有机酸类化感物质的血清蛋白输运机制研究

利用亲和毛细管电泳(Affinity Capillary Electrophoresis,ACE)建立有机酸类化感物质与血清白蛋白(Bovine serum albumin,BSA)结合反应的分析方法。模拟典型有机酸类化感物质与血清白蛋白的结合反应,构建配体(有机酸)-受体(BSA)相互作用体系,采用ACE法研究不同浓度柠檬酸(Citric Acid,CA)/磺基水杨酸(Sulfosalicylic acid,SA)与BSA的结合反应机制并比较不同有机酸作用机理异同。结果表明,有机酸类化感物质CA/SA与BSA发生结合反应形成复合物CABSA和SA-BSA。依据有效淌度变化,理论方程非线性拟合结合反应的表观结合常数KCA-BSA=(1.82±0.11)×104L·mol-1、KSA-BSA=(2.12±0.12)×104L·mol-1,结合反应均为快平衡反应。相关工作阐明了血清蛋白输运有机酸类化感物质的生理作用,为化感物质与生物大分子结合反应的深入研究提供相应理论参考。     

对二甲氨基苯甲酰腙衍生物与金属离子间的相互作用

合成了对二甲氨基苯甲酰缩噻吩(1)、呋喃(2)、苯(3)等腙类衍生物,并通过氢谱、碳谱和质谱进行表征,采用吸收光谱法研究了其与金属离子间的相互作用。结果表明,含噻吩基团的腙衍生物1与Hg2+结合,形成1∶1稳定的配合物,结合常数为9.7×104L·mol-1,噻吩衍生物1对Hg2+响应的线性范围为1.0~18μmol/L,检出限为0.28μmol/L,且Cu2+,Fe3+和Ni2+等共存金属离子不干扰测定。对照化合物中的呋喃衍生物2可1∶1结合Hg2+,Zn2+和Cd2+,其与Hg2+和Zn2+的结合常数分别为6.5×104L·mol-1和5.0×105L·mol-1;而苯衍生物3对测试的金属离子均未显示光谱变化。由此可见,化合物1对Hg2+的高选择性结合作用源于结合基团中不同配位原子的组合,其所含的S和O均参与了与Hg2+的配位作用。     

重金属Cr(Ⅵ)与血清蛋白质组的结合反应性能研究

利用亲和毛细管电泳(Affinity capillary electrophoresis,ACE)方法,基于位点结合模型,构建配体(Cr(Ⅵ))-血清蛋白质组受体(Fetal bovine serum,FBS)结合反应方程,研究重金属离子Cr(Ⅵ)和血清蛋白质组的结合反应性能。结果表明,组学条件下,Cr(Ⅵ)与FBS中高丰度组分白蛋白(Bovine serum albumin,BSA)、转铁蛋白(Bovine apo transferrin,bATF)、免疫球蛋白(Bovine IgG,IgG)之间形成Cr髩-蛋白质络合物;结合反应均为快平衡反应;依据有效淌度的变化,通过结合方程测得结合反应的表观结合常数KCr(Ⅵ)-bATF=1.27×104L.mol-1、KCr(Ⅵ)-IgG=4.91×104L.mol-1、KCr(Ⅵ)-BSA=8.98×104L.mol-1;同时根据实验数据非线性模拟结果表明,Cr髩与FBS中高丰度组分发生相互作用的强度与Cr(Ⅵ)浓度之间存在明显的量效关系。本文有关工作可为重金属离子-蛋白质络合物结合性能等生物无机化学研究内容提供参考。     

均三嗪衍生物应用于Fe3+和Cu2+的选择性识别研究

合成了2,4-二甲基-6-(4’-N,N-二甲氨基苯乙烯基)-1,3,5-均三嗪(1)和2-苯乙烯基-4,6-二甲基-1,3,5-均三嗪(2)两种化合物,并对其进行了1H NMR,MS,元素分析等表征.采用吸收光谱法研究了金属离子与化合物间的相互作用,结果显示:化合物1对Fe3+和Cu2+表现出高选择性光谱响应,其最大吸收波长由393 nm分别红移至525 nm和513 nm,溶液颜色由黄色变为粉红色.化合物1与Fe3+结合形成1∶1型配合物,其结合常数为1.8×104L mol-1;与Cu2+结合形成2∶1型配合物,其结合常数为2.6×1010L mol-1.化合物2仅对Fe3+呈现显著的光谱变化,其最大吸收波长由304nm红移至357 nm,而Cu2+的加入未引起光谱明显变化,2与Fe3+亦形成1∶1型配合物,结合常数为1.0×105L mol-1.结果表明Fe3+可能与化合物1和2中三嗪N配位,而Cu2+与化合物1中甲氨基中的N配位.同时考察了其它金属离子如Li+,K+,Mg2+,Ca2+,Co3+,Ni2+,Ag+,Cd2+,Hg2+和Zn2+等离子对化合物1和2吸收光谱的影响,结果显示两者光谱均无明显变化,据此提出了高选择性Fe3+,Cu2+的识别体系.     

苯并咪唑衍生的单核钴(Ⅱ)和单核镍(Ⅱ)配合物与DNA和蛋白质的结合反应性及细胞毒活性研究

利用紫外吸收光谱、荧光光谱、圆二色光谱(CD)等各种光谱手段对比地研究了由苯并咪唑衍生的单核钴配合物[Co(EDTB)]2+(1)和单核镍配合物[Ni(EDTB)]2+(2)(这里EDTB为N,N,N′,N′-四(2′-苯并咪唑甲基)-1,2-乙二胺)与小牛胸腺DNA(CTDNA)和牛血清白蛋白(BSA)的相互作用。结果表明,在生理条件下,配合物1和2均能通过插入方式较强的与CT-DNA结合,诱导DNA构象的改变;且配合物1对DNA的结合能力略强于2,其结合常数分别为Kb(1)=3.23×104L·mol-1和Kb(2)=2.40×104L·mol-1。配合物与BSA相互作用的研究表明,1和2均能与BSA发生较强的相互作用,结合常数均处在104~105 L·mol-1;该结合引起了BSA微环境和构象发生变化,且使BSA内源荧光被淬灭,淬灭机理为静态淬灭。利用MTT法研究了配合物1和2对小鼠白血病细胞株P388和人非小细胞肺癌细胞株A-549的体外细胞毒活性,实验结果表明,配合物1和2对P388不敏感,对A-549在高浓度(10-4~10-5 mol·L-1)下表现出与顺铂相当的细胞毒活性。     

金属元素组与血清白蛋白的竞争结合反应性能研究

利用亲和毛细管电泳（Affinitycapillaryelectrophoresis,ACE）法研究金属元素组和血清白蛋白（Bovineserumalbumin,BSA）的竞争结合反应性能。基于位点结合模型,构建双金属组[Zn²⁺,Cu²⁺]与血清白蛋白结合反应模型,建立多元金属组与生物大分子竞争结合的理论方程,测定结合参数并解析动力学机制。结果表明,金属元素组[Zn²⁺,Cu²⁺]与BSA发生竞争结合反应形成配合物Zn²⁺-BSA和Cu²⁺-BSA。依据有效淌度变化,通过建立的理论方程非线性拟合竞争结合反应的平均表观结合常数K_Zn²⁺-BSA=4.01×10⁴L·mol^-1、K_Cu²⁺-BSA=7.75×10⁴L·mol^-1。结合反应均为快平衡反应,Cu²⁺对Zn²⁺离子的结合作用有明显拮抗作用。分析ACE谱显示配合物的峰高与配体结合能力大小、配合物稳定性之间存在量效关系。     

壳寡糖及酰化壳寡糖与钕(Ⅲ)配合物的合成及其与牛血清白蛋白相互作用的对比研究

用壳寡糖及酰化壳寡糖与氯化钕反应,合成了壳寡糖-钕和酰化壳寡糖-钕配合物,利用红外光谱(IR)、紫外光谱(UV)手段对其结构进行了表征。在模拟生理条件下,本文采用紫外光谱和荧光光谱研究了两种配合物与牛血清白蛋白(BSA)的相互作用,计算了配合物与BSA的结合常数、结合位点数。荧光光谱结果表明,配合物均可有规律地猝灭BSA的内源荧光,猝灭方式为静态猝灭,壳寡糖-钕和酰化壳寡糖-钕分别与BSA的结合常数为1.33×104L·mol-1和6.95×104L·mol-1,结合位点数为1.05和1.3,说明配合物与BSA均具有较强的结合作用,能够被BSA储存和运输,并且酰化壳寡糖-钕与BSA的结合能力强于壳寡糖-钕。最后采用紫外光谱法对其作用机理进一步确认。因此,酰化壳寡糖-钕可以被BSA存储和运输,有望成为蛋白质荧光探针。     

重金属Cr(Ⅵ)离子与血清白蛋白的结合反应机制研究

电聚法制备聚苯胺修饰电极(PANI/GCE),并通过循环伏安法和电化学交流阻抗法表征修饰电极;利用修饰电极研究Cr(Ⅵ)离子与血清白蛋白(Bovine serum albumin,BSA)的结合反应机制并构建理论模型方程.结果表明,成功制备PANI/GCE修饰电极,修饰电极测定Cr(Ⅵ)-BSA分子相互作用结果显示良好的线性响应关系,利用建立的理论模型方程计算Cr(Ⅵ)与蛋白质的结合参数(β=2.07×104 L.mol-1,n=1.03),说明Cr(Ⅵ)与血清白蛋白存在明显的相互作用.     

新型的去甲斑蝥酸根合镨(Ⅲ)、钐(Ⅲ)配合物的晶体结构、与DNA和BSA的作用及抗增殖活性

合成了两种2-氨甲基苯并咪唑(ambi)和去甲基斑蝥酸(H2DCA=7-氧杂二环[2.2.1]庚烷-2,3-二甲酸)与镨(Ⅲ)、钐(Ⅲ)的配合物。应用元素分析、摩尔电导、红外光谱及X射线单晶衍射法对配合物的组成和结构进行了表征,配合物的组成为:(Hambi)[Ln(DCA)2(H2O)3]·3H2O(Ln=Pr(III)(1),Sm(III))(2);Hambi为质子化的ambi。DCA离子的醚键和羧酸根的氧原子参与配位,为三齿配体,稀土离子的配位数为9。通过紫外光谱法、荧光光谱法和粘度法研究了配合物与DNA和牛血清白蛋白(BSA)的相互作用。结果表明:配合物能通过部分插入模式与DNA发生较强的结合作用(Kb:1.56×104(1)和1.85×104L·mol-1(2))。配合物能与BSA发生强烈的相互作用(KA:9.33×104L·mol-1(1)和1.52×106L·mol-1(2)),结合位点数为1。测试了配合物对人肝癌细胞(SMMC7721)的体外抗增殖活性。结果显示,配合物的抗癌活性较去甲基斑蝥素有明显提高。     

苯乙烯三嗪衍生物的合成及在重金属离子识别中的应用

合成了2,4-二(2-噻吩乙烯基)-6-(4′-N,N-二甲氨基苯乙烯基)-1,3,5-均三嗪(2)并鉴定了其结构。在乙腈-水混合介质中,化合物2在355和416 nm处呈现双吸收峰,加入Cu2+,Hg2+和Fe3+后,均在520 nm附近形成新的吸收峰。化合物2与Cu2+、Hg2+和Fe3+均形成1∶1型配合物,其结合常数分别为1.9×105L·mol-1,6.6×103L·mol-1,2.7×103L·mol-1。对照化合物4与金属离子的光谱响应与化合物2相似,仅吸收峰的位置不同。因此,可认为化合物2和4中三嗪环中的N和噻吩环中的S与Cu2+、Hg2+和Fe3+共同配位形成了稳定的金属配合物。     

Effects of Temperature and Common Ions on Binding of Puerarin to BSA

The effects of temperature and common ions on binding of puerarin to bovine serum albumin (BSA) are investigated. The binding constants (K _a) between puerarin and BSA are 1.13×10⁴ L⋅mol⁻¹ (20 °C) and 1.54×10⁴ L⋅mol⁻¹ (30 °C), and the number of binding sites (n) is (0.95±0.02). However, at a higher temperature (40 °C) the stability of the puerarin–BSA system decreases, which results in a lower binding constant (1.58×10³ L⋅mol⁻¹) and number of binding sites (n=0.73) of the puerarin–BSA system. However, the presence of Cu²⁺ and Fe³⁺ ions increases the binding constants and the number of binding sites in the puerarin–BSA complex.     

Study on Interactions of Phenolic Acid-Like Drug Candidates with Bovine Serum Albumin by Capillary Electrophoresis and Fluorescence Spectroscopy

The interactions of the phenolic acids cinnamic acid (CNA), ferulic acid (FA), caffeic acid (CA) and chlorogenic acid (CLA) with bovine serum albumin (BSA) were investigated and compared using affinity capillary electrophoresis (ACE) and the fluorescence quenching methods. ACE gives binding constants (K _b) and thermodynamic parameters. The thermodynamic parameters show that each of four phenolic acids bind to BSA mainly by hydrogen bonds, electrostatic and hydrophobic interactions. The fluorescence quenching method provided quenching constant K _sv, binding site number n and K _b. The fluorescence results indicate that BSA fluorescence quenching is mainly a static quenching process. The binding constants (K _b) of CNA, FA, CA and CLA were from 2.52×10⁴ to 7.90×10⁴ L⋅mol⁻¹ from ACE experiments and 1.19×10⁴ to 5.21×10⁴ L⋅mol⁻¹ from fluorescence, their increase corresponded to the increase in the number of hydroxyl groups. These results imply that molecular structure and the number of hydroxyl groups of phenolic acids play act key roles in the affinity of natural phenolic acids towards BSA.     

苯并咪唑衍生的单核钴（Ⅱ）和单核镍（Ⅱ）配合物与DNA和蛋白质的结合反应性及细胞毒活性研究

利用紫外吸收光谱、荧光光谱、圆二色光谱（CD）等各种光谱手段对比地研究了由苯并咪唑衍生的单核钴配合物[Co（EDTB）]²⁺（1）和单核镍配合物[Ni（EDTB）]²⁺（2）（这里EDTB为N,N,N’,N’-四（2-苯并咪唑甲基）-1,2-乙二胺）与小牛胸腺DNA（CT-DNA）和牛血清白蛋白（BSA）的相互作用。结果表明,在生理条件下,配合物1和2均能通过插入方式较强的与CT-DNA结合,诱导DNA构象的改变;且配合物1对DNA的结合能力略强于2,其结合常数分别为K_b（1）=3.23×10⁴L·mol^-1和K_b（2）=2.40×10⁴L·mol^-1。配合物与BSA相互作用的研究表明,1和2均能与BSA发生较强的相互作用,结合常数均处在10⁴~10⁵L·mol^-1;该结合引起了BSA微环境和构象发生变化,且使BSA内源荧光被淬灭,淬灭机理为静态淬灭。利用MTT法研究了配合物1和2对小鼠白血病细胞株P388和人非小细胞肺癌细胞株A-549的体外细胞毒活性,实验结果表明,配合物1和2对P388不敏感,对A-549在高浓度（10^-4~10^-5mol·L^-1）下表现出与顺铂相当的细胞毒活性。     

Effect of Hydrogenation on Ring C of Flavonols on Their Affinity for Bovine Serum Albumin

In this paper, the effect of hydrogenation on ring C of flavonols on the affinity for bovine serum albumin was investigated. Two differently substituted B-ring hydroxylation flavonols (myricetin and quercetin) and their dihydrides (dihydromyricetin and dihydroquercetin) were used to study their affinities for BSA by quenching the intrinsic BSA fluorescence in solution. From the spectra, the bimolecular quenching constants, the binding constants, the number of binding sites and the binding distances were calculated. The hydroxylation on ring B and hydrogenation on ring C of flavonols significantly affected the binding/quenching process; in general, the hydroxylation increased the affinity and the hydrogenation decreased the affinity. For myricetin and quercetin, the binding constants (K _a) for BSA were 1.84×10⁸ L⋅mol⁻¹ and 3.83×10⁷ L⋅mol⁻¹. For dihydromyricetin, the binding constant was 1.36×10⁴ L⋅mol⁻¹, while dihydroquercetin hardly quenched the BSA intrinsic fluorescence. These results showed that hydrogen bonding and conjugative effects may play an important role in binding of flavonols to BSA. These results also showed that the properties of flavonols are related to their chemical structure.     

Study on the Interaction of Raloxifene and Bovine Serum Albumin by the Tachiya Model

The interaction of bovine serum albumin (BSA) with raloxifene was assessed via fluorescence spectroscopy. The number of binding sites and the apparent binding constants between raloxifene and BSA were analyzed using the Tachiya model and Stern-Volmer equation, respectively. The apparent binding constant and the number of binding sites at 298 K were 2.33×10⁵ L?mol^?1 and 1.0688 as obtained from the Stern-Volmer equation and 2.00×10⁵ L?mol^?1 and 2.6667 from the Tachiya model. The thermodynamic parameters ΔH and ΔS were calculated to be 69.46 kJ?mol^?1 and 121.12 J?K^?1?mol^?1, respectively, suggesting that the force acting between raloxifene and BSA was mainly a hydrophobic interaction. The binding distance between the donor (BSA) and acceptor (raloxifene) was 4.77 nm according to Förster’s nonradiational energy transfer theory. It was also found that common metal ions such as K⁺, Cu²⁺, Zn²⁺, Mg²⁺ and Ca²⁺ decreased the apparent association constant and the number of binding sites between raloxifene and BSA.     

Probing the Interaction Between a Surfactant–Cobalt(III) Complex and Bovine Serum Albumin

The mechanism of binding of the surfactant–cobalt(III) complex, cis-[Co(phen)₂(C₁₄H₂₉NH₂)Cl](ClO₄)₂⋅3H₂O (phen = 1,10-phenanthroline, C₁₄H₂₉NH₂ = tetradecylamine) with bovine serum albumin (BSA) was investigated by UV–vis absorption, circular dichroism (CD) and fluorescence spectroscopic techniques. The results of fluorescence titration revealed that the surfactant–cobalt(III) complex quenched the intrinsic fluorescence of BSA through a combination of static and dynamic quenching. The apparent binding constant (K _a) and number of binding sites (n) were calculated below and above the critical micelle concentration (CMC). The thermodynamic parameters determined by the van’t Hoff analysis of the constants (ΔH ^∘=14.87 kJ⋅mol⁻¹; ΔS ^∘=152.88 J⋅mol⁻¹⋅K⁻¹ below the CMC and 25.70 kJ⋅mol⁻¹ and 243.14 J⋅mol⁻¹⋅K⁻¹, respectively, above the CMC) clearly indicate that the binding is entropy-driven and enthalpically disfavored. Based on F?rster’s theory of non-radiation energy transfer, the binding distance, r, between donor (BSA) and the acceptor (surfactant–cobalt(III) complex) was evaluated. UV–vis, CD and synchronous fluorescence spectral results showed that the binding of the surfactant–cobalt(III) complex to BSA induced conformational changes in BSA.     

Studies of the interaction between apigenin and bovine serum albumin by spectroscopic methods

The interaction between apigenin (Ap) and bovine serum albumin (BSA) in physiological buffer (pH = 7.4) is investigated by fluorescence quenching technique and UV-vis absorption spectra. The results reveal that Ap could strongly quench the intrinsic fluorescence of BSA. The quenching mechanism of Ap for BSA varies with the change of Ap concentration. when Ap concentration is lower, it is a static quenching procedure, when Ap concentration is higher, a combined quenching (both static and dynamic) would operate. The apparent binding constants Ka and number of binding sites n of Ap with BSA are obtained by fluorescence quenching method. The thermodynamic parameters, enthalpy change (Δ_r H ^m and entropy change (Δ_r S ^m ), are calculated to be −15.382 kJ mol⁻¹ K⁻¹ < 0 and 104.888 J mol⁻¹ K⁻¹ > 0, respectively, which indicate that the interaction of Ap with BSA is driven mainly by hydrogen bonding and hydrophobic interactions. The distance r between BSA and Ap is calculated to be 1.89 nm based on F?rster’s non-radiative energy transfer theory. The results of synchronous fluorescence spectra show that binding of Ap with BSA cannot induce conformational changes in BSA.