Interaction of flavonoids with bovine serum albumin: a fluorescence quenching study

The interaction between four flavonoids (catechin, epicatechin, rutin, and quercetin) and bovine serum albumin (BSA) was investigated using tryptophan fluorescence quenching. Quenching constants were determined using the Stern-Volmer equation to provide a measure of the binding affinity between the flavonoids and BSA. The binding affinity was strongest for quercetin and ranked in the order quercetin > rutin > epicatechin = catechin. The pH in the range of 5-7.4 does not affect significantly (p < 0.05) the association of rutin, epicatechin, and catechin with BSA, but quercetin exhibited a stronger affinity at pH 7.4 than at lower pH (p < 0.05). Quercetin has a total quenching effect on BSA tryptophan fluorescence at a molar ratio of 10:1 and rutin at approximately 25:1. However, epicatechin and catechin did not fully quench tryptophan fluorescence over the concentration range studied. Furthermore, the data suggested that the association between flavonoids and BSA did not change molecular conformation of BSA and that hydrogen bonding, ionic, and hydrophobic interaction are equally important driving forces for protein-flavonoid association.     

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.     

Probing the binding of the flavonoid diosmetin to human serum albumin by multispectroscopic techniques

The binding mechanism of molecular interaction between diosmetin and human serum albumin (HSA) in a pH 7.4 phosphate buffer was studied using atomic force microscopy (AFM) and various spectroscopic techniques including fluorescence, resonance light scattering (RLS), UV-vis absorption, circular dichroism (CD), and Fourier transform infrared (FT-IR) spectroscopy. Fluorescence data revealed that the fluorescence quenching of HSA by diosmetin was a static quenching procedure. The binding constants and number of binding sites were evaluated at different temperatures. The RLS spectra and AFM images showed that the dimension of the individual HSA molecules were larger after interaction with diosmetin. The thermodynamic parameters, ΔH° and ΔS° were calculated to be -24.56 kJ mol(-1) and 14.67 J mol(-1) K(-1), respectively, suggesting that the binding of diosmtin to HSA was driven mainly by hydrophobic interactions and hydrogen bonds. The displacement studies and denaturation experiments in the presence of urea indicated site I as the main binding site for diosmetin on HSA. The binding distance between diosmetin and HSA was determined to be 3.54 nm based on the F?rster theory. Analysis of CD and FT-IR spectra demonstrated that HSA conformation was slightly altered in the presence of diosmetin.     

Stability to light, heat, and hydrogen peroxide at different pH values and DPPH radical scavenging activity of acylated anthocyanins from red radish extract

The stability of red radish extract to light, heat, and hydrogen peroxide at different pH values (3, 5, and 7) was examined, in which major anthocyanins were pelargonidin glycosides acylated with a combination of p-coumaric, ferulic, or caffeic acids. The light irradiation (fluorescence light, 5000 lx; at 25 degrees C) indicated that the red radish extract was more stable at lower pH than at higher pH. The HPLC analyses revealed that diacylated anthocyanins in the extract were more stable to light at pH 3 than monoacylated anthocyanins. No significant difference in degradation rates of acylated anthocyanins at pH 5 was observed, whereas anthocyanins acylated with p-coumaric or ferulic acids were more stable at pH 7 than ones with caffeic acids. The stability to heat (at 90-95 degrees C) showed a tendency similar to that for light. The number of intramolecular acyl units contributes to stability to light and heat at lower pH, whereas the characteristics of intramolecular acyl units influence the stability at higher pH. The degradation behavior of red radish extract to H2O2 were almost the same to those of light and heat, depending on the pH. However, HPLC analyses revealed that the stability of individual acylated anthocyanins were independent of the pH. These data suggest that the characteristics, the number, and the binding site of intramolecular acyl units affect the stability of anthocyanin to H2O2. DPPH radical scavenging activity of all acylated anthocyanins was higher than those of pelargonidin and perlargonidin-3-glucoside. The activity of acylated anthocyanins mostly depended on the activity of intramolecular acyl units (caffeic acid > ferulic acid > p-coumaric acid). However, the activity was highly affected by the binding site of intramolecular acyl units even if anthocyanins have common acyl units.     

Influence of B-ring hydroxylation on interactions of flavonols with bovine serum albumin

The B-ring substitution pattern of flavonols is a significant structural feature for their function as free radical scavengers and antioxidants. In this paper, four differently substituted B-ring hydroxylation flavonols (galangin, kaempferol, quercetin, and myricetin) and a flavonol glycoside (quercitrin) were studied for their ability to bind BSA by quenching the protein intrinsic fluorescence. From the spectra obtained, the biomolecular quenching constants, the apparent static binding constants, and the binding site values were calculated. The B-ring hydroxylation of flavonols significantly affected the binding/quenching process; in general, the binding affinity increased with the number of hydroxyl groups on the B-ring. The binding constants ( Ka) were determined as myricetin (4.90 x 10(8) L/mol) > quercetin (3.65 x 10(7) L/mol) > kaempferol (2.57 x 10(6) L/mol) > galangin (6.43 x 10(5) L/mol). The glycoside substitute at the C-ring position decreased the binding affinity. The chromatographic retention factor ( K') and logarithms of apparent partition coefficient (log Kow) were linear to the logarithms of apparent binding constants (log Ka) for flavonols with increasing hydroxyl groups on the B-ring. These results showed that the hydrogen bond force play an important role in binding flavonols to BSA. These results are also in agreement with the generally accepted structure-dependent free radical scavenger and antioxidant abilities of flavonols.     

Sensitive determination of DNA based on the interaction between norfloxacin-Tb3+ complex and DNA

The fluorescence intensity of the norfloxacin (NFX)-Tb3+ complex enhanced by DNA was studied. Therefore, a sensitive fluorescence method for the determination of DNA was developed. The optimal conditions of the method were as follows: the hexamethylenamine (HMA)-HCl buffer was adopted for adjusting the pH to 6.5 +/- 0.1, the concentrations of NFX and Tb3+ were both fixed in 1.0 x 10(-6) mol L(-1), and the excitation and emission wavelengths were selected at 290 and 545 nm, respectively. Under the optimal conditions, the enhanced fluorescence intensity was in proportion to the concentration of DNA in the same range of 5.0 x 10(-9) - 1.0 x 10(-6) g mL(-1) for hsDNA and thermally denatured ctDNA. The detection limits (S/N = 3) were 0.9 and 0.6 ng mL(-1), respectively. In addition, the interaction between NFX-Tb3+ and DNA was discussed in detail. The experimental results from UV absorption spectra, fluorescence spectra, and the salt effect study indicated that the interaction between norfloxacin-Tb3+ complex and DNA had at least two different binding modes: the electrostatic binding and the intercalation binding. The mechanism of the fluorescence enhancement effect was also discussed.     

Calf thymus DNA-binding ability study of anthocyanins from purple sweet potatoes ( Ipomoea batatas L.)

A total of 10 anthocyanin compounds were identified from five purple sweet potato ( Ipomoea batatas L.) varieties, Qunzi, Zishu038, Ji18, Jingshu6, and Ziluolan, by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) to assess their calf thymus DNA-binding ability in vitro. The interaction between anthocyanins and calf thymus DNA in Tris-HCl buffer solution (pH 6.9) was evaluated by fluorescence spectroscopy. Using ethidium bromide (EB) as a fluorescence probe, fluorescence quenching of the emission peak was seen in the DNA-EB system when anthocyanins were added, indicating that the anthocyanins bound with DNA. The acylated groups influenced the ability of the interaction with DNA. Anthocyanins from purple sweet potato with more acylated groups in sorphorose have a stronger binding ability with DNA.     

Binding studies of oxovanadium(V) with ovalbumins (OA)

The effect of protein oxovanadium(V) ion concentration and pH on the ratio of diffusion current (id/id₀) was studied in vanadium(V) ovalbumin-S and denatured ovalbumin systems. In both the cases marked decrease in diffusion current was observed at the respective pH values, indicating that binding takes place with cationic groups of the proteins. The binding sites (n) were found to be pH dependent. The uniformity of logK and ΔG ⁰ value at all pH values indicated the involvement of same sites in interaction. Furthermore, the linear scatchard plots in both the systems supported the involvement of single class of independent sites in oxovanadium(V) anion interaction. The difference in binding sites (n) has been attributed to the folded structure of ovalbumin-S while unfolded one of denatured ovalbumin.     

二苯-α-吡喃酮类链格孢霉毒素和人血清白蛋白的相互作用及机理探究

为探究链格孢酚单甲醚(AME)和链格孢酚(AOH)这两种二苯-α-吡喃酮类链格孢霉毒素与人血清白蛋白(HSA)之间的分子相互作用及机理,本研究模拟血液生理pH值,采用稳态荧光光谱、同步荧光光谱、3D荧光光谱以及圆二色光谱等方法研究两者之间的相互作用。结果表明,AME和AOH与HSA相互作用均发生了静态猝灭,具有较高亲和力,以氢键和范德华力(ΔH<0,ΔS<0)结合形成1:1复合物,互作过程中这两种类毒素破坏了人血清白蛋白中稳定二级结构的氢键网络,使蛋白二级结构展开,且AME和AOH使人血清白蛋白中的α-螺旋结构含量从48.93%分别减少至39.41%和44.01%,并使色氨酸残基极性降低、疏水性增加,但酪氨酸残基极性变化不大,即结合位点可能位于色氨酸残基所在的空腔(即结合位点I);AME对HSA的猝灭程度、猝灭常数(K_sv)及结合常数(K_a)均大于AOH,结合距离也更小(r_AME=2.56 nmAOH=2.60 nm)。本研究结果为进一步探究二苯-α-吡喃酮类链格孢霉毒素的毒代动力学和药代动力学,完善丰富其毒理学相关资料,以及进行风险评估和在食品中限量标准的制定提供了参考依据。     

Effect of supramolecular structures on thermoplastic zein-lignin bionanocomposites

The effect of alkaline lignin (AL) and sodium lignosulfonate (LSS) on the structure of thermoplastic zein (TPZ) was studied. Protein structural changes and the nature of the physical interaction between lignin and zein were investigated by means of X-ray diffraction and Fourier transform infrared (FT-IR) spectroscopy and correlated with physical properties. Most relevant protein structural changes were observed at low AL concentration, where strong H-bondings between the functional groups of AL and the amino acids in zein induced a destructuring of inter- and intramolecular interactions in α-helix, β-sheet, and β-turn secondary structures. This destructuring allowed for an extensive protein conformational modification which, in turn, resulted in a strong improvement of the physical properties of the bionanocomposite.     

土地利用方式对西南喀斯特土壤碳、氮、磷化学计量特征及酶活性的影响

  目的  探究西南喀斯特土壤碳（C）、氮（N）、磷（P）化学计量特征和酶活性对不同土地利用方式的响应,有利于为西南喀斯特的土地利用调控与生态修复提供决策支持。  方法  本研究以喀斯特高原峡谷（贵州省关岭县花江研究区）亚热带森林（SUF）、疏林（SPF）、灌木林（SHF）、草地（GL）、玉米地（CL）、裸地（BL）及弃荒地（AL）的土壤为研究对象,通过采集其0 ~ 15 cm土层样品,分析土壤C、N、P化学计量特征及酶活性的差异。  结果  ①土壤有机碳含量（SOC）和全氮（TN）含量表现为SPF > AL > BL > SHF > CL > SUF > GL,土壤全磷（TP）含量表现为AL > BL > CL > SPF > GL > SHF > SUF。C∶N表现为SUF > AL > SPF > SHF > CL > BL > GL,C∶P表现为SPF > SUF > SHF > CL > AL > GL > BL,N∶P表现为SPF > SHF > SUF > CL > GL > AL > BL。土壤微生物量碳（MBC）含量则是SPF > SHF > CL > AL > SUF > BL > GL。②脲酶（URE）活性表现为SUF > CL > SPF > SHF > AL > GL > BL,蔗糖酶（SUC）活性表现为BL > AL > SPF > CL > SHF > GL > SUF,碱性磷酸酶（ALP）活性表现为SUF > BL > SHF > SPF > AL > CL > GL,过氧化氢酶（CAT）活性则是CL > AL > BL > SHF > SPF > GL > SUF。③URE与C∶P、N∶P、MBC极显著正相关,与C∶N显著正相关,与TP显著负相关;SUC与TP极显著正相关,与SOC、TN显著正相关;ALP与C∶N、C∶P显著正相关;CAT与TP极显著正相关,与TN显著正相关,与C∶P显著负相关。④在前两个排序轴中土壤理化因子累计解释了土壤酶活性变化的84.83%,按重要性排序依次为pH > 土壤温度 > TP > C∶P > TN > 容重 > SOC > C∶N > N∶P > MBC。  结论  不同土地利用方式土壤C、N、P化学计量特征及酶活性存在显著差异,土壤N∶P < 14表明土壤养分主要受氮限制。影响酶活性的主要土壤理化因子为土壤pH、土壤温度和土壤全磷。     

壳聚糖与牛血清白蛋白作用机制及其复合物乳化性的研究

为探究蛋白质与多糖之间的相互作用及其复合物的研究乳化性质,本试验以牛血清白蛋白(BSA)-水溶性壳聚糖(WCS)复合体系为研究对象,采用紫外吸收光谱、荧光发射光谱、电位粒径分析、乳液储藏稳定性观察等方法研究在微观环境下蛋白质与多糖的相互作用机理,并进一步探究两者对水包油型乳液乳化性能的影响。结果表明,多糖和蛋白质的相互作用随着pH值和BSA/WCS复合比的变化而不同,混合溶液的紫外吸收峰和荧光发射峰发生偏移,且当复合比小于1∶2时发生荧光淬灭。混合溶液的Zeta电位和平均粒径测定结果表明,BSA与WCS之间主要通过静电相互作用形成复合物,且该复合物的电荷性质、尺寸大小具有pH值和复合比依赖性。研究混合溶液乳化性发现,当pH值为3.0、4.0、复合比为4∶1~1∶1时,形成的乳液液滴粒径较小,性质稳定;当pH 值大于5.0时,乳液液滴粒径大,出现相分离、乳液高度不稳定现象。本研究为设计具有可预测稳定性或刺激响应性的乳液配方及新型功能特性材料提供了理论依据。     

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.     

Effect of heat treatment on bovine beta-lactoglobulin A, B, and C explored using thiol availability and fluorescence.

Dilute solutions of beta-lactoglobulin (beta-Lg) A, B, and C were heated at temperatures between about 40 and 94 degrees C for 10 min, cooled, and analyzed using Trp fluorescence and extrinsic fluorescence spectra of the probe 1,8-anilinonaphthalene sulfonate (ANS). Thiol availabilities using 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) were determined using a separate set of samples. The normalized ANS fluorescence emission intensity and the thiol availability results showed a 1:1 relationship with the loss of nativelike but not SDS-monomeric protein, as determined by PAGE analysis. The normalized Trp emission intensity results did not show a comparable 1:1 relationship with the loss of nativelike protein, indicating that the Trp intensity arose from consequential disulfide bond reorganization and not the initial unfolding reaction. The results were also analyzed in terms of two-state models, and the midpoint temperatures (T(mid)) for the proteins were generally beta-Lg C > beta-Lg A > beta-Lg B, and the slopes at the midpoint temperatures for the A variant were generally less than those for the B and C variants indicating that beta-Lg A may denature by a different mechanism from that of beta-Lg B or beta-Lg C. The T(mid) parameters derived from the ANS fluorescence intensity results were similar to those for thiol availability and both were lower than the T(mid) values for Trp emission intensity showing that creation of an ANS binding site on a beta-Lg molecule was linked to the irreversible exposure of a thiol group and the loss of native beta-Lg but preceded the decrease in Trp(61) fluorescence quenching. These results for the differences between the behavior of the A and B or the C variants involved the creation of a destabilizing cavity by the Val(118)Ala (A --> B) substitution and the changed charge distribution within the CD loop caused by the Asp(64)Gly (A --> B) substitution.     

Biological relevance of the interaction between procyanidins and trypsin: a multitechnique approach

The interactions between the digestive protease trypsin type IX-S from porcine pancreas and grape seed procyanidins were monitorized by fluorescence quenching, dynamic light scattering, nephelometry, circular dichroism, and enzymatic inhibition assay. This work reports that the inhibition of trypsin activity by grape seed procyanidins and the respective quenching of intrinsic protein fluorescence are closely related. These two phenomena increase with the molecular weight of the tested procyanidins. The interaction between procyanidins and enzyme was shown to involve a specific interaction as inferred from the fluorescence assays. It was also shown by fluorescence spectroscopy that the binding of procyanidin molecules to the enzyme does not induce significant structural modifications. A relationship between aggregate formation, using dynamic light scattering and nephelometry, and fluorescence quenching was observed with maxima achieved for similar stoichiometric ratios. The binding of procyanidins to trypsin affects only slightly protein structure as seen by circular dichroism.     

Optical spectroscopic exploration of binding of Cochineal Red A with two homologous serum albumins

Cochineal Red A is a negatively charged synthetic azo food colorant and a potential carcinogen. We present here the study of binding of Cochineal Red A with two homologous serum albumins, human (HSA) and bovine (BSA), in aqueous pH 7.4 buffer by optical spectroscopic techniques. Protein intrinsic fluorescence quenching by Cochineal Red A occurs through ground-state static interaction and its binding with BSA is stronger than with HSA. The magnitudes of thermodynamic parameters suggest that dye binding occurs principally via electrostatic complexation. Site-marker competitive binding shows that Cochineal Red A binds primarily to site I of serum albumins. Circular dichroic spectra indicate that dye binding results in some conformational modification of serum albumins. Increased ionic strength of the medium results in lowering of binding. This study provides an important insight into possible means of removal of dye toxicity.     

Investigation of the mechanism of enhanced effect of EGCG on huperzine A's inhibition of acetylcholinesterase activity in rats by a multispectroscopic method

The mechanism of enhanced effect of (-)-epigallocatechin-3-gallate (EGCG) on huperzine A's (HUP) inhibition of acetylcholinesterase (AChE) activity in rats was investigated. The inhibitory effects of HUP at 10 and 5 microg/kg on AChE activity were quite weak in the whole phase. In contrast, upon addition of EGCG (100 mg/kg) to the HUP 10 and 5 microg/kg groups, remarkably enhanced inhibitory effects with maximum inhibitory percentages of 90.94 and 88.13% were observed under the same conditions. EGCG also can greatly prolong the inhibitory time. The mechanism of the enhanced effects of EGCG on HUP's inhibition of AChE activity was investigated by steady fluorescence spectroscopy, infrared spectroscopy, and ultraviolet spectroscopy. HUP hardly interacted with the main transport protein, whereas there was a very strong binding interaction between EGCG and bovine serum albumin. The enhanced transport of HUP is a possible cause of the enhanced effect of EGCG on HUP bioactivity.     

Binding of 2-nonanone and milk proteins in aqueous model systems

Interactions of the model flavor compound 2-nonanone with individual milk proteins, whey protein isolate (WPI), and sodium caseinate in aqueous solutions were investigated. A method to quantify the free 2-nonanone was developed using headspace solid-phase microextraction followed by gas chromatography with flame ionization detection. Binding constants (K) and numbers of binding sites (n) for 2-nonanone on the individual proteins were calculated. The 2-nonanone binding capacities decreased in the order bovine serum albumin > beta-lactoglobulin > alpha-lactalbumin > alpha s1-casein > beta-casein, and the binding to WPI was stronger than the binding to sodium caseinate. All proteins appeared to have one binding site for 2-nonanone per molecule of protein at the flavor concentrations investigated, except for bovine serum albumin, which possessed two classes of binding sites. The binding mechanism is believed to involve predominantly hydrophobic interactions.     

Comparison of Two Soil Phosphorus Analytical Methods in Croatia

Abstract

The purpose of this article was to compare soil phosphorus (P) extraction by sodium bicarbonate solution (Olsen P) and by ammonium lactate (AL P) and to create a model for prediction of Olsen P using ordinary soil‐fertility control data. The soils data used in this study included Olsen P, pH_KCl, pH_H2O, organic matter, AL P, and AL K. Soil pH_KCl ranged from 3.5 to 8, organic matter up to 5%, AL K up to 400 mg kg^?1, and AL P up to 200 mg kg^?1. Olsen P and AL P were significantly correlated, and the difference between them was influenced by soil pH. Regression models included all soil data grouped by soil pH range, which significantly decreased the difference between predicted and measured Olsen P. The validation of the model was conducted on new data sets from field fertilization trials. The results show that Olsen P can be related to AL P and used for fertilizer recommendations instead of AL P.     

Beta-lactoglobulin structure and retinol binding changes in presence of anionic and neutral detergents

Bovine beta-lactoglobulin (beta-LG) in vivo (in milks) has been found in complexes with lipids such as butyric and oleic acids. To elucidate the still unknown structure-function relationship in this protein, the structural changes of beta-lactoglobulin variant A (beta-LG A) in the presence of anionic surfactant such as sodium n-dodecyl sulfate (SDS) and in the presence of nonionic surfactant such as Triton X-100 have been investigated. Subsequently, the retinol binding by beta-LG has been investigated in the presence of various amounts of these surfactants as its binding indicator. The results of UV-vis and fluorescence studies show a higher denaturating effect of SDS at acid pH that can be due to greater positive charges of beta-LG at this pH indicating also the nonspecific hydrophobic interactions of Triton X-100 with beta-LG at all studied pHs. Isothermal titration calorimetry (ITC) measurements indicate the endothermic nature of beta-LG/SDS interactions and the exothermic nature of Triton X-100/beta-LG interactions. The analysis of the binding data demonstrates the absence of considerable changes in retinol binding properties of beta-LG in the presence of various amounts of these surfactants. This implies that surfactant binding does not change the conformation of beta-LG in the regions defining the retinol-binding site.