荧光和圆二色光谱法用于低密度脂蛋白氧化的研究

氧化修饰低密度脂蛋白（oxidized low density lipoprotein,oxLDL）被认为是动脉硬化的关键致病因素。oxLDL的性能与其唯一的组成蛋白——载脂蛋白B-100（apoB-100）的二级结构即构象密切相关。荧光光谱和圆二色（CD）光谱是研究蛋白质构象的有力手段,因此在oxLDL结构的研究中也有广泛的应用。本文综述了近年来荧光光谱和圆二色光谱在LDL氧化研究方面的应用,并对今后的研究方向做了展望。     

光谱法研究核壳量子点CdTe/CdS与牛血清白蛋白的相互作用

应用荧光光谱、圆二色光谱和紫外吸收光谱等技术研究核壳量子点CdTe/CdS与牛血清白蛋白(BSA)相互作用的结果表明,CdTe/CdS对BSA的荧光猝灭机理为静态猝灭。根据不同温度下量子点对BSA的荧光猝灭作用计算了结合常数、热力学参数,证明了量子点与BSA相互作用力主要是范德华力或氢键作用力。探讨了量子点对BSA构象的影响。     

光谱法及分子模拟法研究杨梅素与ct-DNA的相互作用

采用紫外光谱、荧光光谱结合亚甲基蓝荧光探针(MB)、圆二色谱(CD)以及分子模拟等技术研究了生理条件下杨梅素与小牛胸腺DNA(ct-DNA)的相互作用,探索了其可能的结合位点与作用机制。热力学参数显示ΔH0、ΔS0,说明杨梅素与ct-DNA通过氢键和范德华力发生相互作用。光谱研究显示,ct-DNA能够猝灭杨梅素的内源荧光,猝灭方式为静态猝灭,两者的结合常数为1.86×103mol-1,结合位点数为1。在竞争性实验中杨梅素未能将MB从MB-DNA复合体系中游离出来,说明其与ct-DNA的作用方式为沟槽结合。圆二色光谱研究表明,杨梅素的加入未能破坏ct-DNA的双螺旋结构,两者的作用方式为沟槽结合。进一步通过分子模拟研究得到杨梅素与DNA结合的最低能量构象,杨梅素A环7-O和B环3’-O分别与DNA的碱基DA18和DA6在边缘小沟处通过氢键结合,周围富含碱基A和T,与光谱学研究结果一致,结合距离分别为2.061和1.918。     

氨基黑10B与牛血清白蛋白作用的光谱法研究

用紫外、荧光、圆二色光谱法研究了氨基黑10B与牛血清白蛋白(BSA)结合反应的光谱特性.结果表明氨基黑10B对牛血清白蛋白的荧光有猝灭作用,其猝灭类型属于静态猝灭;得到了不同温度下的结合常数和结合位点数;利用Gibbs-Helmholtz方程计算得到该猝灭反应的热力学参数,表明氨基黑10B主要以氢键和范德华力与BSA相互作用;圆二色和同步荧光光谱显示氨基黑10B对BSA构象产生了影响.     

基于大环多胺的新型线型聚合物的合成及与DNA相互作用的研究

设计合成了一种新型线形大环聚合多胺（POGEC）,该聚合物可通过1,3-丙二醇二缩水甘油基醚和1,7-二（二乙氧磷酰基）-1,4,7,10-四氮杂环十二烷反应得到．琼脂糖凝胶电泳和电子扫描光谱（SEM）显示POGEC与DNA有很强的结合能力．当生理浓度的氯化钠溶液加入到已经形成的POGEC／DNA复合物中,DNA可被有效释放．通过荧光光谱测量了POGEC与DNA的结合能力及紧缩能力．圆二色光谱表明POGEC的强结合力并未改变DNA的构象．     

肝素诱导人细胞型朊蛋白构象变化的光谱表征

粘多糖在朊病毒病中所发挥的作用目前仍存在争议.以肝素钠作为粘多糖的代表,通过共振光散射光谱、荧光光谱和圆二色光谱的变化研究了肝素钠与人重组细胞型朊蛋白(rhPrPC23-231)的相互作用.结果表明,肝素钠与朊蛋白相互作用后光散射和荧光信号均得到增强,并且使朊蛋白的荧光寿命有一定程度的延长.圆二色光谱表明肝素钠能诱导朊蛋白从富含α-螺旋的构象向富含β-折叠的构象转变.     

光谱法研究萘酰亚胺类衍生物与牛血清白蛋白的相互作用

采用荧光光谱、紫外-可见吸收光谱、红外光谱和圆二色光谱研究了3-氨基-4-二甲氨基-N-丁基-1.8-萘酰亚胺(ADBN)与牛血清白蛋白(BSA)结合反应的特征。荧光光谱与紫外-可见吸收光谱表明,ADBN使BSA荧光猝灭的机理为静态猝灭。根据热力学参数判断ADBN与BSA主要是通过氢键和范德华力结合,结合距离为4.08 nm。同步荧光光谱、圆二色光谱以及红外光谱表明,结合过程中BSA的构象发生了变化。     

光谱法研究两种抗菌类药物对牛血清白蛋白结构变化的影响

利用荧光猝灭光谱、同步荧光光谱、三维荧光光谱、紫外-可见吸收光谱及圆二色谱法研究了磺胺甲恶唑(Sulfamethoxazole,SMX)、磺胺二甲嘧啶(Sulfamethazine,SMZ)对牛血清白蛋白(Bovine serum albumin,BSA)结构的影响。荧光猝灭实验结果表明SMX和SMZ可以使BSA发生荧光猝灭;通过同步荧光光谱、三维荧光光谱和紫外-可见光谱实验数据定性的证实了SMX和SMZ的加入会使BSA的构象发生变化,并且通过紫外-可见光谱的实验结果可知SMX和SMZ与BSA之间的作用机理为静态猝灭。此外,由圆二色谱法得到的定量结果可知:当SMX或SMZ与BSA之间的摩尔浓度比为4∶1时,SMX可使BSA的α-螺旋结构含量由53.77%降低到51.82%;SMZ可使BSA的α-螺旋结构含量由53.77%降低到47.59%。     

分子内电荷转移荧光探针1-酮-2-（对二甲氨基苯亚甲基）四氢萘与人血清白蛋白作用

采用荧光及紫外光谱研究了1-酮-2-（对二甲氨基苯亚甲基）-四氢萘（KDTN）与人血清白蛋白（HSA）相互作用的光谱特性。 结果表明,静态猝灭和非辐射能量转移是导致KDTN对HSA荧光猝灭的主要原因。 测得17、27和37 ℃ 3个温度下的结合常数K_A分别为1.633×10⁸、0.7998×10⁸和0.347×10⁸ L/mol,结合位点数n分别为1.7、1.6和1.7;据Forster偶极 偶极非辐射能量转移理论,计算得到KDTN与HSA在3个温度下的作用距离r分别为2.64、2.59和2.64 nm;能量转移效率E分别为0.5100、0.4797和0.4210。 热力学参数表明,二者主要以范德华力或氢键结合;用同步荧光技术研究了KDTN对HSA构象的影响,结果表明,KDTN的加入对HSA构象影响不大。     

光谱法研究石墨烯-TiO_2与溶菌酶的相互作用及对酶活性的影响

在模拟人体生理条件下,采用紫外光谱、荧光光谱、同步荧光光谱、三维荧光光谱和圆二色谱等方法,以纳米Ti O2作对照,研究GR-Ti O2与溶菌酶(LYSO)的相互作用,通过酶活性实验测定GR-Ti O2对LYSO活性的影响。荧光光谱结果表明GR-Ti O2与LYSO发生了相互作用,导致LYSO内源荧光猝灭,猝灭机制为静态猝灭,作用力类型为氢键和范德华力,与纳米Ti O2研究结果一致。同步荧光与圆二色谱结果进一步表明GR-Ti O2对LYSO的构象影响更大。酶活性实验结果表明GR-Ti O2对LYSO的活性抑制作用强于纳米Ti O2。     

CD36, a scavenger receptor implicated in atherosclerosis

CD36 is a membrane glycoprotein that is present on various types of cells, including monocytes, macrophages, microvascular endothelial cells, adipocytes and platelets. Macrophage CD36 participates in atherosclerotic arterial lesion formation through its interaction with oxidized low-density lipoprotein (oxLDL), which triggers signaling cascades for inflammatory responses. CD36 functions in oxLDL uptake and foam cell formation, which is the initial critical stage of atherosclerosis. In addition, oxLDL via CD36 inhibits macrophage migration, which may be a macrophage-trapping mechanism in atherosclerotic lesions. The role of CD36 was examined in in vitro studies and in vivo experiments, which investigated various functions of CD36 in atherosclerosis and revealed that CD36 deficiency reduces atherosclerotic lesion formation. Platelet CD36 also promotes atherosclerotic inflammatory processes and is involved in thrombus formation after atherosclerotic plaque rupture. Because CD36 is an essential component of atherosclerosis, defining the function of CD36 and its corresponding signaling pathway may lead to a new treatment strategy for atherosclerosis.     

Apolipoprotein B100 analysis in microchip with electrochemical detection

Apolipoprotein B100(apoB-100) is a major protein of the cholesterol-rich low-density lipoprotein(LDL) and reflects a better assessment of total atherogenic burden to the vascular system than LDL.In this work,a simple and sensitive method has been developed to determine picoliter apoB-100s using the PMMA microfluidic chip coupled with electrochemical detection system. This method performs very well with a detectable linear range of 1-800 pg/mL and a detection limit of 1 pg/mL.A real serum sample has further been detected by this microchip-based biosensor.The results show that this kind of method is practicable and has the potential application in clinical analysis and diagnosis.     

Oxidation-dependent effects of oxidized LDL: proliferation or cell death

Oxidized low-density lipoprotein (oxLDL) induces a wide range of cellular responses to produce atherosclerotic lesion, but key factors determining the response are not understood. In this study, purified LDL was oxidized with copper sulfate, and its physical properties and the related biological responses were investigated. The average hydrodynamic diameter of the lightly oxidized LDL was approximately 25 nm and its Rf value relative to nLDL on agarose gel was between 1.0 and 1.25. The diameter of the extensively oxidized LDL was over 30 nm, the Rf value was over 2.0. A 24 h-exposure of resting RAW264.7 macrophage cells to 100 microg/ml of the lightly oxidized LDL induced proliferation and macrophage activation whereas the extensively oxidized LDL induced cell death at the same concentration. In contrast, 200 microg/ml of oxLDL caused cell death regardless of oxidation degree. Short incubation (4-6 h) of the highly oxidized LDL (100 microg/ml) also resulted in cell proliferation. OxLDL-induced cell death showed mixed characteristics of apoptosis and/or necrosis depending on the strength and duration of the insult. These results suggest that cellular responses induced by oxLDL be dependent on the oxidation degree, the duration of exposure, and the concentration of oxLDL.     

Fabrication of a label-free electrochemical immunosensor of low-density lipoprotein

The silver chloride@polyaniline (PANI) core-shell nanocomposites (AgCl@PANI) combined with Au nanoparticles (AuNPs) were used to prepare the AuNPs-AgCl@PANI hybrid material. A novel sensitive label-free low-density lipoprotein (LDL) electrochemical biosensor was fabricated by adsorption of antibody to apolipoprotein B-100 (aopB-100) on an AuNPs-AgCl@PANI-modified glassy carbon (GC) electrode. The hybrid material could provide surface for high antibody loading due to its large surface-to-volume ratio. Since each LDL has an apoB-100 on its phospholipids coat, they could be bonded to the electrode surface through the specific antibody-antigen reaction. Electrochemical impedance spectroscopy (EIS) was used to characterize the recognition of LDL. The negative charges carried by LDL phospholipids coat would block the electron transfer of the [Fe(CN)6]3-/4- redox couple severely. In addition, the conductivity of LDL is very poor, so small amounts of LDL on the electrode could result in great change in the electron-transfer resistance (Ret). The biosensor exhibited a highly sensitive response to LDL with a detection limit of 0.34 pg/mL, and some factors that would affect the performance of the biosensor were studied, such as incubation time and temperature.     

Quartz crystal microbalance, a valuable tool for elucidation of interactions between apoB-100 peptides and extracellular matrix components

Atherosclerosis has received wide attention as a primary cause of premature death in developed countries. The retention of low-density lipoprotein (LDL) particles in the intima, the inner layer of the capillaries, has been imputed as the main cause of the development of atherosclerotic plaques. The entrapment of LDL is mainly due to the specific interaction between the lysine-rich site on apolipoprotein B-100 (apoB-100), a major apolipoprotein of LDL, and extracellular matrix (ECM) components such as collagen, proteoglycans, and glycosaminoglycans (GAGs). Although valuable techniques already exist for studies on apoB-100 and ECM interactions, there is continued need for miniaturized tools that can complement the tools already available and even provide totally new data. This work explores the applicability of the quartz crystal microbalance (QCM) for interaction studies between apoB-100 peptide fragments and various components of the ECM. Two positive peptide fragments, PP and PP₂, and two components of the ECM, collagen I and a selected GAG, chondroitin 6-sulfate (C6S), were immobilized on polystyrene and carboxyl sensor chips. C6S was injected as analyte for PP- and PP₂-coated surfaces, while PP was the analyte for collagen I and C₆S surfaces. The estimated dissociation constant (K _D) indicates that the interactions occur via the positive residues, lysine and arginine, of apoB-100. The continuous-flow QCM system employed in this study is shown to be an excellent tool for the elucidation of interactions between these types of biomolecules.

低密度脂蛋白结构研究进展

本文对低密度脂蛋白（LDL）的形成、结构做了简单概述。LDL是由极低密度脂蛋白（VLDL）演化而成,对于其结构,目前为人们普遍接受的是乳状液颗粒模型。作为LDL中的主要载脂蛋白apoB-100,其一级结构已经为人们所知,而高级结构的研究也得出了不少有益的结论。此外,本文还对LDL上受体结合部位和LDL受体上LDL结合部位等方面做了简单叙述。     

Native and unfolded cytochrome c--comparison of dynamics using 2D-IR vibrational echo spectroscopy

Unfolded vs native CO-coordinated horse heart cytochrome c (h-cyt c) and a heme axial methionine mutant cyt c552 from Hydrogenobacter thermophilus ( Ht-M61A) are studied by IR absorption spectroscopy and ultrafast 2D-IR vibrational echo spectroscopy of the CO stretching mode. The unfolding is induced by guanidinium hydrochloride (GuHCl). The CO IR absorption spectra for both h-cyt c and Ht-M61A shift to the red as the GuHCl concentration is increased through the concentration region over which unfolding occurs. The spectra for the unfolded state are substantially broader than the spectra for the native proteins. A plot of the CO peak position vs GuHCl concentration produces a sigmoidal curve that overlays the concentration-dependent circular dichroism (CD) data of the CO-coordinated forms of both Ht-M61A and h-cyt c within experimental error. The coincidence of the CO peak shift curve with the CD curves demonstrates that the CO vibrational frequency is sensitive to the structural changes induced by the denaturant. 2D-IR vibrational echo experiments are performed on native Ht-M61A and on the protein in low- and high-concentration GuHCl solutions. The 2D-IR vibrational echo is sensitive to the global protein structural dynamics on time scales from subpicosecond to greater than 100 ps through the change in the shape of the 2D spectrum with time (spectral diffusion). At the high GuHCl concentration (5.1 M), at which Ht-M61A is essentially fully denatured as judged by CD, a very large reduction in dynamics is observed compared to the native protein within the approximately 100 ps time window of the experiment. The results suggest the denatured protein may be in a glassy-like state involving hydrophobic collapse around the heme.     

Lysosomal cholesterol accumulation inhibits subsequent hydrolysis of lipoprotein cholesteryl ester.

Human macrophages incubated for prolonged periods with mildly oxidized LDL (oxLDL) or cholesteryl ester-rich lipid dispersions (DISP) accumulate free and esterified cholesterol within large, swollen lysosomes similar to those in foam cells of atherosclerosis. The cholesteryl ester (CE) accumulation is, in part, the result of inhibition of lysosomal hydrolysis due to increased lysosomal pH mediated by excessive lysosomal free cholesterol (FC). To determine if the inhibition of hydrolysis was long lived and further define the extent of the lysosomal defect, we incubated THP-1 macrophages with oxLDL or DISP to produce lysosome sterol engorgement and then chased with acetylated LDL (acLDL). Unlike oxLDL or DISP, CE from acLDL normally is hydrolyzed rapidly. Three days of incubation with oxLDL or DISP produced an excess of CE in lipid-engorged lysosomes, indicative of inhibition. After prolonged oxLDL or DISP pretreatment, subsequent hydrolysis of acLDL CE was inhibited. Coincident with the inhibition, the lipid-engorged lysosomes failed to maintain an acidic pH during both the initial pretreatment and subsequent acLDL incubation. This indicates that the alterations in lysosomes were general, long lived, and affected subsequent lipoprotein metabolism. This same phenomenon, occurring within atherosclerotic foam cells, could significantly affect lesion progression.     

Mass Spectrometry for the Monitoring of Lipoprotein Oxidations by Myeloperoxidase in Cardiovascular Diseases

Oxidative modifications of HDLs and LDLs by myeloperoxidase (MPO) are regularly mentioned in the context of atherosclerosis. The enzyme adsorbs on protein moieties and locally produces oxidizing agents to modify specific residues on apolipoproteins A-1 and B-100. Oxidation of lipoproteins by MPO (Mox) leads to dysfunctional Mox-HDLs associated with cholesterol-efflux deficiency, and Mox-LDLs that are no more recognized by the LDL receptor and become proinflammatory. Several modification sites on apoA-1 and B-100 that are specific to MPO activity are described in the literature, which seem relevant in patients with cardiovascular risk. The most appropriate analytical method to assess these modifications is based on liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). It enables the oxidized forms of apoA-1and apoB-100 to be quantified in serum, in parallel to a quantification of these apolipoproteins. Current standard methods to quantify apolipoproteins are based on immunoassays that are well standardized with good analytical performances despite the cost and the heterogeneity of the commercialized kits. Mass spectrometry can provide simultaneous measurements of quantity and quality of apolipoproteins, while being antibody-independent and directly detecting peptides carrying modifications for Mox-HDLs and Mox-LDLs. Therefore, mass spectrometry is a potential and reliable alternative for apolipoprotein quantitation.     

Expression of a supramolecular complex at a multivalent interface

The multivalent binding of a supramolecular complex at a multivalent host surface by combining the orthogonal beta-cyclodextrin (CD) host-guest and metal ion-ethylenediamine coordination motifs is described. As a heterotropic, divalent linker, an adamantyl-functionalized ethylenediamine derivative was used. This was complexed with Cu(II) or Ni(II). The binding of the complexes to a CD self-assembled monolayer (SAM) was studied as a function of pH by means of surface plasmon resonance (SPR) spectroscopy. A heterotropic, multivalent binding model at interfaces was used to quantify the multivalent enhancement at the surface. The Cu(II) complex showed divalent binding to the CD surface with an enhancement factor higher than 100 relative to the formation of the corresponding divalent complex in solution. Similar behavior was observed for the Ni(II) system. Although the Ni(II) system could potentially be trivalent, only divalent binding was observed at the CD SAMs, which was confirmed by desorption experiments.