动静态光散射技术在蛋白质研究中的应用进展

动静态光散射技术是研究蛋白质等大分子物质尺寸、分布、形态及构象的一种重要手段,以其快速、便捷、对样品无干扰等特点,逐步深入到生化、物理、医药病理等领域,具有良好的应用前景。本文简要阐述了光散射技术的发展,主要介绍了动静态光散射技术的原理,从流体力学半径、重均分子量、均方根回旋半径以及渗透第二维里系数4个重要的物理参数出发,对近年来动静态光散射技术在蛋白质研究中的应用进行了调研和综述,并对该技术的发展进行了展望。     

单分子荧光共振能量转移用于生物大分子构象动态变化过程研究进展

生物大分子参与生命活动的各个过程,在单分子水平上实时观测和分析生物大分子自身的结构动态以及生物大分子相互作用的动态过程,对于深入理解生物大分子的作用机制具有重要意义。自提出以来,单分子荧光共振能量转移技术逐渐展现出其在研究生物大分子构象变化和相互作用过程等方面的巨大潜力,一系列新的作用机理陆续被提出。本文对单分子荧光共振能量转移技术在蛋白质与核酸分子构象动态变化、蛋白质-蛋白质相互作用以及蛋白质-核酸相互作用等方面取得的研究进展进行了综述。     

探针体在蛋白质大分子上Langmuir吸附聚集及应用──蛋白质/荧光桃红B(PB)结合反应研究

蛋白质化学是生物化学家当今感兴趣的前沿研究领域. 研究小分子(离子)在生物大分子上的聚集形式以及机理, 帮助人们弄清其污染物及毒物在生物大分子上的结合, 在病理分析、临床检测以及基因变异有重要意义. 常用光谱分析方法包括分光光度法[1～6]、荧光法[7,8]和共振光散射光谱技术(RRS)等[9～12]. 但是, 大分子与探针分子间的结合机理仍存在一些尚未解决的问题[13,14]. 蛋白质分子由于复杂的立体构象形成弱微静电场[15,16], 在其作用下, 带电荷的小分子以单分子层形式被吸附到微相电场表面.     

有机染料作为光散射探针在分析应用中的研究进展

结合本实验室的研究工作,对有机染料作为光散射探针在蛋白质、核酸和金属离子测定中的应用进行了系统的评述。结合光散射原理及有机染料的共振光散射增强理论,对实验中出现的现象作出解释,并对可能作为蛋白质和核酸光散射分析的有机染料探针的结构特点进行预测。     

共振散射相关光谱一种新的单颗粒探测方法

基于共焦构型构建了共振散射相关光谱新方法, 阐明了共振散射相关光谱的原理, 并利用纳米金的共振散射特性, 将纳米金标记到生物分子上. 考察了该系统的重现性以及溶液粘度、粒径、浓度和激光能量对金纳米粒子在溶液中扩散行为的影响. 结果表明, 共振散射相关光谱可以替代荧光相关光谱, 应用于生物分析和某些生物系统研究.     

刚果红共振光散射法测定蛋白质

本文研究了生物染料刚果红(Congo red)与人血清白蛋白(HSA)作用的共振光散射光谱，pH为4．35的溶液中，刚果红与人血清白蛋白作用导致在575m处共振光散射明显增强，且共振光散射信号值与蛋白质的浓度具有线性关系。     

依波西隆蓝与蛋白质作用的共振光散射光谱及其分析应用

微量蛋白质对依波西隆蓝的共振光散射产生增强作用，且增强程度与蛋白质浓度有良好的线性关系，由此建立了测定蛋白质的灵敏共振光散射分析方法。利用该方法测定合成样品和血清样品中的蛋白质，均获得了可靠的分析结果。     

光散射技术在蛋白质晶体生长研究中的应用和进展

光散射技术广泛应用于生物大分子的晶体生长研究中,它包括静态光散射和动态光散射两种。利用静态光散射可以测定蛋白质溶液渗透的第二维里系数;利用动态光散射可以测定蛋白质溶液的平动扩散系数,获得溶液中蛋白质粒子的流体力学半径及分布情况,分离蛋白质结晶的成核与生长过程,研究大分子的聚集行为和晶体生长的动力学。借助光散射技术可以实现蛋白质晶体生长过程的动态控制。近些年光散射仪器向着小型化、轻便化的方向发展,光散射技术不断得到改进,日益完善,不仅用于地面实验,也应用于空间领域蛋白质晶体生长的研究中。     

三种偶氮化合物的荧光光谱研究

近年来 ,一些重要的有机染料探针在生物大分子光度分析、荧光分析和共振光散射分析方面得到了日益广泛地应用[1,2 ] ,深入研究这些有机染料的结构和光谱特性 ,探讨这些有机染料与生物大分子之间的作用 ,对于筛选性能优良的分子光谱探针 ,设计更加灵敏简便的分子光谱检测器件 ,是现代分析化学十分重要的课题。本文测定了偶氮胂Ⅱ (Ａｒｓｅｎａｚｏ)、铬黑Ｔ(ＥｒｉｏｃｈｒｏｍｅｂｌａｃｋＴ)、铬蓝黑 (ＥｒｉｏｃｈｒｏｍｅｂｌｕｅｂｌａｃｋＲ)等 3种化合物的荧光光谱 ,并用量子化学半经验方法ＡＭ1和ＰＭ3对3种化合物分子进行包括构型…     

亮黄共振光散射法测定微量蛋白质

基于蛋白质对生物染色剂亮黄的共振光散射的增强效应,拟定了一种测定蛋白质的共振光散射法。在pH 2.5的Britton Robison缓冲溶液中,亮黄在510 nm处的共振光散射增强与蛋白质浓度呈线性关系。对牛血清白蛋白,线性范围为0.25~11.5 mg·L-1,检出限48μg·L-1。方法用于合成样品和人尿样品中蛋白质的测定,结果满意。     

DNA-inspired hierarchical polymer design: electrostatics and hydrogen bonding in concert

Nucleic acids and proteins, two of nature's biopolymers, assemble into complex structures to achieve desired biological functions and inspire the design of synthetic macromolecules containing a wide variety of noncovalent interactions including electrostatics and hydrogen bonding. Researchers have incorporated DNA nucleobases into a wide variety of synthetic monomers/polymers achieving stimuli-responsive materials, supramolecular assemblies, and well-controlled macromolecules. Recently, scientists utilized both electrostatics and complementary hydrogen bonding to orthogonally functionalize a polymer backbone through supramolecular assembly. Diverse macromolecules with noncovalent interactions will create materials with properties necessary for biomedical applications.     

High-performance liquid chromatography packing materials for the analysis of small molecules in biological matrices by direct injection

The increasing demand on high-performance liquid chromatography to resolve mixtures of closely related components in complex biological matrices in less time with higher precision has led to the development of a variety of new high-performance liquid chromatography columns, which eliminate the need for sample preparation. These packings isolate small molecules from biological macromolecules on direct sample injection by exerting two separation mechanisms. They allow elution of all sample macromolecules with high recovery in one peak at the extraparticulate void, because of size-exclusion interactions with hydrophilic outer particulate surfaces. Simultaneously, these packings allow permeation and partitioning of small molecules on bonded-phases which are protected from contamination by macromolecules. The names given to these new packings include "internal surface reversed-phase", "shielded hydrophobic phase", "semipermeable surface", "dual zone material" and "mixed-functional phases". The fundamental principles behind each of the design concepts are reviewed, and applications are cited.     

Non-covalent polyvalent ligands by self-assembly of small glycodendrimers: a novel concept for the inhibition of polyvalent carbohydrate-protein interactions in vitro and in vivo

Polyvalent carbohydrate-protein interactions occur frequently in biology, particularly in recognition events on cellular membranes. Collectively, they can be much stronger than corresponding monovalent interactions, rendering it difficult to control them with individual small molecules. Artificial macromolecules have been used as polyvalent ligands to inhibit polyvalent processes; however, both reproducible synthesis and appropriate characterization of such complex entities is demanding. Herein, we present an alternative concept avoiding conventional macromolecules. Small glycodendrimers which fulfill single molecule entity criteria self-assemble to form non-covalent nanoparticles. These particles-not the individual molecules-function as polyvalent ligands, efficiently inhibiting polyvalent processes both in vitro and in vivo. The synthesis and characterization of these glycodendrimers is described in detail. Furthermore, we report on the characterization of the non-covalent nanoparticles formed and on their biological evaluation.     

Non‐Covalent Polyvalent Ligands by Self‐Assembly of Small Glycodendrimers: A Novel Concept for the Inhibition of Polyvalent Carbohydrate–Protein Interactions In Vitro and In Vivo

Polyvalent carbohydrate–protein interactions occur frequently in biology, particularly in recognition events on cellular membranes. Collectively, they can be much stronger than corresponding monovalent interactions, rendering it difficult to control them with individual small molecules. Artificial macromolecules have been used as polyvalent ligands to inhibit polyvalent processes; however, both reproducible synthesis and appropriate characterization of such complex entities is demanding. Herein, we present an alternative concept avoiding conventional macromolecules. Small glycodendrimers which fulfill single molecule entity criteria self‐assemble to form non‐covalent nanoparticles. These particles—not the individual molecules—function as polyvalent ligands, efficiently inhibiting polyvalent processes both in vitro and in vivo. The synthesis and characterization of these glycodendrimers is described in detail. Furthermore, we report on the characterization of the non‐covalent nanoparticles formed and on their biological evaluation.     

Selective stabilization of self-assembled hydrogen-bonded molecular capsules through π-π interactions

Subtle noncovalent forces such as π-π interactions play an import role in the folding of biological macromolecules such as DNA and proteins. We describe here a system where such interactions on the outside of a molecular capsule trigger a selective change of its structure as a self-assembled receptor.     

Method for macromolecular colocalization using atomic recombination in dynamic SIMS

Localizing two or more components of assemblies in biological systems requires both continued development of fluorescence techniques and invention of entirely new techniques. Candidates for the latter include dynamic secondary ion mass spectrometry (D-SIMS). The latest generation of D-SIMS, the Cameca NanoSIMS 50, permits the localization of specific, isotopically labeled molecules and macromolecules in sections of biological material with a resolution in the tens of nanometers and with a sensitivity approaching in principle that of a single protein. Here we use two different systems, crystals of glycine and mixtures of proteins, to show that the formation of recombinant CN secondary ions under Cs bombardment can be exploited to create a new colocalization technique. We show experimentally that the formation of the recombinant (13)C(15)N secondary ion between (13)C- and (15)N-labeled macromolecules is indeed an indicator of the distance between the interacting macromolecules and on their shape. We build up a convolution model of the mixing-recombination process in D-SIMS that allows quantitative interpretations of the distance-dependent formation of the recombinant CN. Our results show that macromolecules can be colocalized if they are within 2 nm of one another. We discuss the potential advantages of this new technique for biological applications.     

云母晶面上准外延生长的胶原蛋白纳米线阵列的性质研究

胶原蛋白纤维与同样具有周期性结构表面的羟磷灰石等矿物质晶体能够通过多种作用在介观尺度上相互识别,从而有效地进行规范骨骼、牙齿等器官生长的生物矿化.在本研究中,反相利用生物矿化原理,开发出一种与热壁外延生长(Hot wall epitaxy)技术效果相似的生物大分子纳米线阵列的简单制备技术,成功地将5～10 μg/mL的天然I型鼠尾胶原蛋白单体溶液在云母晶体的(001)晶面上培育成取向单一且长程有序的胶原蛋白纳米线阵列.原子力显微镜实验结果表明,纳米线阵列随胶原蛋白单体浓度增大而变得致密,但是单条纳米线的宽度与高度较为稳定,分别约为60.0和1.5 nm.有纳米线覆盖的云母晶面亲水性好,晶面接触角由25.8°降为9.5°.基于电子背面散射衍射和透射电子显微镜的分析表征结果,纳米线的取向应沿云母[110]方向, 更详实地验证了胶原蛋白纳米线的准外延生长机理.     

Zwitterionic Moieties from the Huisgen Reaction: A Case Study with Amphiphilic Dendritic Assemblies

Supramolecular nano‐assemblies that reduce nonspecific interactions with biological macromolecules, such as proteins, are of great importance for various biological applications. Recently, zwitterionic materials have been shown to reduce nonspecific interactions with biomolecules, owing both to their charge neutrality and their ability to form a strong hydration layer around zwitterions via electrostatic interactions. Here, new triazole‐based zwitterionic moieties are presented that are incorporated as the hydrophilic functionalities in facially amphiphilic dendrons. The amphiphilic zwitterionic dendrons spontaneously self‐assemble in aqueous solutions forming micelle‐type aggregates, which were confirmed by DLS, TEM, and fluorescence techniques. The structural and functional characteristics of the zwitterionic dendrons are also compared with the corresponding charge‐neutral PEG‐based dendrons and anionic carboxylate‐based dendrons. Surface‐charge measurements, temperature sensitivity and evaluation of interactions of these assemblies with proteins form the bases for these comparisons.     

Electrostatic field-adapted molecular fractionation with conjugated caps for energy calculations of charged biomolecules

An electrostatic field-adapted molecular fractionation with conjugated caps (EFA-MFCC) approach is implemented for treating macromolecules with several charge centers. The molecular fragmentation is performed in an "electrostatic field," which is described by putting point charges on charge centers, directly affecting the Hamiltonians of both fragments and conjugated caps. So the present method does not need truncation during the calculation of electrostatic interactions. Our test calculations on a series of charged model systems and biological macromolecules using the HF and B3LYP methods have demonstrated that this approach is capable of describing the electronic structure with accuracy comparable to other fragment-based methods. The EFA-MFCC approach is an alternative way for predicting the total energies of charged macromolecules with acyclic, loop, and intersectional loop structures and interaction energies between two molecules.