Interaction of beta-lactoglobulin with small hydrophobic ligands as monitored by fluorometry and equilibrium dialysis: nonlinear quenching effects related to protein--protein association

Although a thorough characterization of binding parameters is essential for application of beta-lactoglobulin as a carrier for a variety of small hydrophobic ligands, the binding parameters derived in various studies using various techniques are inconsistent. The bindings of several small ligands as detected by fluorometry and equilibrium dialysis were compared. Fluorescence spectroscopy showed that beta-ionone, retinol, and fatty acid lactones all bound in the vicinity of a tryptophan residue. Retinol and fatty acid lactone competed for the same binding site. Exclusively for ligands that quench the beta-lactoglobulin fluorescence through a resonance energy transfer mechanism, fluorometry yielded a systematically higher binding affinity than equilibrium dialysis. The binding overestimation in fluorometric measurements can be explained by oligomer formation of protein, together with an underestimation of the limiting quenching level at saturating ligand concentrations due to the use of a limited set of data points.     

Investigation of binding behavior of alpha- and beta-ionones to beta-lactoglobulin at different pH values using a diffusion-based NOE pumping technique

Diffusion-based NMR techniques were employed to study effects of pH on beta-lactoglobulin (BLG) conformation and binding affinity to alpha- and beta-ionone. In the first part of the study, the influence of pH on the diffusion coefficient of BLG in D(2)O solution was investigated using a stimulated-echo NMR experiment. The diffusion coefficient of BLG decreased with increasing pH values. A significant decrease in the diffusion coefficient observed at pH 11 may be due to total unfolding (denaturation) of the protein, resulting in hydrophobically driven self-aggregation. A diffusion-based NOE pumping technique was then applied to determine the relative binding affinities between alpha- and beta-ionones and BLG at pH values varying from 3 to 11. An increase in signal intensities for beta-ionone with increasing molar concentration ratios between beta-ionone and BLG was observed at all pH ranges studied. The increased signal intensities reflect increased relative binding affinity. The greatest binding affinity occurred at pH 9 and the lowest at pH 11. alpha-Ionone showed binding evidence only at pH 9, and the binding was significantly weaker than that obtained for beta-ionone at the same pH. The high affinity observed for both aroma compounds at pH 9 may be due to a flexible conformation of BLG at this pH so that the flavor ligand accessibility increases. Conversely, alkaline denaturation occurring at pH 11 gives rise to relatively lower binding affinity compared to that observed at the other pH values.     

Interactions between beta-lactoglobulin and aroma compounds: different binding behaviors as a function of ligand structure

Interactions between beta-lactoglobulin (BLG) in its monomeric form and a wide range of aroma compounds were investigated by Fourier transform infrared (FT-IR) and 2D nuclear magnetic resonance (NMR) spectroscopies. A screening of the ligands was carried out by FT-IR through the amide I region changes of BLG upon binding. The location of two binding sites was determined by 2D NMR from the study of 10 selected ligands with different structures. All of the data suggest at least two binding behaviors as a function of the chemical class, the hydrophobicity, or the structure of the ligands. The binding of the elongated aroma compounds, such as 2-nonanone or ethyl pentanoate, within the central cavity involves residues located at the entrance of the calyx and Trp19. The binding onto the protein surface of aroma compounds that have or adopt a compact structure occurs in a site located between strand beta-G, alpha helix, and strand beta-I.     

Pressure-induced denaturation of monomer beta-lactoglobulin is partially irreversible: comparison of monomer form (highly acidic pH) with dimer form (neutral pH)

This study was conducted to assess the effect of high hydrostatic pressure on monomer beta-lactoglobulin (BLg) at acid pH by fluorescence spectroscopy under pressure and by circular dichroism (CD) and (1)H NMR spectroscopies after release of pressure. The intrinsic (tryptophan) fluorescence measurement and the study of 8-anilinonaphthalene-1-sulfonate (ANS) binding to BLg indicated that at pH 2.0 the recovery of center of spectral mass or ANS fluorescence was almost complete upon pressure release. No difference in (1)H NMR spectra was observed between pressurized and unpressurized BLg. In addition, NMR detection of the H/D exchange of aromatic protein indicated that the conformation of the vicinity of tryptophan residues could be refolded almost completely after release of pressure. These results seemingly confirm that the pressure-induced denaturation of BLg at pH 2.0 is reversible. However, cis-parinaric acid binding ability of pressurized BLg was largely lost, although its retinol binding ability was the same as its unpressurized one. Furthermore, CD spectra of the far-UV region and 2D NMR spectra evidently revealed the difference in the conformation of the molecule between unpressurized and pressurized BLg. These results are interpreted as an existence of partially fragile structure in the BLg molecule by high pressure.     

High-resolution NMR and diffusion-ordered spectroscopy of port wine

The use of high-resolution NMR and high-resolution diffusion-ordered spectroscopy (DOSY) for the characterization of selected Port wine samples of different ages with the aim of identifying changes in composition is described. Conventional 1D and 2D NMR methods enabled the identification of about 35 compounds, including minor components such as some medium-chain alcohols, amino acids, and organic acids. High-resolution (HR) DOSY extended sample characterization, increasing the number of compounds identified and NMR assignments made, by providing information on the relative molecular sizes of the metabolites present. Port wines of different ages were found to differ mainly in their content of (a) organic acids and some amino acids, (b) an unidentified possible disaccharide, and (c) large aromatic species. The relative amount of these last high Mw aromatics is seen to decrease significantly in the oldest wine, as expected from the known formation and precipitation of anthocyanin-based polymers during red wine aging.     

Study of the compositional changes of mango during ripening by use of nuclear magnetic resonance spectroscopy

Liquid-state NMR spectroscopy was used to follow the compositional changes in mango juice during ripening, whereas MAS and HR-MAS techniques enabled resolved (13)C and (1)H NMR spectra of mango pulps to be recorded. Spectral assignment enabled the identification of several organic acids, amino acids, and other minor components, and the compositional changes upon ripening were followed through the changes in the spectra. In pulps, sucrose was found to predominate over fructose and glucose at most ripening stages, and citric acid content decreased markedly after the initial ripening stages while alanine increased significantly. Other spectral changes reflect the complex biochemistry of mango ripening and enabled the role played by some compounds to be discussed. Some differences observed between the composition of juices and pulps are discussed. This work shows that NMR spectroscopy enables the direct characterization of intact mango pulps, thus allowing the noninvasive study of the overall biochemistry in the whole fruit.     

Identification of amino acids in wines by one- and two-dimensional nuclear magnetic resonance spectroscopy

Amino acids are minor compounds in wines, but they have a profound influence on wine quality, and amino acids composition can be used to differentiate wines according to the vine variety, geographical origin, and year of production. The NMR signals of amino acids in NMR spectra are overlapped by the signals of other compounds present and especially by the signals of dominant compounds such as water, ethanol, and glycerol. In this work we used 1D (1)H and (13)C, 2D homonuclear COSY, TOCSY, and 2D heteronuclear HSQC and HMQC pulse sequences, also with an incorporated WET pulse sequence element that allows the simultaneous suppression of several frequencies. Complete (1)H and (13)C NMR assignments for 17 amino acids commonly present in wine and of gamma-aminobutyric acid at pH 3 have been achieved in wine sample of Sauvignon from the Coastal wine-growing region of Slovenia, vintage 1994.     

Nondestructive quantification of organic compounds in whole milk without pretreatment by two-dimensional NMR spectroscopy

In this study, various organic compounds in commercial whole milk were quantified simultaneously by 1H 1D and 1H - 13C HSQC 2D NMR spectra without any pretreatment. 2D NMR spectroscopy was applied to quantification of milk compounds for the first time. Milk fat content was easily determined to be 3.6 +/- 0.1%, and the lactose content was 47.8 +/- 1.0 mg/mL by 1H NMR spectra. From 1H-13C HSQC spectra, the concentrations of citrate, N-acetylcarbohydrates, and trimethylamine were determined to be 3.2 +/- 0.2, 2.9 +/- 0.1, and 4.0 +/- 0.6 mM, respectively. The latter two compounds were quantified in milk for the first time. Butyric acid, total monounsaturated fatty acids, and total polyunsaturated fatty acids of triacylglycerols were 6.2 +/- 0.5, 9.1 +/- 0.9, and 2.9 +/- 0.3 mM, respectively. The fatty acid compositions (mol %) of triacylglycerols were then calculated and were observed to be in good agreement with reference values. The results indicated that 1H 1D and 1H-13C HSQC 2D NMR spectroscopy is useful for the rapid and nondestructive determination of various compounds in milk.     

Determination of apparent binding constants for aroma compounds with beta-lactoglobulin by dynamic coupled column liquid chromatography

Apparent binding constants of aroma compounds limonene, alpha- and beta-ionone, and terpenyl acetate, with beta-lactoglobulin (BLG), were determined, using dynamic coupled column liquid chromatography, for pH values varying from 3 to 11. K(a) values varied from 2.61 to 3.21 x 10(3) M(-1) for limonene, indicating a strong interaction with BLG. Similarly, significant and close apparent binding constants were obtained for alpha- and beta-ionone, 1.7 x 10(2) and 4.5 to 5.4 x 10(2) M(-1), respectively. These data indicated that a similar mechanism is involved for the binding of these two molecules. The weaker values obtained at low pH, for alpha-ionone relative to beta-ionone, can be explained by the existence of steric hindrance. An increase of the apparent binding constant was observed, for all the compounds studied, when the pH was increased from 3 to 9. At this pH, an apparent binding constant was obtained for terpenyl acetate (1.04 x 10(2) M(-1)), whereas this determination was not possible at pH 3 and 6. The apparent binding constant increase was in agreement with the decrease of aroma compound relative activity coefficient in the presence of BLG, previously observed at this pH. It indicated a best accessibility to the same binding site. The binding constants of all the aroma compounds studied decreased at pH 11 as a result of the important release of the BLG structure previously reported.     

Effects of ionic strength and denaturation time on polyethyleneglycol self-diffusion in whey protein solutions and gels visualized by nuclear magnetic resonance

Pulsed field gradient NMR spectroscopy was used to determine the poly(ethylene glycol) (PEG) self-diffusion coefficient (D(PEG)) as a function of NaCl concentration (C(NaCl)) and denaturation time (t(D)) in whey protein solutions and gels. D(PEG) in the gel decreased with increasing C(NaCl) concentrations and increased with increasing t(D); the increase ceased for all PEGs when the gel was fixed. This increase was more pronounced for the 82250 g/mol PEG than the 1080 g/mol PEG. Moreover, the diffusion coefficient of nonaggregated whey protein was measured and an increase for longer t(D) was also observed. Scanning electron microscopy images and (1)H spectra demonstrated that D(PEG) were related to the structure changes and to the percentage of beta-lactoglobulin denaturation.     

Identification of synthetic regioisomeric lutein esters and their quantification in a commercial lutein supplement

Synthetic mixtures of 24 mono- and diesters of the asymmetric hydroxylated carotenoid lutein with lauric, myristic, palmitic, and stearic acids were analyzed by liquid chromatography-ultraviolet/visible spectroscopy (LC-UV-vis) and characterized by LC-mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR). These compounds were then used for identifying the composition of a commercial lutein supplement. This is the first report of chromatographic separation of mixed fatty acid lutein diesters. Preferential MS loss of fatty acids or water occurred initially at the 3'-hydroxy position in the epsilon-ionone ring and subsequently at the 3-hydroxy position in the beta-ionone ring. This selective fragmentation leads to facile assignment of the specific fatty acids to the appropriate regioisomeric ionone ring. A commercial lutein supplement contained low levels of two pairs of regioisomeric monoesters and nearly equal levels of three homogeneous diesters and five pairs of mixed diesters. Palmitic acid was the predominant fatty acid, with lower amounts of myristic, stearic, and lauric acids.     

Raman spectral analysis in the C-H stretching region of proteins and amino acids for investigation of hydrophobic interactions.

Raman spectra of amino acids showed complexity in the C-H stretching region (2800-3100 cm(-)(1)) attributed to diversity of CH, CH(2), and CH(3) groups in the side chains, ionization state, and microenvironment. The involvement of specific amino acids in the C-H stretching region of selected proteins, namely, lysozyme, alpha-lactalbumin, beta-lactoglobulin, and their binary mixtures, was investigated by deconvolution using maximum likelihood techniques. The main protein band near 2940 cm(-)(1) was attributed not only to aromatic and aliphatic amino acids but also to many other amino acids. A band near 3065 cm(-)(1) was assigned to aromatic residues, whereas bands near 2880 and 2900 cm(-)(1) corresponded primarily to aliphatic amino acids. Heating at 90 degrees C increased relative intensity near 2940 cm(-)(1) and decreased relative intensity at 2895-2902 cm(-)(1) for lysozyme and its mixtures with alpha-lactalbumin or beta-lactoglobulin. Additional bands at 2812 or 2838 and 3003 cm(-)(1) were observed after heating or in 8 M deuterated urea, reflecting changes upon denaturation.     

Two-dimensional 1H-13C nuclear magnetic resonance (NMR)-based comprehensive analysis of roasted coffee bean extract

Coffee was characterized by proton and carbon nuclear magnetic resonance (NMR) spectroscopy. To identify the coffee components, a detailed and approximately 90% signal assignment was carried out using various two-dimensional NMR spectra and a spiking method, in which authentic compounds were added to the roasted coffee bean extract (RCBE) sample. A total of 24 coffee components, including 5 polysaccharide units, 3 stereoisomers of chlorogenic acids, and 2 stereoisomers of quinic acids, were identified with the NMR spectra of RCBE. On the basis of the signal assignment, state analyses were further launched for the metal ion-citrate complexes and caffeine-chlorogenate complexes. On the basis of the signal integration, the coffee components were successfully quantified. This NMR methodology yielded detailed information on RCBE using only a single observation and provides a systemic approach for the analysis of other complex mixtures.     

Amino acid and protein scavenging of radicals generated by iron/hydroperoxide system: an electron spin resonance spin trapping study

Reduction of free radicals generated by Fe(II)/cumene-hydroperoxide (CumOOH) by amino acids (Gly, Cys, Met, His, and Trp) and proteins (bovine serum albumin (BSA), beta-lactoglobulin, and lactoferrin) was followed by electron spin resonance spectroscopy using alpha-phenyl-N-tert-butylnitrone (PBN), 2-methyl-2-nitrosopropane (MNP), and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as spin traps. The radical species detected were mostly carbon-centered radicals from CumOOH fragmentation (methyl/*H3 and ethyl/*H2CH3), although carbon-centered radicals originated from amino acids could be formed in the presence of Cys, Met, His, or Trp. All proteins and amino acids, except Cys, were effective at inhibiting generation of radicals from the Fe(II)/CumOOH system. Trp was the amino acid with the highest antiradical activity, followed by His > Gly approximately Met. Lactoferrin was the protein showing the most efficient inhibition of radical formation from the Fe(II)/CumOOH system, and BSA and beta-lactoglobulin were not significantly different in their antiradical activities. These results suggest that proteins with higher inhibitory activity on lipid oxidation promoted by transition metal catalytic decomposition of hydroperoxides should be those with elevated metal-chelating and radical-scavenging properties as well as low concentration and accessibility of reducing groups from amino acids capable of activating metals, such as sulfhydryl groups.     

Interaction of alkylsulfonate ligands with beta-lactoglobulin AB from bovine milk.

The interaction of alkyl sulfonate ligands (AL) with bovine beta-lactoglobulin AB was measured using Trp fluorescence enhancement. One binding site per protein molecule was observed. The location of this site was related with the dimer formation and could be coincident with the fatty acids and SDS binding site. The apparent binding constants for AL were in the range of 10(-)(6) M, at pH 6.8. At pH 3.0 no binding was observed by this fluorescence method. The strength of the interaction was decreasing in the following way: AL16 > AL12 > AL14 > AL10. Other sites on the monomer were evidenced by the protective action of the AL toward the urea unfolding of the protein.     

Characterization of sinapyl derivatives in pineapple (Ananas comosus [L.] Merill) juice.

Three previously unidentified phenolic compounds were found in pineapple (Ananas comosus [L.] Merill) juice in substantial concentrations and were isolated by semipreparative reverse phase HPLC. The structures were elucidated from UV spectra, acid hydrolysis, and subsequent amino acid analysis, mass spectrometry, and two-dimensional NMR spectroscopy. The compounds are identified as S-sinapyl-L-cysteine, N-L-gamma-glutamyl-S-sinapyl-L-cysteine, and S-sinapylglutathione.     

紫色水稻土胡敏酸的形成——稻草腐解试验

土壤腐植酸类物质的形成是土壤固碳的重要过程,但对腐植酸类物质形成过程的了解仍不甚清楚,为了丰富土壤腐植酸类物质形成理论,采用富立叶变换红外光谱和固态交叉极化-魔角旋转^13C-核磁共振光谱技术分析了紫色水稻土稻草腐解过程中胡敏酸的波谱学特征。结果表明,稻草腐解的前期,胡敏酸的红外光谱所有吸收峰（3364、2933、1653、1599、1508、1461、1421、1331、1225、1126、1033cm^-1）强度皆有明显减弱,核磁共振光谱的烷基、多羟基和芳基的共振峰明显减弱且甲氧基的共振峰显著增强,即表明提取的胡敏酸为类胡敏酸的木质素;随着腐解的进行,胡敏酸的红外光谱的吸收峰强度皆显著增强,核磁共振光谱的烷基、芳基和羰基的共振峰增强,即表明此时的胡敏酸已是以木质素残体为核心并结合烷基、酰胺以及糖类物质反应形成的高分子聚合体;稻草腐解的后期,胡敏酸的红外光谱的2933cm^-1处的吸收峰强度减弱,1651、1599、1508、1461、1422和1224cm^-1处的吸收峰小幅增强,核磁共振光谱的烷基共振峰减弱,甲氧基共振峰增强,表明此时的胡敏酸发生脱烷基（主要是甲基）过程。因此,红外光谱吸收峰强度与核磁共振光谱共振峰强度的规律性变化反映了稻草腐解过程紫色水稻土胡敏酸的形成过程具有阶段性,紫色水稻±胡敏酸的形成过程符合木质素学说。     

Characterization of the key aroma compounds in apricots (Prunus armeniaca) by application of the molecular sensory science concept

An aroma extract dilution analysis applied on an aroma distillate prepared from fresh apricots revealed (R)-gamma-decalactone, (E)-beta-damascenone, delta-decalactone, and (R/S)-linalool with the highest flavor dilution (FD) factors among the 26 odor-active compounds identified. On the basis of quantitative measurements performed by application of stable isotope dilution assays, followed by a calculation of odor activity values (OAVs), beta-ionone, (Z)-1,5-octadien-3-one, gamma-decalactone, (E,Z)-2,6-nonadienal, linalool, and acetaldehyde appeared with OAVs >100, whereas in particular certain lactones, often associated with an apricot aroma note, such as gamma-undecalactone, gamma-nonalactone, and delta-decalactone, showed very low OAVs (<5). An aroma recombinate prepared by mixing the 18 most important odorants in concentrations as they occurred in the fresh fruits showed an overall aroma very similar to that of apricots. Omission experiments indicated that previously unknown constituents of apricots, such as (E,Z)-2,6-nonadienal or (Z)-1,5-octadien-3-one, are key contributors to the apricot aroma.     

辣椒泛素结合酶变体类似蛋白基因克隆及其与氮吸收利用的相关性分析

UEVs蛋白是泛素结合酶E2的变体,可通过与E2形成复合物催化底物泛素化。为探究UEVs蛋白在氮吸收利用中的作用,本研究参考辣椒低氮胁迫相关转录组数据,利用反转录PCR方法从辣椒叶片中克隆了泛素结合酶变体类似蛋白基因,命名为CaUev1D-L,对其进行生物信息和系统进化分析,并对转CaUev1D-L基因烟草进行低氮胁迫后的表型和生理特性分析。结果表明,CaUev1D-L开放阅读框长402 bp,编码133个氨基酸,具有E2结合酶UBC结构域,比辣椒、烟草、番茄和马铃薯等茄科作物的Uev1D蛋白氨基酸序列末端少14个氨基酸,与Uev1D蛋白的UBC结构域存在6个氨基酸的差异,与辣椒等作物的Uev1D蛋白不在同一进化分支。将CaUev1D-L转入烟草,在正常氮素水平下,转基因植株的茎叶干重显著小于野生型植株;在低氮水平下,转基因烟草植株的茎叶干重和根干重均显著大于野生型植株。本研究结果为氮吸收利用分子机制研究和相关基因的发掘利用提供了参考。     

Identification of three novel polyphenolic compounds, origanine A-C, with unique skeleton from Origanum vulgare L. using the hyphenated LC-DAD-SPE-NMR/MS methods

Identification of new compounds especially those with new skeletons from plant kingdom has long been a vital aspect for understanding phytochemistry, plant metabolisms and discovering new bioactive compounds. In this study, we identified and isolated three novel polyphenolic compounds, origanine A-C, from a well-researched plant Origanum vulgare L. using the hyphenated LC-DAD-SPE-NMR/MS methods. Based on the combined information from UV-visible, accurate mass and 2D NMR spectra together with computational calculations, we found that these compounds all had a novel skeleton of cyclohexenetetracarboxylic acids attached with some well-known bioactive moieties including 3,4-dihydroxyphenyl, 4-(β-d-glucopyranosyloxy)benzyl alcohol (gastrodin), and 3-(3,4-dihydroxyphenyl)lactic acid (danshensu) residues. These findings provided crucial information to fill the gaps in our knowledge in terms of the plant secondary metabolism. This study also indicated the necessity for further research in plant secondary metabolism for even well-studied plants and demonstrated the powerfulness of the hyphenated LC-DAD-SPE-NMR/MS methods for comprehensive analysis of plant metabolites in particular for discovering new natural compounds.