filters, and PUF substrates. Quinone species were measured using O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine derivatization in conjunction with gas chromatography-mass spectrometry and high-performance liquid chromatography-mass spectrometry. Nine quinones were observed, ranging from C6 to C16. New species identified in motor vehicle exhaust include methyl-1,4-benzoquinone, 2-methyl-1,4-naphthoquinone (MNQN), and aceanthrenequinone. Gas-phase motor vehicle emissions of quinones are also reported for the first time. Six gas-phase quinones were quantified with emission rates of 2-28 000 microg L(-1) fuel consumed. The most abundant gas-phase quinones were 1,4-benzoquinone (BON) and MNQN. The gas-phase fraction was > or = 69% of quinone mass for light-duty gasoline emissions, and > or = 84% for heavy-duty diesel emissions. Eight particle-phase quinones were observed between 2 and 1600 microg L(-1), with BQN the most abundant species followed by 9,10-phenanthrenequinone and 1,2-naphthoquinone. Current particle-phase quinone measurements agree well with the few available previous results. Further research is needed concerning the gas-particle partitioning behavior of quinones in ambient and combustion source conditions.     

壳聚糖与阳离子淀粉接枝共聚物作为造纸助剂的研究

研究了壳聚糖和阳离子淀粉接技共聚反应。共聚物作为造纸增强剂加入浆中，其应用效果在用量相同时，增强效果优于复配物，达到相同的使用效果时，用量少于复配物。共聚物在纤维间产生了离子键结合，增加形成氢键的能力，是提高纸页强度的主要机理。     

Geometry of 1,2-dihydroxy-1,2-disulphonates

An evaluation of the probability of hydrogen bonding between sulphonate and hydroxyl groups on adjacent carbon atoms of 1,2-dihydroxy-1,2-disulphonates is reported. Using published bond lengths and angles, and allowing free rotation about CC, CO and CS bonds, 2·9% of the possible conformations lead to OH....dO and H....O distances conducive to hydrogen bonding. The significance of the calculated OH....O and SO....H bond angles is also considered. These results are discussed in terms of the observation that 1,2-dihydroxy-1,2-disulphonates appear to be more labile than 1-hydroxy-2-oxosulphonates, and the relevance of this result to the inhibition of non-enzymic browning by sulphur dioxide in foods is appraised.     

Hydroxyl radical generation mechanism during the redox cycling process of 1,4-naphthoquinone

Airborne quinones contribute to adverse health effects of ambient particles probably because of their ability to generate hydroxyl radicals (·OH) via redox cycling, but the mechanisms remain unclear. We examined the chemical mechanisms through which 1,4-naphthoquinone (1,4-NQ) induced ·OH, and the redox interactions between 1,4-NQ and ascorbate acid (AscH(2)). First, ·OH formation by 1,4-NQ was observed in cellular and acellular systems, and was enhanced by AscH(2). AscH(2) also exacerbated the cytotoxicity of 1,4-NQ in Ana-1 macrophages, at least partially due to enhanced ·OH generation. The detailed mechanism was studied in an AscH(2)/H(2)O(2) physiological system. The existence of a cyclic 1,4-NQ process was shown by detecting the corresponding semiquinone radical (NSQ·-) and hydroquinone (NQH(2)). 1,4-NQ was reduced primarily to NSQ·- by O2·- (which was from AscH(2) reacting with H(2)O(2)), not by AscH(2) as normally thought. At lower doses, 1,4-NQ consumed O2·- to suppress ·OH; however, at higher doses, 1,4-NQ presented a positive association with ·OH. The reaction of NSQ·- with H(2)O(2) to release ·OH was another important channel for OH radical formation except for Haber-Weiss reaction. As a reaction precursor for O2·-, the enhanced ·OH response to 1,4-NQ by AscH(2) was indirect. Reducing substrates were necessary to sustain the redox cycling of 1,4-NQ, leading to more ·OH and a deleterious end point.     

15N NMR investigation of the covalent binding of reduced TNT amines to soil humic acid,model compounds,and lignocellulose

The five major reductive degradation products of TNT-4ADNT (4-amino-2,6-dinitrotoluene), 2ADNT (2-amino-4,6-dinitrotoluene), 2,4DANT (2,4-diamino-6-nitrotoluene), 2,6DANT (2,6-diamino-4-nitrotoluene), and TAT (2,4,6-triaminotoluene)-labeled with 15N in the amine positions, were reacted with the IHSS soil humic acid and analyzed by 15N NMR spectrometry. In the absence of catalysts, all five amines underwent nucleophilic addition reactions with quinone and other carbonyl groups in the soil humic acid to form both heterocyclic and nonheterocyclic condensation products. Imine formation via 1,2-addition of the amines to quinone groups in the soil humic acid was significant with the diamines and TAT but not the monoamines. Horseradish peroxidase (HRP) catalyzed an increase in the incorporation of all five amines into the humic acid. In the case of the diamines and TAT, HRP also shifted the binding away from heterocyclic condensation product toward imine formation. A comparison of quantitative liquid phase with solid-state CP/MAS 15N NMR indicated that the CP experiment underestimated imine and heterocyclic nitrogens in humic acid, even with contact times optimal for observation of these nitrogens. Covalent binding of the mono- and diamines to 4-methylcatechol, the HRP catalyzed condensation of 4ADNT and 2,4DANT to coniferyl alcohol, and the binding of 2,4DANT to lignocellulose with and without birnessite were also examined.     

Mechanisms and products of surface-mediated reductive dehalogenation of carbon tetrachloride by Fe(II) on goethite

Natural attenuation processes of chlorinated solvents in soils and groundwaters are increasingly considered as options to manage contaminated sites. Under anoxic conditions, reactions with ferrous iron sorbed at iron(hyro)xides may dominate the overall transformation of carbon tetrachloride (CCl4) and other chlorinated aliphatic hydrocarbons. We investigated mechanisms and product formation of CCl4 reduction by Fe(II) sorbed to goethite, which may lead to completely dehalogenated products or to chloroform (CHCl3), a toxic product which is fairly persistent under anoxic conditions. A simultaneous transfer of two electrons and cleavage of two C-Cl bonds of CCl4 would completely circumvent chloroform production. To distinguish between initial one- or two-bond cleavage, 13C-isotope fractionation of CCl4 was studied for reactions with Fe(II)/ goethite (isotopic enrichment factor epsilon = -26.5% percent per thousand) and with model systems for one C-Cl bond cleavage and either single-electron transfer (Fe(II) porphyrin, epsilon = -26.1 percent per thousand) or partial two-electron transfer (polysulfide, epsilon = -22.2 percent per thousand). These epsilon values differ significantlyfrom calculations for simultaneous cleavage of two C-Cl bonds (epsilon approximately equal to -50 percent per thousand), indicating that only one C-Cl bond is broken in the critical first step of the reaction. At pH 7, reduction of CCl4 by Fe(II)/ goethite produced approximately 33% CHCl3, 20% carbon monoxide (CO), and up to 40% formate (HCOO-). Addition of 2-propanol-d8 resulted in 33% CDCl3 and only 4% CO, indicating that both products were generated from trichloromethyl radicals (*CCl3), chloroform by reaction with hydrogen radical donors and CO by an alternative pathway likely to involve surface-bound intermediates. Hydrolysis of CO to HCOO-was surface-catalyzed by goethite butwastoo slow to account for the measured formate concentrations. Chloroform yields slightly increased with pH at constant Fe(II) sorption density, suggesting that pH-dependent surface processes direct product branching ratios. Surface-stabilized intermediates may thus facilitate abiotic mineralization of CCl4, whereas the presence of H radical donors, such as natural organic matter, enhances formation of toxic CHCl3.     

Comparative Effects of Ohmic,Induction Cooker,and Electric Stove Heating on Soymilk Trypsin Inhibitor Inactivation

During thermal treatment of soymilk, a rapid incorporation of Kunitz trypsin inhibitor (KTI) into protein aggregates by covalent (disulfide bond, SS) and/or noncovalent interactions with other proteins is responsible for its fast inactivation of trypsin inhibitor activity (TIA). In contrast, the slow cleavage of a single Bowman–Birk inhibitor (BBI) peptide bond is responsible for its slow inactivation of TIA and chymotrypsin inhibitor activity (CIA). In this study, the effects of Ohmic heating (220 V, 50 Hz) on soymilk TIA and CIA inactivation were examined and compared to induction cooker and electric stove heating with similar thermal histories. It was found that: (1) TIA and CIA inactivation was slower from 0 to 3 min, and faster after 3 min as compared to induction cooker and electric stove. (2) The thiol (SH) loss rate was slower from 0 to 3 min, and similar to induction cooker and electric stove after 3 min. (3) Ohmic heating slightly increased protein aggregate formation. (4) In addition to the cleavage of one BBI peptide bond, an additional reaction might occur to enhance BBI inactivation. (5) Ohmic heating was more energy‐efficient for TIA and CIA inactivation. (6) TIA and CIA inactivation was accelerated with increasing electric voltage (110, 165, and 220 V) of Ohmic heating. It is likely that the enhanced inactivation of TIA by Ohmic heating is due to its combined electrochemical and thermal effects.     

Using 19F NMR spectroscopy to determine trifluralin binding to soil

Trifluralin is a widely used herbicide for the control of broad leaf weeds in a variety of crops. Its binding to soil may result in significant losses in herbicidal activity and a delayed pollution problem. To investigate the nature of soil-bound trifluralin residues, 14C-labeled herbicide was incubated for 7 weeks with four soils under anoxic conditions. As determined by radiocounting, trifluralin binding ranged between 10 and 53% of the initial 14C depending on the soil tested. 19F NMR analyses of the methanol extracts and different fractions of the extracted soil suggested that bound residue formation largely involved reduced metabolites of the herbicide. A 2,6-diamino product of trifluralin reduction with zero-valent iron (Fe-TR), and the standard of a 1,2-diaminotrifluralin derivative (TR6) formed covalent bonds with fulvic acid (FA), as indicated by the 19F NMR spectra taken periodically over a 3-week contact time. At short contact times, TR6 and Fe-TR formed weak physical bonds with FA, as the respective spin-lattice relaxation times (T1) decreased from the range 1300-1831 ms for TR6 or Fe-TR analyzed in the absence of FA to the range 150-410 ms for TR6/FA or Fe-TR/FA mixtures. In general, the results indicated that trifluralin immobilization involved a variety of mechanisms (covalent binding, adsorption, sequestration), and with time it became increasingly stable.     

香云纱工艺中晒莨工序的染色机制

采用高效液相色谱、红外光谱等测试手段,考察香云纱工艺中晒莨工序前后薯莨单宁组分与结构的变化、纤维材料结构的变化情况,结合植物多酚化学理论,探讨晒莨工序的染色作用机理。认为：一、在晒莨工序中,曝晒使薯莨单宁氧化形成醌式结构而显棕色,因此随着浸染、曝晒次数的增多,晒坯表面的颜色不断加深,最终变为棕色或棕红色。二、疏水键-氢键多点结合可能是薯莨单宁与丝、棉、麻等纤维结合的共同方式;化学结构的不同使得各种纤维与薯莨单宁结合的数量、强度差异很大,因而影响染色牢度。蚕丝结构中含有疏水性氨基酸以及众多的氢键结合点,可形成大量的疏水键-氢键结合,因此可获得良好的加工效果。三、丝绸加工效果好的另外两个可能的原因是薯莨单宁与蚕丝蛋白形成共价键结合,以及蚕丝丰富的原纤结构,使得薯莨单宁与蚕丝的结合点显著多于其他纤维。     

Understanding trichloroethylene chemisorption to iron surfaces using density functional theory

This research investigated the thermodynamic favorability and resulting structures for chemical adsorption of trichloroethylene (TCE) to metallic iron using periodic density functional theory (DFT). Three initial TCE positions having the plane defined by HCC atoms parallel to the iron surface resulted in formation of three different chemisorption complexes between carbon atoms in TCE and the iron surface. The Cl-bridge initial configuration with the HCC plane of TCE perpendicular to the iron surface did not result in C-Fe bond formation. The most energetically favorable complex formed at the C-bridge site where the initial configuration had the C=C bond in TCE at a bridge site between adjacent iron atoms. In the C-bridge complex, one C atom formed two a bonds to different Fe atoms, while the second C atom formed a sigma bond with a second Fe atom. Surface complexation atthe C-bridge site resulted in scission of all three C-Cl bonds and also resulted in a shortening of the C==C bond to a distance intermediate between a double and a triple bond. Initial configurations with the C==C bond adsorbed at top or hollow sites on the iron surface resulted in formation of C-Fe a bonds between a single C and two adjacent Fe atoms, and the scission of only two C==Cl bonds. Bond angles and bond lengths indicated that there were no changes in bond order of the C==C bond for top and hollow adsorption. Chemisorption at the C-bridge site had an activation energy of 49 kJ/mol and an early transition state where all three C-CI bonds were activated. The early transition state and the loss of all three Cl atoms upon chemisorption are consistent with most experimental observations that TCE undergoes complete dechlorination in one interaction with the iron surface. The absence of chemisorption and scission of only two C--Cl bonds at the Cl-bridge site is consistent with experimental observations that trace amounts of chloroacetylene may also be produced from reactions of TCE with iron.     

Effect of solution pH on solubility and some structural properties of soybean protein isolate films

Changes in solubility and molecular properties of protein films obtained from soy protein isolate (SPI) solutions at different pH values (2, 8 and 11) were investigated to study protein behavior during film formation. Proteins retained their native conformation in films at pH 8, but were partially or extensively denatured at pH 11 and 2. Although film protein networks were maintained by the same type of interactions at different pH values—covalent (disulfide bonds) and non‐covalent bonds (especially hydrophobic interactions and hydrogen bonds)—the intensity of each type of interaction (predicted from solubility tests in buffers with different chemical action) depended on the pH of the initial solution. Films obtained at pH 8 presented the highest solubility in all the buffers, whereas films at formed pH 2 presented the lowest, except in the buffer of pH 8 that contained urea, SDS and 2‐mercaptoethanol, which totally dissolved 100% of the film proteins. Films prepared at extreme pH values had a denser microstructure than those at pH 8. SDS–PAGE patterns indicated that films were mainly formed by β‐conglycinin and glycinin, which aggregated in different forms during film formation, depending on the pH of the initial solutions. Some of these proteins remained weakly bonded and/or were held by the network of films. These differences in the protein networks structure would affect the physical, mechanical and barrier properties of the films. Copyright © 2006 Society of Chemical Industry     

高密度CO2处理过程中虾肌球蛋白分子间作用力的变化

高密度CO2（dense phase carbon dioxide,DPCD）是一种非常有前景的非热食品加工技术,能诱导蛋白 质发生变性并进行自组装形成凝胶。为了探讨DPCD诱导蛋白质自组装形成凝胶的机制,以凡纳滨对虾肌球蛋 白为对象,研究DPCD处理压力和温度对肌球蛋白分子间作用力的影响。结果表明：与未处理的相比,DPCD处 理能使肌球蛋白的氢键和离子键贡献降低（P＜0.05）,而使疏水相互作用、二硫键和非二硫共价键的贡献增加 （P＜0.05）;在相同DPCD处理温度下,压强（5～25 MPa）对肌球蛋白分子间作用力无显著影响（P＞0.05）;在 相同DPCD处理压强下,随着温度（40～60 ℃）升高,氢键和二硫键贡献无显著变化（P＞0.05）,非二硫共价键 贡献呈增加趋势（P＜0.05）,疏水相互作用呈降低趋势（P＜0.05）,离子键贡献则先降低后升高（P＜0.05）。因 此,疏水相互作用、二硫键和非二硫共价键是DPCD诱导肌球蛋白形成凝胶过程中的主要分子间作用力,而氢键和 离子键不是其主要分子间作用力。研究结果为阐明DPCD诱导蛋白质形成凝胶的机制提供了基础数据。     

Covalent interactions between amino acid side chains and oxidation products of caffeoylquinic acid (chlorogenic acid)

BACKGROUND: Physicochemical properties and digestibility of proteins can be modified by covalent interactions with oxidized phenolic compounds, i.e., quinones. In order to control these interactions in food products, the covalent interactions between quinones from caffeoylquinic acid (CQA) and amino acid side chains were studied with mass spectrometry using N‐terminally protected amino acids. RESULTS: The addition of two molecules of CQA, presumably in the form of a pre‐formed dimer, was observed for lysine, tyrosine, histidine and tryptophan. A monomer of CQA seemed to be able to react with histidine and tryptophan, whereas no interaction with a CQA monomer was observed for lysine and tyrosine. Serine and threonine showed no covalent interactions with CQA. Cross‐linking between CQA and the side chains of two molecules of lysine is likely to occur also in proteins. The results show that protein cross‐linking may also be expected to occur via two tyrosine residues in the absence of other phenolic substrates. The side chains of lysine and tyrosine are more reactive than that of histidine and tryptophan. CONCLUSIONS: These results show that covalent protein modification by oxidized phenolics occurs preferentially via an initial dimerization and encompasses not only lysine and cysteine residues. Copyright © 2008 Society of Chemical Industry     

Molecular and Supermolecular Structure of Commercial Pyrodextrins

Size exclusion chromatography with triple detection as well as infrared spectroscopy studies of commercially available pyrodextrins proved that these molecules are characterized not only by significantly lower molecular mass, in comparison to that of native starch, but also by increased branching. Therefore, pyrodextrins adopt a very compact structure in solution and show Newtonian behavior under shear in spite of their molecular masses of tens of thousands Daltons. The results also indicate that 50% reduction of digestibility of pyrodextrins is, to a minor extent, caused by formation of low‐molecular color compounds containing carbonyl functional groups. The main reason is, as postulated in the literature, transglycosidation that leads to decreased occurrence of α‐1,4‐glycoside bonds in the molecular structure. In the process of dextrinization starch also undergoes changes in supermolecular structure, which, however, have no influence on digestibility. Likewise, the effect of formation of low‐molecular colorful compounds containing carbonyl groups is not crucial.     

Effects of malondialdehyde-induced protein oxidation on the structural characteristics of rice protein

Malondialdehyde (MDA) was selected to represent a secondary by-product of lipid peroxidation during rice ageing. This study aimed to investigate the effects of MDA modification on the structural characteristics of rice protein. The results showed that as MDA concentration increased, rice protein carbonyl and disulphide groups increased, but sulphydryl content decreased. The blue shift of maximum fluorescence peak, the decrease of rice protein intrinsic fluorescence intensity and the reduction of surface hydrophobicity indicated the formation of protein aggregates caused by MDA oxidative modification. The results of molecular weight distribution and particle size distribution showed that MDA modification resulted in the formation of soluble protein aggregates, and the decrease of rice protein solubility indicated that insoluble protein aggregates were formed. Results of protein electrophoresis showed that MDA modification contributed to rice protein aggregation via non-disulphide covalent bonds. The results showed that rice protein gradually aggregated with increasing MDA concentration.     

花青素与大豆分离蛋白的不同共价交联法对蛋白结构的影响

分别运用生物酶法和化学碱法,研究不同质量浓度花青素与大豆分离蛋白（soy protein isolate,SPI）发生共价相互作用后对蛋白结构的影响。采用共价结合率测定和游离巯基含量测定方法对花青素和SPI在不同共价交联方式下的结合强度进行比较,后采用傅里叶变换红外光谱、紫外-可见吸收光谱和荧光光谱对不同共价交联方式下的花青素-SPI共价聚合物的结构及构象进行解析。结果表明：随着花青素添加量的升高,花青素与SPI的结合率逐渐提升,游离巯基含量持续下降。光谱测定显示花青素对SPI的共价交联可以改变蛋白的二级结构,使蛋白多肽链解折叠,并且改变蛋白芳香族氨基酸残基所处的微环境,进而使蛋白的构象发生改变。此外,相较于化学碱法,生物酶法共价交联的花青素-SPI聚合物结合率更高,巯基含量下降更显著,结构及构象的变化更为明显,这表明花青素与SPI在生物酶法处理下,共价结合能力强于化学碱法处理。