超氧阴离子自由基诱导蛋白质氧化模拟体系的研究

研究超氧阴离子自由基氧化体系中,不同质量浓度的邻苯三酚(0～100mmol/L)对模拟体系中牛血清蛋白(BSA)巯基及活性巯基、表面疏水性、羰基含量及蛋白质的二级结构的影响。试验结果表明:氧化后,牛血清蛋白中总巯基及活性巯基均有着不同程度的变化,表面疏水性和羰基含量均呈现增加的趋势。傅里叶红外(FT-IR)测定表明蛋白质的二级结构发生了改变。超氧阴离子自由基对蛋白质的理化性质及结构均有影响。     

Protein-binding and antioxidant potential of phenolics of mangosteen fruit (Garcinia mangostana)

Phenolics were extracted from mangosteen fruit parts with 70% (v/v) aqueous acetone. The yield of crude extracts of phenolics (CP) ranged from 5.8% to 7.9%. The total phenolics (TPH) content ranged from 9.3 mg to over 250 mg of gallic acid equivalents per g of extract in the edible aril and skin, respectively. The formation of phenolic–protein complexes was assayed by both the dye-labelled bovine serum albumin (BSA) and the fluorescence quenching methods. Phenolics from peel and rind displayed a strong protein-precipitating potential. On the other hand, phenolics from edible aril exhibited greater affinity for BSA, as determined by the fluorescence quenching assay. The static quenching was the dominant mode of quenching of BSA fluorescence by mangosteen fruit phenolics. Mangosteen phenolics occupied two binding sites on BSA molecules located most likely in or near both tryptophan residues in the BSA molecule acting as an intrinsic fluorescence probe.     

Effects of caffeic acid and bovine serum albumin in reducing the rate of development of rancidity in oil-in-water and water-in-oil emulsions

The antioxidant properties of caffeic acid and bovine serum albumin in oil-in-water and water-in-oil emulsions were studied. Caffeic acid (5 mmol/kg emulsion) showed good antioxidant properties in both 30% sunflower oil-in-water (OW) and 20% water-in-sunflower oil emulsions (WO), pH 5.4, during storage at 50 °C. Although bovine serum albumin (BSA) (0.2%) had a slight antioxidant effect, the combination of caffeic acid and BSA showed a synergistic reduction in the rate of development of rancidity, with significant reductions in concentration of total volatiles, peroxide value (PV) and p-anisidine value (PA) for both emulsion types. The synergistic increase in stability of the OW and WO emulsions containing BSA and caffeic acid was 102.9% and 50.4% respectively based on total oxidation (TOTOX) values, which are calculated as 2PV + PA, with greater synergy calculated if based on formation of headspace volatiles. The OW emulsion was more susceptible to the development of headspace volatiles by oxidation than the WO emulsion, even though the degree of oxidation assessed by the TOTOX value was similar.     

Facilitation of cleaning of alumina surfaces fouled with heat-treated bovine serum albumin by ozone treatment

Facilitation of cleaning of alumina (A12O3) particles fouled with heat-treated bovine serum albumin (BSA), which contains sulfhydryl groups on the molecule, by gaseous ozone was studied. With increasing temperature of heat treatment, the amount of adsorbed BSA onto A12O3 surfaces increased, whereas the rate of BSA desorption during alkali cleaning decreased markedly, resulting in the larger amounts of BSA remaining on 12O3 surfaces. No significant amounts of BSA were removed from A12O3 surfaces by alkali cleaning alone when treated at temperatures above 120 degrees C. Before alkali cleaning, the heat-treated, BSA-fouled AI2O3 at 150 degrees C were treated with 0.05 to 0.30% (vol/vol) gaseous ozone at room temperature. Ozone pretreatment markedly accelerated the rate of BSA desorption during subsequent alkali cleaning. The effect of ozone pretreatment on BSA removal depended on the concentration of ozone and treatment time and hence on the total amount of ozone supplied. The molecular weight (MW) of desorbed BSA during alkali cleaning without ozone pretreatment coincided with the MW of the native BSA, whereas the MW of desorbed BSA during the combined ozone-alkali cleaning was lower than the MW of the native BSA. This indicated that the heat-treated BSA molecules adsorbed on A12O3 were partially decomposed into some fragments by ozone pretreatment, resulting in the facilitation of the removal of BSA during alkali cleaning.     

Effect of amidation on the foaming and physico-chemical properties of bovine serum albumin

Amidation of bovine serum albumin (BSA) was achieved by a water-soluble carbo-diimide, ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)-mediated reaction using ammonium chloride as the nucleophile. Partial and substantial amidation of 0.5% (w/v) BSA in 5.5 m ammonium chloride solution with 1 times 10-²mmol EDC over 120 min and 1 times 10^-1mmol EDC over 10 min respectively was achieved on a large scale using diafiltration for rapid termination of the reaction and purification. Residual ammonium chloride otherwise enhanced foaming properties. The amidated proteins were characterized by isoelectric focusing, electrophoresis and hydrophobicity and disulphide- and sulphydryl-group measurements. Compared with native BSA, partially amidated BSA (PA-BSA) produced enhanced foam expansion and foam stability values. This was attributed to minimal denaturation and to the presence of both acidic and basic components (pI range 5.25–7.50) within the single protein. In contrast, substantially amidated BSA (SA-BSA) (pI range 7–9.1) had similar foaming properties to those of the ultrafiltered BSA control which were slightly lower than those of native BSA. However SA-BSA interacted synergistically with native BSA producing enhanced foaming properties particularly at the 1:1 ratio through electrostatic interactions, conformational changes and increased hydrophobicity.     

Conformation of adsorbed bovine serum albumin governing its desorption behavior at alumina-water interfaces

The mode of initial adsorption of bovine serum albumin (BSA) onto positively charged Al2O3 particles was studied as a function of surface coverage (theta). The adsorption isotherm of BSA exhibited saturation (theta = 1) and the existence of an inflection point at theta of 0.82. The relative numbers of ionic groups on a BSA molecule interacting with the Al2O3 surface at various theta were monitored by measuring the relative adsorption density of H+ and OH-, ([gamma(H+) - gamma(OH-)]), for BSA-adsorbed Al2O3 using potentiometric titration. The [gamma(H+) - gamma(OH-)] curves for Al2O3, BSA, and BSA-adsorbed Al2O3 at various KNO3 concentrations showed a common intersection point (cip) which was the pH giving the acid-base equivalence point, respectively. Compared with the cip's of Al2O3 (5.6) and BSA (5.2), the cip's of BSA-adsorbed Al2O3 were situated at points corresponding to more alkaline pH values over the theta range of 0.13 to 1.0. These results suggested that negatively charged groups, mainly carboxyl groups, on the BSA molecule electrostatically interacted with the Al2O3 surface. The degree of shift in the cip increased gradually with increasing theta from 0.13 to 0.70, while it decreased markedly over the theta range of 0.82 to 1.0. The variation in the cip reflected the change in the total number of ion pairs formed between BSA molecules and Al2O3. The initial rates of BSA desorption during alkali cleaning were low and almost constant over the theta range of 0.13 to 0.70, but increased markedly at theta higher than 0.82. It is suggested that the conformational changes of BSA adsorbed on Al2O3, involving changes in the relative magnitude of electrostatic interaction forces, occur discretely at theta of approximately 0.8.     

Improvement of functional properties of bovine serum albumin through phosphorylation by dry-heating in the presence of pyrophosphate

ABSTRACT:  Bovine serum albumin (BSA) was phosphorylated by 2 methods. One is dry-heating in the presence of pyrophosphate, and the other is conjugation with maltopentaose through the Maillard reaction and subsequent dry-heating in the presence of pyrophosphate. The phosphorus content of BSA was increased to approximately 0.45% by dry-heating at pH 4.0 and 85 °C for 5 d in the presence of pyrophosphate, and approximately 0.91% by glycation and subsequent phosphorylation. The circular dichroism spectra showed that the change of secondary structure in the BSA molecule by phosphorylation was mild. However, tryptophan fluorescence intensity of BSA decreased by phosphorylation. The differential scanning calorimetry thermograms of BSA showed a disappearing of the 1st peak and a lowering of the 2nd peak denaturation temperature by phosphorylation. These results indicated molten (partially unfolded) conformations of BSA formed by both phosphorylation methods. The functional properties of BSA such as heat stability and calcium phosphate solubilizing ability were improved by phosphorylation alone and further by phosphorylation after glycation. Transparent gels of BSA with relatively high water-holding capacity were obtained by phosphorylation alone, and the immunogenicity of BSA was reduced significantly by glycation and phosphorylation, respectively.     

大叶冬青皂苷与牛血清蛋白的相互作用研究

为研究不同温度下大叶冬青皂苷G（Latifoloside G）、大叶冬青皂苷C（Latifoloside C）及苦丁皂苷G（Kudinoside G）与牛血清白蛋白（BSA）的相互作用情况，为大叶冬青皂苷在体内运输及作用机制研究提供实验依据，本文采用荧光光谱法研究小分子与蛋白的结合机制、结合模式、结合常数和结合位点等，采用圆二色谱法研究蛋白质的构象变化。结果表明:三种皂苷均能有效猝灭BSA内源荧光，Kudinoside G为静态猝灭，Latifoloside G、Latifoloside C为动态猝灭。随着三种药物小分子浓度的增加，BSA的内源荧光强度降低，两种温度下BSA的最大发射峰皆发生轻微蓝移，三种皂苷的发射波长皆由347 nm蓝移到345 nm，三种皂苷与BSA结合能力的顺序为Latifoloside G>Latifoloside C>Kudinoside G。Kudinoside G与BSA之间主要作用力类型为氢键和范德华力，Latifoloside G及Latifoloside C与BSA之间主要作用力为疏水作用。发现Latifoloside G和Kudinoside G两种小分子与BSA的结合能力与C-28位所连的极性基团有关，且齐墩果烷型的Latifoloside C比Kudinoside G更易于插入到BSA的疏水腔中。圆二色光谱表明，三种皂苷与BSA的结合均可使BSA内部结构环境发生改变，α-螺旋含量增加，微环境极性减小，疏水性增加。     

黑豆皮花色苷的降血糖作用及其机理的研究

目的:观察黑豆皮花色苷(Black Soybean Anthocyanins,BSA)的降血糖效果,并对其机理进行初步研究。方法:对不同组的正常小鼠和利用四氧嘧啶造模的实验性糖尿病小鼠分别进行相应浓度的黑豆皮花色苷腹腔注射,并测定血糖、超氧化物歧化酶(SOD)、丙二醛(MDA)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-PX)以及胰岛素含量。结果:黑豆皮花色苷可以降低糖尿病小鼠的血糖和血清中MDA与CAT的含量,提高血清中SOD和GSH-PX的含量,同时提高了血清中胰岛素的含量。结论:黑豆皮花色苷具有降低糖尿病小鼠高血糖的作用,而它的作用机理可能是通过促进胰岛素分泌和清除自由基及抗脂质过氧化过程实现的。     

Comparison of the Precipitation of Alfalfa Leaf Protein and Bovine Serum Albumin by Tannins in the Radial Diffusion Method

The precipitation of protein by condensed and hydrolysable tannins was evaluated with the radial diffusion method of Hagerman (1987) using bovine serum albumin (BSA) and isolated leaf protein from fresh alfalfa (Medicago sativa). Alfalfa leaf protein (AALP) was included at two concentrations, 25 and 156 mg N litre^-1, at pH 6·8 and 39°C to simulate rumen conditions. The condensed tannins were purified from lyophilised samples of Arachis pintoi, Desmodium ovalifolium, Gliricidia sepium, Manihot esculenta and quebracho (Schinopsis balansae). Hydrolysable tannins from tannic acid (TA) were used as well. There was a significant interaction (P<0·001) between tannin and protein source, and protein level on protein precipitation. Most purified condensed tannins (CTs) precipitated more AALP than BSA when protein was included at the same level. Purified CT from quebracho and hydrolysable tannin from TA failed to precipitate AALP at both protein levels. In a second experiment, tannins from crude plant extracts were studied in the radial diffusion method using BSA and two levels of AALP. The crude plant extracts were obtained from lyophilised plant samples of A pintoi, Centrosema macrocarpum, Clitoria ternatea, D ovalifolium, Erythrina berteroana, E poepigiana, G sepium, M esculenta, Pueraria montana and P phaseoloides. The protein precipitated by soluble tannins in the plant samples was correlated to the total phenolic content and to the soluble CT estimated by the acid butanol assay or by the radial diffusion method. Tannins from different plant species precipitated different amounts of BSA and AALP. Therefore, the measures of the biological activity of tannins based on BSA precipitation may not reflect the ability of tannins to precipitate proteins of plant origin such as those commonly found in the diets of herbivores. The present study offers the possibility of using the radial diffusion method with plant proteins at precipitation conditions similar to those in the rumen. © 1997 SCI.     

亚甲基蓝及其代谢产物与牛血清白蛋白相互作用的研究

采用多种光谱法研究了亚甲基蓝(MB)及其代谢产物天青B(AZB)与牛血清白蛋白(BSA)之间的共同作用机制。实验结果表明,MB和AZB对BSA的荧光猝灭均为静态猝灭。298K时MB和AZB与BSA的结合常数分别为1.71×10⁵ L/mol和8.83×10⁴ L/mol。由熵和焓推断,MB和AZB与BSA的作用力类型均主要为静电作用。位点竞争实验结果表明MB和AZB均结合在BSA的SiteⅠ。紫外-可见吸收光谱法实验结果表明,MB和AZB诱导BSA的构象改变。三者共存时,AZB会与MB竞争结合BSA,减小MB与BSA作用的猝灭常数和结合常数,改变MB与BSA的作用力类型。     

Study on the interaction of food colourant quinoline yellow with bovine serum albumin by spectroscopic techniques

The interaction of a food colourant, quinoline yellow (Qy), and bovine serum albumin (BSA) was investigated by spectrophotometry, spectrofluorometry, FT-IR and circular dichroism (CD) techniques. The experimental results indicated that the quenching mechanism of BSA by the dye was a static procedure. Various binding parameters were evaluated. The negative value of ΔH, negative value of ΔS and the negative value of ΔG indicated that van der Waals force and hydrogen bonding play major roles in the binding of Qy and BSA. Based on Forster's theory of non-radiation energy transfer, the binding distance, r, between the donor (BSA) and acceptor (Qy) was evaluated. The results of CD and UV-vis spectroscopy showed that this dye could bind to BSA and the conformation of BSA changed.     

Optimisation of the Amido Black assay for determination of the protein content of grape juices and wines

While the heat/chill stability of white wines cannot be predicted from the total protein concentration, this information is useful in assessing the effectiveness of fining treatments. We have adapted the Amido Black assay for use in grape juice and wine. The response of bovine serum albumin (BSA) was similar in water, model wine and model juice. The linear range of the assay (<100 mg l^?1) extends beyond protein concentrations typically found in grape juices and wines. The limit of quantification was 11.1 mg l^?1 for BSA in model wine and 7.6 mg l^?1 in model juice, while the limit of detection was found to be less than 3 mg l^?1 in either solution. In contrast to other methods of protein determination, this assay is not affected by common wine phenolic and pectic compounds or by glutathione. The mean response of BSA was similar in model juice and red and white grape juices. The mean response of BSA in red wine was lower than in white wine and model wine (p < 0.001), and there was considerable variation in response among wines. Protein concentrations determined using this method were reproducible (CV < 10%). This optimised assay can be used to monitor protein levels in grape juices and wines. © 2001 Society of Chemical Industry     

Changes in protein-protein and protein-polysaccharide interactions induced by high pressure

β-Lactoglobulin and bovine serum albumin (BSA) protein solutions (0.1%, 0.2% and 2.5%), when subjected to high pressure treatment (800 MPa for 20 min) at neutral pH, were denatured and some aggregates formed. The total calorimetric enthalpy of 2.5% solutions of the pressure-treated proteins decreased to virtually zero for both β-lactoglobulin and BSA following pressure treatment. Isoelectric focussing patterns (IEF) indicated that aggregation occurred in both proteins and there was a concomitant loss of sulphydryl groups (42% for β-lactoglobulin and 55% for BSA), suggesting that protein aggregation after high pressure processing was caused, at least in part, by the formation of -S-S- bridges. The surface hydrophobicity of the two proteins was modified, increasing (40%) with β-lactoglobulin and decreasing (41%) with BSA. Pressure treatment of 1:1 mixtures of BSA and dextran sulphate (DS) yielded structures with a significant enthalpy. However, addition of DS to β-lactoglobulin had little effect on the thermograms, suggesting that the DS either protects the protein against pressure induced unfolding or enables the pressure-denatured protein to regain some secondary structure.     

牛血清蛋白对纤维素酶水解小麦秸秆的影响

在以分别被稀硫酸和氢氧化钠处理的小麦秸秆为底物进行纤维素酶解时添加牛血清蛋白（BSA）来评估BSA对酶解的影响,当接入纤维素酶40Ug秸秆,添加牛血清蛋白0.04g时,反应48h,还原糖得率分别提高30%和22%,添加牛血清蛋白后酸处理的秸秆水解速率更快,酶解液中酶失活趋势减小,以滤纸为底物测定纤维素酶活力时添加牛血清蛋白能将酶活提高1倍以上,从而推断牛血清蛋白可以提高纤维素酶的稳定性并减少酶因吸附木质素而失活,提高酶的水解效率。     

Multispectroscopic studies on the interaction of maltol,a food additive,with bovine serum albumin

The interaction between maltol, a food additive, and bovine serum albumin (BSA) under simulated physiological conditions was investigated by fluorescence, UV–Vis absorption, circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy. The results suggested that the fluorescence quenching of BSA by maltol was a static procedure forming a maltol–BSA complex. The positive values of enthalpy change and entropy change indicated that hydrophobic interactions played a predominant role in the interaction of maltol with BSA. The competitive experiments of site markers revealed that the binding of maltol to BSA mainly took place in subdomain IIA (Sudlow site I). The binding distance between maltol and BSA was 3.01 nm based on the Förster theory of non-radioactive energy transfer. Moreover, the results of UV–Vis, synchronous fluorescence, CD and FT-IR spectra demonstrated that the microenvironment and the secondary structure of BSA were changed in the presence of maltol.     

Organ distribution of bovine serum albumin administered intragastrically in the rat

Radioimmunoassay was employed to examine distribution of antigenic structures of bovine serum albumin (BSA), absorbed by the gastrointestinal tract, in the blood serum and organs of intact rats. It was shown that 3 h after administering 3H-BSA an appreciable amount of its antigenic structures could be identified in the blood serum, liver, spleen, and carcass of the animals. The total amount of antigenic determinants of BSA which got into the internal environment of the body from the intestine amounted to about 0.2% of the dose administered. The highest specific content of antigenic structures of BSA supplied via the intestinal barrier was detected in the spleen.     

Effect of the attachment of valine residues on the physicochemical and functional properties of bovine serum albumin

The effects of attaching a hydrophobic amino acid residue, valine, to the ?-amino groups (lysine residues) of bovine serum albumin (BSA) on the physicochemical and functional properties were assessed. The valyl groups were attached using an N-carboxyvaline anhydride derivative. The valine content of BSA was increased from 27 mol mol^?1 protein to 47·91 or 53·72 mol mol^?1. The number of lysine residues acylated was dependent on the pH of the reaction mixture as was the degree of polyvalylation. Attachment of polyvalyl chains resulted in improved whipping and gelling properties compared with a control sample of ultrafiltered BSA, and interfered with the formation of α-helices. Hydrophobicity measurements using the fluorescent probe cis-parinaric acid revealed increased hydrophobicity values only after the modified samples had been heat denatured.     

荧光光谱法探究pH值对叶黄素与牛血清白蛋白相互作用的影响

采用荧光光谱法研究不同pH值（3.0、5.2、7.4）条件下叶黄素与牛血清白蛋白（bovine serum albumin，BSA）的相互作用，利用数学模型计算得到二者的猝灭常数和热力学参数等，分析叶黄素与BSA的荧光猝灭现象及蛋白构象的变化，最后通过位点竞争实验和分子模拟技术确定了二者的结合位点。荧光光谱表明，不同pH值条件下叶黄素对BSA均产生荧光猝灭效应，在298 K时，pH 7.4的猝灭常数最大，结合常数较大；热力学参数和分子对接结果表明，叶黄素与BSA间的作用力主要是疏水相互作用；同步荧光光谱表明BSA的构象在叶黄素与pH值的共同作用下发生改变；位点竞争性实验和分子对接实验表明，pH 7.4和pH 5.2时，二者的结合位点在亚结构域IIA和IIIA之间，但更接近于Sudlow’s site II；pH 3.0时，结合位点不在亚结构域IIA及IIIA附近。结果表明，pH值对叶黄素和BSA的相互作用有影响，这为蛋白质与生物活性成分的相互作用提供一定的理论依据。     

Effect of Condensed Grape Tannins on the In Vitro Activity of Digestive Proteases and Activation of Their Zymogens

Only at concentrations substantially higher than those likely to occur in human diets did grape tannins have a significant adverse effect on the in virro digestion of bovine serum albumin (BSA). The activities of pepsin and chymotrypsin were at such levels (concentrations greater than 0.1%) significantly reduced. In contrast, that of trypsin increased markedly due to denaturation of BSA by the tannins. Tannin concentrations in excess of 0.5% strongly inhibited the activation of chymotrypsinogen, while the activation of trypsinogen by enterokinase was drastically reduced at concentrations as low as 0.05% due to precipitation of the substrate. BSA digestion was markedly reduced in sequential multizymogen experiments at tannin concentrations of 0.5% but not at 0.1%.