新型含芘荧光探针分子与蛋白质相互作用的光谱研究

利用荧光发射光谱和紫外-可见吸收光谱,研究了新型含芘荧光探针分子芘丁酰谷氨酸(PLE)和芘甲酰谷氨酸(PYE)与溶菌酶(Lyso)和牛血清白蛋白(BSA)的相互作用,分析了结合过程和机理。结果显示:不同间隔链长度的探针分子在与Lyso作用时,表现的光谱性质差别不大,这主要是由于探针分子结合到了Lyso的表面;而不同间隔链长度的探针分子在与BSA作用过程中,却表现不同的光物理性质,这可能是探针分子作用于BSA空腔时其结合位点或者结合方式的不同引起的。目前的研究对揭示蛋白质分子的识别位点和定位切割具有重要的意义。     

分子吸收光谱在生物大分子研究中的应用

综述了近年来分子吸收光谱法在生物大分子结构研究中的应用,重点介绍了利用分子吸收光谱法研究蛋白质、核酸结构及其与小分子的相互作用。分子吸收光谱法因其简便、快速的特点而广泛应用于生物大分子结构的测定。紫外光谱法常用于从分子水平上研究小分子如抗癌药物与核酸作用的机理,并应用于小分子作为光谱探针测定蛋白质、核酸构象的变化。傅里叶变换红外光谱法主要应用于蛋白质二级结构的测定,运用傅里叶自卷积将重叠的酰胺带进行处理,获取蛋白质二级结构的含量信息,从而使傅里叶变换红外光谱法成为揭示蛋白质构象变化的重要工具。     

超快多维红外光谱学:分子结构的时空分辨

利用时间分辨的超快二维红外(2DIR)光谱、稳态一维红外光谱等手段,本文探讨了从溶液中的小分子体系到生物大分子体系的超快振动特性及所反映的分子结构动力学过程。研究了五羰基溴化锰在四氯化碳中的时间分辨2DIR光谱,发现了分子对称性增强的13 CO配体的红外吸收信号,并利用2DIR对角峰和非对角峰表征了其与12 CO配体振动态的相互作用和分子内能量传递过程;实现了钌羰基配合物在光敏黄色蛋白突变体M100A上的定点标记,并研究了该外源标记物的羰基配体的结构动力学,发现了探针分子的振动光谱指纹对其所处的空间位置的敏感性,还发现该探针分子的振动扩散过程对水相中的蛋白质结构涨落具有灵敏性。     

染料探针在生物分析中的应用研究进展

评述近年来染料探针在生物分析中用于蛋白质的定量测定及蛋白质、核酸与小分子相互作用等方面的研究进展。列出了一些重要体系以及分析特征。     

红外光谱的检测灵敏度和选择性研究进展

红外光谱和二维红外光谱已被广泛应用于各种多肽和蛋白质的结构和动力学的研究中。但在推广到生物大分子体系的研究时却面临着很多困难,主要原因是目前红外光谱的选择性不够好和灵敏度不够高。因此,在多数情况下人们无法通过生物分子自身的振动模式来获得位点选择的蛋白质结构和动力学信息。为了解决这些困难,我们发展了非天然氨基酸红外光谱探针并把它们结合到蛋白质中去进行位点选择的光谱研究,我们也发展了提高二维红外光谱检测灵敏度的实验方法。我们深入研究了一些非天然氨基酸红外光谱探针如氰基吲哚及其位置异构体。研究表明,不同氰基吲哚的寿命和对环境的灵敏度都不相同,这意味着氰基吲哚是一类非常有前景的蛋白质红外光谱探针。     

镧系配合物的荧光光谱

对镧系配合物荧光光谱的研究进展进行了评述,并对其光物理、光化学进行了归纳、分类,特别是对目前研究非常活跃的镧系离子荧光探针法在配合物和生物分子体系结构探测中的应用情况进行了总结。     

用光波导光模光谱技术研究DNA-DNA结合蛋白相互作用

探讨了应用光波导光模光谱(Optical waveguide lightmode spectroscopy,OWLS)技术研究DNA-DNA结合蛋白相互作用的可行性和灵敏性。以固定在传感器芯片表面的DNA探针为捕捉分子,溶液中同时含有探针结合序列和NF—κB结合位点序列的寡核苷酸与NF-κB亚单位p50同源二聚体形成的DNA-蛋白质复合物为检测分子,用光波导光模光谱检测技术建立非标记DNA-DNA结合蛋白相互作用检测研究体系。利用这一体系对不同样品中NF-κB p50浓度和具不同NF-κB结合位点序列的寡核苷酸与NF-κB p50亲合和力进行检测。样品中低至0．33 nmol/1的NF-κB p50被光波导光模光谱检测出,不同的NF-κB结合序列与NF-κB p50亲合力有显著的差异。研究发现,光波导光模光谱技术可以用于DNA-DNA结合蛋白相互作用研究,所建立的非标记检测研究体系可以进行样品中结合蛋白含量高灵敏检测和核酸序列与结合蛋白的亲合力的检测研究。     

SERS传感器间接检测蛋白质的研究进展

蛋白质在生命体中发挥着重要的作用,蛋白质的检测对于了解生命体系及在生物医学领域都有基础性的应用价值。表面增强拉曼光谱(SERS)技术对研究化学和生物传感应用前景很有优势,是由于其高灵敏度以及很好的选择性,并且已被运用于生物分子检测,特别是在蛋白质检测方面。综述了SERS探针技术用于蛋白质检验的研究现状,如免疫胶体金技术、基于纳米材料探针的SERS研究、基于酶促技术和基于试剂技术的蛋白质研究进展。     

光与生物分子相互作用时间特性的研究

应用辐射与物质相互作用时间特性的研究结论（ＲＭＴ）来研究光与生物分子相互作用。ＲＭＴ对视紫质分子和菌紫质分子的光化学异构化的成功应用说明了ＲＭＴ对光生物学的适用性。此文应用ＲＭＴ从时间的角度比较了ＤＮＡ和含有二硫键蛋白质的ＵＶ损伤和暗修复，定性解释了表征损伤的作用光谱。     

金属阳离子与β-肽骨架相互作用的振动光谱特征

盐溶液中,金属阳离子(如Na+和Mg2+等)与肽分子骨架的相互作用,在酰胺基团的振动光谱中能得到体现。本工作利用红外光谱实验、分子动力学模拟和量化计算方法,以酰胺-Ⅰ带和酰胺-Ⅱ带为探针,研究了上述金属阳离子与?-肽模型分子-氮乙基丙酰胺(N-ethylpropionamide,NEPA)分子骨架的作用。研究表明在盐溶液中,阳离子/阴离子/水分子之间形成复合物。作为阳离子配体的水分子与NEPA中的羰基氧之间存在动态相互作用,从而使得酰胺-Ⅰ带和酰胺-Ⅱ带的光谱发生劈裂。我们也讨论了酰胺NH与水合离子团簇的相互作用。     

Analytical Techniques Used to Detect DNA Binding Modes of Ruthenium(II) Complexes with Extended Phenanthroline Ring

This review describes the analytical techniques used to detect DNA-probes such as Ru(II) complexes with hetero cyclic imidazo phenanthroline (IP) ligands. Studies on drug-DNA interactions are useful biochemical techniques for visualization of DNA both in vitro and in vivo. The interactions of small molecules that binds to DNA are mainly classified into two major classes, one involving covalent binding and another non-covalent binding. Covalent binding in DNA can be irreversible and may leads to inhibition of all DNA processes which subsequently leads to cell death. Usually, covalent interactions leads to permanent changes in the structure of nucleic acids. The non-covalent interaction of molecules with DNA can be due to electrostatic interaction, intercalation and groove binding. These interactions of DNA probes can be explored by various spectroscopic techniques viz. UV–visible, emission, emission quenching spectroscopy, viscosity and thermal denaturation measurements.     

Fiber optic probes for linear and nonlinear Raman applications – Current trends and future development

This review focuses on fiber optic probes for linear and nonlinear Raman spectroscopy, especially for medical applications. It aims at providing an overview over contemporary technology, recent first clinical trials, and helps identifying future developments necessary to bring the emerging technology to clinical end users. After a short introduction to linear and nonlinear Raman spectroscopic modalities, general design considerations will be discussed and compared to common fiber probe setups. Subsequently, examples for medical applications of fiber optic Raman probes will be given concentrating on probes for linear Raman spectroscopy as these devices are technologically more mature compared to their counterparts based on nonlinear Raman spectroscopy. The review also includes a brief summary of first multimodal fiber optic probes and highlights their benefits for clinical applications. Finally, probes are introduced which employ either nonlinear Raman spectroscopy or surface enhanced spectroscopy.     

Binding of an Anti-inflammatory Drug Lornoxicam with Blood Proteins: Insights from Spectroscopic Investigations

The interaction between an anti-inflammatory drug, lornoxicam (LXM) and protein (human serum albumin and bovine serum albumin) was studied by spectroscopic techniques (Fluorescence, synchronous, FT-IR, UV-vis absorption and circular dichroism). The quenching mechanism of fluorescence of the protein by the drug was discussed. Based on the interaction studies carried out at different temperatures by spectrofluorometry, the binding constant and the number of binding sites for drug on protein have been evaluated. The nature of binding force operating between the drug and protein was proposed to be electrostatic and hydrophobic based on thermodynamic parameters. The distance r between the donor (protein) and acceptor (drug) was determined based on the Förster’s theory of non-radiation energy transfer and found to be 2.38 nm and 2.56 nm for LXM-BSA and LXM-HSA respectively. Displacement studies with different site probes revealed that the drug bound to the hydrophobic pocket located in sub domain IIA; that is to say, Trp-214 was near or within the binding site. Circular dichroism data of protein in the presence of drug revealed the decreased α-helicity and hence changes in secondary structure of protein. The effects of some common ions were also investigated.     

Fluorescence methods to study lipid-protein association: The interaction of protein kinase C with lipid-loaded mixed micelles

The interaction of protein kinase C with lipids was studied in a mixed micellar system. Two fluorescence spectroscopic methods are presented with a different but complementary information content. Diffusion monitored by fluorescence correlation spectroscopy provides information on the interaction of the protein with the whole lipid aggregate. Resonance energy transfer from tryptophans to pyrene-labeled lipids monitored by time-correlated single-photon counting supplies information on the interaction of the protein with specific lipid cofactors within the micelle. The results can be extended to postulate new mechanisms for the activation of protein kinase C by the signal transduction cascades in the cell. Both fluorescence spectroscopic methods can be easily applied to other protein systems which interact with lipids.     

共振光散射探针在核酸分析中的应用

核酸是生命现象的物质基础,核酸的分析测定在医学和生物学研究中有重要的意义.共振光散射技术是研究小分子物质与核酸发生作用并形成聚集体后的灵敏探测技术.本综述引用了32篇文献,介绍了近年来共振光散射探针在核酸分析中的研究进展,并评价了各种方法的优劣.     

Complexation of fluoroquinolone antibiotics with human serum albumin: A fluorescence quenching study

Mechanism of interaction and detailed physico-chemical characterization of the binding of four fluoroquinolones: levofloxacin, sparfloxacin, ciprofloxacin HCl and enrofloxacin with human serum albumin has been studied at physiological pH (7.4) using fluorescence spectroscopic technique. The stoichiometry of interaction was found to be 1:1 for all the drugs used. The association constants for the interaction were of the order of 10⁴ in most cases. At low drug:protein ratios, a significant fraction of the added drug was bound. The predominant interactions involved are hydrogen bonding and Van der Waal’s interactions in the case of levofloxacin, hydrophobic interactions in the case of ciprofloxacin hydrochloride and enrofloxacin and hydrogen bonding, hydrophobic and electrostatic interactions in the case of sparfloxacin.The drug binding region did not coincide with that of the hydrophobic probe, 1-anilinonaphthalene-8-sulfonate (ANS). From the displacement of site-specific probes and site-marker drugs, it was concluded that ciprofloxacin hydrochloride is site II-specific while enrofloxacin is a site I-specific drug. Levofloxacin binds at both site I and site II with equal affinity. Sparfloxacin had higher affinity for site II than site I. It is also possible that sparfloxacin binds at the interface between site I and site II. Stern-Volmer analysis of the data showed that the quenching mechanism is predominantly collisional for the binding of ciprofloxacin HCl and enrofloxacin while both static and collisional quenching mechanisms are operative in the case of levofloxacin and sparfloxacin. High magnitude of the rate constant for quenching showed that the process is not entirely diffusion controlled. Circular dichroism (CD) spectroscopic studies showed that the presence of drugs did not cause any major changes in the secondary structure of HSA.     

Fluorescence spectroscopic study to characterize and monitor TEOS based sol–gel process for development of optical biosensors

Development of optical biosensors is an active area of research in the field of medical technology. Sol–gel matrices made from alkoxide silicates, tetraethyl orthosilicate (TEOS) appear to be suitable glassy host matrix for the sensing system. However, the major problem in the TEOS based sol–gel matrices is stability. So it is important to study dopant–matrix interaction as a function of time. In the present study, we report fluorescence emission and excited state lifetime measurements on fluorescent probes entrapped in TEOS sol–gel for monitoring the physico-chemical processes for characterization and monitoring of local environment (pores) of dopant molecule (fluorescent probes) for construction of sensing layer for optical transducer. Different types of fluorescent probes viz., Hoechst 33258 (H258) and pyranine (PY) were used. Sol–gels containing these probes were prepared at pH=6.0 and the physical and spectroscopic parameters were monitored as a function of storage time (days). The emission intensity from entrapped H258 has shown relatively higher extent of decrease during aging. The excited state fluorescence lifetime measurements on these probes depicted single exponential decay component at 5.4 ns (PY) and 3.6 ns (H258) in fresh sol–gels. After a few days of storage the sol–gel containing H258 revealed an additional short decay component whereas no such alteration could be observed with the probe molecule PY. Further confirmation of multicomponents decay was obtained by distribution analysis of lifetime of H258 where an increase in width of mean lifetime was observed with storage whereas no such change was indicated from PY. Thus it appears that H258 is a better probe molecule for characterizing and monitoring local environment of pores in sol–gel.     

Investigation of Novel Quantum Dots/Proteins/Cellulose Bioconjugate Using NSOM and Fluorescence

We investigated the engineered bioconjugate of cadmium selenide core/zinc sulfide shell, (CdSe)ZnS, quantum dots (QDs) with genetically modified proteins using fluorescence spectroscopy, near-field scanning optical microscopy (NSOM) and spectroscopy (NSOS). The protein polymer was allowed to self-assemble to the bacterial microcrystalline cellulose surface through the cellulosic binding domain. Results from the sample containing the QDs/protein/cellulose assemblies suggest that QDs were arrayed along the cellulose surface. The spectroscopic change of spectroscopic properties of the QDs upon bioconjugation, indicating the interaction among the immobilized QDs and between the constructed protein and QDs.     

FTIR用于变性蛋白质二级结构的研究进展

蛋白质是最重要的生命物质之一,有关蛋白质的各类研究也是人们比较感兴趣的课题。蛋白质的二级结构在维持蛋白质生理活性过程中发挥着重要的作用。随着科技的发展和研究方法的改进,对变性蛋白质微观结构的研究也越来越深入。近年来,国内外学者利用各种物理和化学方法对其作了大量的工作,而傅里叶红外光谱法(FTIR)以其独特的优越性,在研究蛋白质二级结构中发挥着不可替代的作用。文章就这方面的研究进行了初步概述,重点介绍了变性蛋白质二级结构的红外光谱学研究内容、谱学特点及其分析方法的研究进展。