五氯酚对人胎盘碱性磷酸酶抑制的研究

监测人胎盘碱性磷酸酶在不同浓度五氯酚溶液中活力与荧光光谱的变化;测定五氯酚对其抑制的类型及pH对其抑制的影响.结果显示：低浓度的五氯酚(＜1.0 mmol/L)使酶活力及其荧光强度迅速下降,发射峰位明显红移;继续提高五氯酚浓度,其活力与荧光强度逐渐下降,发射峰位仍在红移;五氯酚浓度为5.0 mmol/L时,荧光淬灭,但酶仍保留51.4%的活力;五氯酚浓度高达10.0 mmol/L时,酶剩余活力为30.0%.进一步实验表明五氯酚使L-色氨酸的荧光强度降低,并伴有发射峰位的红移,当五氯酚浓度为5.0 mmol/L时,L-色氨酸的荧光淬灭.五氯酚是人胎盘碱性磷酸酶的反竞争性抑制剂,其抑制常数(K_i)为3.86 mmol/L.其抑制也受溶液pH影响,pH＜7.5时,对酶无抑制;pH为7.5～10.5时,随pH的增加,抑制作用逐渐增强.提示五氯酚能够进入酶分子内部使其荧光淬灭,并抑制了酶活力;表明酶活力抑制与五氯酚的解离状态有关.     

人肌和兔肌肌酸激酶在低pH条件下再折叠的研究

利用蛋白质内源荧光和远紫外ＣＤ光谱研究了在低ｐＨ条件下人肌肌酸激酶和兔肌肌酸激酶的构象状态。结果表明,在低离子强度下,随着酸的加入人肌和兔肌肌酸激酶去折叠,至ｐＨ２．０附近几乎都达到充分去折叠。它们的色氨酸荧光发射峰位红移至３５０ｎｍ,这表明了内埋的色氨酸残基已经完全暴露到极性溶剂之中,它们的远紫外ＣＤ光谱表明二级结构也遭破坏,但是仍保留有相当部分的二级结构。而且在高离子强度低ｐＨ条件下由Ｇｏｔｏ等人首先发现的中间体构象状态（融球结构状态）在我们的实验中也被观察到了。它具有与天然酶类似的二级结构。色氨酸荧光发射光谱表明与天然酶类似,它的最大发射峰位也为３３５ｎｍ表明了蛋白质已经完全折叠,然而其荧光强度远低于天然酶,表明这种结构状态具有较大运动性而导致荧光的动态淬灭。上述结果支持了Ｇｏｔｏ等人的发现,说明了融球中间体结构可能是蛋白质折叠过程需经历的一个中间态。     

各种因子对固定化烟草核酮糖1,5-二磷酸羧化酶/加氧酶解离作用的影响

pH,温度、离子强度及效应剂等对固定化烟草RuBP羧化酶在2.5mol/L尿素处理下的解离作用有各种不同的影响。在pH6.0时,仅小亚基从大亚基核(L_8)解离,当pH为中性偏碱时,大亚基核也解离。低温和低离子强度均促进酶的解离,而温度和离子强度对大亚基之间的解离的影响显著大于对大、小亚基之间的影响。这表明酶的亚基之间存在着不同的极性和疏水作用,而大亚基之间的疏水作用比大、小亚基之间的强。6-PG对大、小亚基之间解离的抑制作用表明大亚基上的催化位置与小亚基之间有一定的密切关系。     

盐离子作用下组蛋白变体H2A.Z增强核小体结构的稳定性

真核生物染色质的基本结构组成单元是核小体,基因组DNA被压缩在染色质中,核小体的存在通常会抑制转录、复制、修复和重组等发生在DNA模板上的生物学过程。组蛋白变体H2A.Z可以调控染色质结构进而影响基因的转录过程,但其详细的调控机制仍未研究清楚。为了比较含有组蛋白变体H2A.Z的核小体和常规核小体在盐离子作用下的稳定性差异,本文采用Förster共振能量转移的方法检测氯化钠、氯化钾、氯化锰、氯化钙、氯化镁等离子对核小体的解聚影响。实验对Widom 601 DNA序列进行双荧光Cy3和Cy5标记,通过荧光信号值的变化来反映核小体的解聚变化。Förster共振能量转移检测结果显示:在氯化钠、氯化钾、氯化锰、氯化钙和氯化镁作用下,含有组蛋白变体H2A.Z的核小体解聚速度相比于常规核小体要慢,且氯化钙、氯化锰和氯化镁的影响更明显。电泳分析结果表明,在75℃条件下含有组蛋白变体H2A.Z的核小体的解聚速率明显低于常规核小体。采用荧光热漂移检测(fluorescence thermal shift analysis , FTS)进一步分析含有组蛋白变体H2A.Z核小体的稳定性,发现两类核小体的荧光信号均呈现2个明显的增长期,含有组蛋白变体H2A.Z核小体的第1个荧光信号增速期所对应的温度明显高于常规核小体,表明核小体中H2A.Z/H2B二聚体的解聚变性温度要高于常规的H2A/H2B二聚体,含有组蛋白变体H2A.Z核小体的热稳定性高。研究结果均表明,含有组蛋白变体H2A.Z的核小体的结构比常规核小体的结构稳定。     

荧光共振能量转移效率的实时定量测量

荧光共振能量转移（FRET）广泛用于研究分子间的距离及其相互作用,与荧光显微镜结合,可定量获取有关生物活体内蛋白质、脂类、DNA和RNA的时空信息。随着绿色荧光蛋白（GFP）的发展,FRET荧光显微镜有可能实时测量活体细胞内分子的动态性质。提出了一种定量测量FRET效率以及供体与受体间距离的简单方法,仅需使用一组滤光片和测量一个比值,利用供体和受体的发射谱肖除光谱间的串扰。该方法简单快速,可实时定量测量FRET的效率和供体与受体间的距离,尤其适用于基于GFP的供体－受体对。     

人肌肌酸激酶在SDS溶液中失活与构象变化的比较研究

应用紫外差吸收光谱、荧光发射光谱、ＣＤ先谱等监测手段,研究了ＳＤＳ溶液滴定人肌肌酸激酶时的构象与活力变化的关系。结果表明酶的活力丧失先于以紫外差吸收先谱、荧先发射谱和巯基暴露数目所监测到的构象变化。ＳＤＳ滴定时引起的酶的荧光发射光谱的变化在低滴定度阶段随着ＳＤＳ滴定量的增加,荧光强度下降,发射峰位红移,当ＳＤＳ浓度达到２．１ｍｍｏｌ／Ｌ时,荧光强度增大,继续增加ＳＤＳ滴定量,荧光强度又降低,发射峰位红移直至终态。紫外差吸收光谱随着ＳＤＳ溶液的加入,２８１ｎｍ．２８７ｎｍ和２９２ｎｍ的负差吸收峰增大。ＣＤ光谱结果表明在本实验所用的ＳＤＳ浓度范围内,ＳＤＳ对人肌肌酸激酶的二级结构几乎没有影响。上述结果支持了酶的活性部位构象柔性的观点。     

基于石英表面荧光性吖啶橙纳米自组装膜测定土壤中铬（Ⅵ）

研究了在石英表面上吖啶橙纳米金自组装膜的制备及其荧光特性,发现所构建的吖啶橙自组装膜具有强的荧光信号。在pH为7．60的KH2PO4^-NaOH缓冲溶液中,Cr^6＋离子能使吖啶橙自组装膜在λ=528nm处的荧光增强,增强的强度（△F）与Cr^6＋的浓度在一定浓度范围内呈线性关系,该方法的线性范围为0．00～36．00×10^-8μg／mL,检出限（DL）为2．47×10^-11μg／mL,将构建的吖啶橙自组装膜测定土壤中的Cr^6＋,回收率为94．91％～96．24％,相对标准偏差（RSD）为0．31％～0．70％。由此可知吖啶橙自组装膜适用于超痕量铬离子的界面荧光测定。实验结果表明该膜具有较好的可逆再生性能,自组装膜法检测限低、灵敏度高、反应速度快、稳定性好。     

姜黄素与DNA相互作用的电化学研究

目的:利用电化学方法研究姜黄素与小牛胸腺双链DNA的相互作用,考察离子强度对姜黄素与DNA相互作用方式的影响.探讨姜黄素与DNA相互作用的机理.方法:在5 mmol·L-1 Tris-HCl(pH 7.0)溶液中,在不同离子强度下,考察姜黄素在ds-DNA修饰玻碳电极上的姜黄素氧化峰电流及式电位的变化情况.结果:在低离子强度下,姜黄素主要通过静电作用与DNA结合.高离子强度下,姜黄素主要通过嵌入作用与DNA结合.结论:电化学方法是研究姜黄素与电极表面固定化小牛胸腺双链DNA的相互作用的一种简单的方法.     

蓝藻藻胆体与类囊体膜之间光能传递的研究

从嗜热蓝藻优雅粘囊藻（Ｍｙｘｏｓａｒｃｉｎａ ｃｏｎｃｉｎｎａ Ｐｒｉｎｔｚ．）中分离到具有放氧活性的藻胆体－类囊体膜复合物。它的吸收峰位于６８０、６２８、４９０、４３８和４２０ ｎｍ ,在低离子强度（０．１～０．４ ｍ ｏｌ／Ｌ）磷酸缓冲液中的６６４ ｎｍ 荧光发射峰随离子强度的降低而升高,７１８ ｎｍ 荧光发射峰与此相反（７７ 。Ｋ,Ｅｘ＝ ５８０ ｎｍ ）。当把游离的藻胆体和已解离去藻胆体的类囊体膜在蔗糖磷酸缓冲液中重组时,随时间延长（０～６０ ｍ ｉｎ）,７１８ ｎｍ 荧光发射峰逐渐升高,６８５ ｎｍ 荧光发射峰逐渐下降（７７ 。Ｋ,Ｅｘ ＝ ５８０ ｎｍ ）,表明藻胆体与类囊体之间的解离和结合对光能传递的影响     

对果蝇S期细胞内组蛋白基因的原位定量分析

利用微型计算机控制的荧光显微镜、荧光强度检测仪和图像记录装置并结合荧光原位杂交法对果蝇细胞核内组蛋白基因的复制时期进行了研究,从而建立了一套细胞内直接定量分析的方法。根据果蝇胚胎原代培养细胞核的DAPI染色强度确定处于S期的细胞。用杂交信号的荧光强度与细胞核荧光强度的相关关系来反映组蛋白基因的复制时期。结果表明果蝇组蛋白基因的复制是在DNA合成早期进行的。这套方法至少可直接在细胞上对每套基因组100以上拷贝数的熏复DNA序列进行有效的定量分析。     

The nature of forces stabilizing nucleosome structure. Dissociation of histone octamers from DNA

We have used the measurements of the histone fluorescence parameters to study the influence of the ionic strength on histone-DNA and histone-histone interactions in reconstructed nucleosomes. The ionic strength increase lead to the two-stage nucleosome dissociation. The dimer H2A-H2B dissociates at the first stage and the tetramer (H3-H4)2 at the second one. The dimer H2A-H2B dissociation from nucleosome is a two-stage process also. The ionic bonds between (H2A-H2B) histone dimer and DNA break at first and then the dissociation of dimer from histone tetramer (H3-H4)2 occurs. According to the proposed model the dissociation accompanying a nucleosome "swelling" and an increase of DNA curvature radius. It was shown that the energy of electrostatic interactions between histone dimer and DNA is sufficiently less than the energy of dimer-tetramer interaction. We propose that the nucleosome DNA ends interact with the dimer and tetramer simultaneously. The calculated number (approximately 30 divided by 40) of ionic bonds between DNA and histone octamer globular part practically coincides with the number of exposed cationic groups on the surface of octamer globular head. On this basis we have assumed that the spatial distribution of these groups is precisely determined, which explains the high evolutionary conservatism of the histone primary structure.     

Comparing mono- and divalent DNA groove binding cyanine dyes -- binding geometries, dissociation rates, and fluorescence properties

The unsymmetrical cyanine dyes BOXTO-PRO and BOXTO-MEE were derived from the DNA groove binder BOXTO, by adding a positively charged or a non-ionic hydrophilic tail to BOXTO, respectively. The main objective was to obtain more efficient DNA probes, for instance in electrophoresis and microscopy, by slowing down the dissociation of BOXTO from DNA. The interactions with mixed sequence DNA was studied with fluorescence and absorbance spectroscopy, stopped-flow dissociation and gel electrophoresis. Both the derivatives are groove bound as BOXTO, and have similar fluorescence properties when bound to mixed sequence DNA in free solution. BOXTO-PRO exhibits a slower dissociation than BOXTO from DNA, whereas the dissociation rate for BOXTO-MEE is faster and, unexpectedly independent of the ionic strength. During gel electrophoresis both BOXTO-PRO and BOXTO-MEE exhibit a faster dissociation rate than BOXTO. Still, BOXTO-PRO seems to be a good alternative as DNA probe, especially for applications in free solution where the dissociation is slower than for the corresponding intercalator TOPRO-1.     

Reversibility of Structural Rearrangements in Mononucleosomes Induced by Ionic Strength

Using fluorescence microscopy of single particles with Förster resonance energy transfer recording, structural rearrangements that occurred in nucleosomes formed on the 603 DNA template at high ionic strength were studied. Within the range of 0.7–1.3 M KCl, large-scale changes occurred in the nucleosome structure, including the formation of at least two states differing in the degree of DNA unwrapping from the histone octamer and affecting from 13 to 35 and more pairs of nucleotides. Content of the fraction of nucleosomes with modified structure varied from 60% at 0.7 M KCl to 100% at 1.3 M KCl. Preservation of the association between core histones and DNA in the new conformational states ensured reversibility of structural changes when KCl concentration was reduced to a physiological level. Reversibility was ~100% upon transition from 0.7 M to 0.15 KCl and decreased to ~50% upon transition from 1.3 M to 0.15 KCl.     

Histone Hl-DNA interaction. Influence of phosphorylation on the interaction of histone Hl with linear fragmented DNA.

By measuring the fluorescence polarization of fluorescent histone H1 derivatives complexed with DNA, binding of the histone to DNA was studied as a function of ionic strength in the solution prior to and after the H1 phosphorylation on Ser-37 residue. Fluorescent labels were covalently linked either specifically to Tyr-72 residues or unspecifically to lysine residues in the H1 polypeptide chain. The values of the corresponding rotational relaxation times showed that at low ionic strength all the segments of the H1 molecule were immobilized on binding to DNA. The gradual increasing NaC1 concentration in the solution of H1-DNA complex was accompanied at first by additional retardation of the histone mobility in the complex, and then by progressive release of histone H1 from from the complex which was completed at 0.5-0.6 M NaC1 irrespective of phosphorylation. tat the same time the phosphorylation of histone H1 led to removal of the central and, presumably, N-terminal regions of H1 from DNA.     

Labelling of histone H5 and its interaction with DNA. 2. Cooperative binding of histone H5 to DNA as probed by steady-state fluorescence and diffusion-enhanced energy transfer

The interaction of histone H5 labelled with fluorescein isothiocyanate (FITC) with DNA has been studied by fluorescence titration, and diffusion-enhanced fluorescence energy transfer (DEFET) measurements with Tb(III) lanthanide chelates as donors. Analysis of the binding data by the model of Schwarz and Watanabe (J.Mol.Biol. 163, 467-484 (1983)) yielded a mean stoichiometry of 60 nucleotides per H5 molecule, independently of ionic strength, in the range of 3 to 300 mM NaCl, at very low DNA concentration (6 microM in mononucleotide). It ensues an approximate electroneutrality of the saturated complexes. Histone H5 molecules appeared to be clustered along the DNA lattice in clusters containing on average 3 to 4 H5 molecules separated by about 79 base pairs, at mid-saturation of the binding sites. The interaction process was found highly cooperative but the cooperativity parameter was also insensitive to ionic strength in the above range. DEFET experiments indicated an important decrease of accessibility of the FITC label to the TbHED3A and TbEDTA- chelates with ionic strength in the 0 to 100 mM NaCl range. In the presence of DNA, H5 appears already folded at low ionic strength so that the FITC probe is also not accessible to the donor chelate. The present study constitutes an indispensable preliminary step to further studies on the localization of histone H5 in condensed chromatin structures.     

A new fluorescence resonance energy transfer approach demonstrates that the histone variant H2AZ stabilizes the histone octamer within the nucleosome

Nucleosomes are highly dynamic macromolecular complexes that are assembled and disassembled in a modular fashion. One important way in which this dynamic process can be modulated is by the replacement of major histones with their variants, thereby affecting nucleosome structure and function. Here we use fluorescence resonance energy transfer between fluorophores attached to various defined locations within the nucleosome to dissect and compare the structural transitions of a H2A.Z containing and a canonical nucleosome in response to increasing ionic strength. We show that the peripheral regions of the DNA dissociate from the surface of the histone octamer at relatively low ionic strength, under conditions where the dimer-tetramer interaction remains unaffected. At around 550 mm NaCl, the (H2A-H2B) dimer dissociates from the (H3-H4)(2) tetramer-DNA complex. Significantly, this latter transition is stabilized in nucleosomes that have been reconstituted with the essential histone variant H2A.Z. Our studies firmly establish fluorescence resonance energy transfer as a valid method to study nucleosome stability, and shed new light on the biological function of H2A.Z.     

Index to Subjects,Volume 5

Abstract

The interaction of histone H5 labelled with fluorescein isothiocyanate (FITC) with DNA has been studied by fluorescence titration, and diffusion-enhanced fluorescence energy transfer (DEFET) measurements with Tb(III) lanthanide chelates as donors.

Analysis of the binding data by the model of Schwarz and Watanabe (J. Mol. Biol. 163, 467-484 (1983)) yielded a mean stoichiometry of 60 nucleotides per H5 molecule, independently of ionic strength, in the range of 3 to 300 mM NaCl, at very low DNA concentration (6 μM in mononucleotide). It ensues an approximate electroneutrality of the saturated complexes. Histone H5 molecules appeared to be clustered along the DNA lattice in clusters containing on average 3 to 4 H5 molecules separated by about 79 base pairs, at mid-saturation of the binding sites. The interaction process was found highly cooperative but the cooperativity parameter was also insensitive to ionic strength in the above range.

DEFET experiments indicated an important decrease of accessibility of the FITC label to the TbHED3A° and TbEDTA^? chelates with ionic strength in the 0 to 100 mM NaCl range. In the presence of DNA, H5 appears already folded at low ionic strength so that the FITC probe is also not accessible to the donor chelate. The present study constitutes an indispensable preliminary step to further studies on the localization of histone H5 in condensed chromatin structures.     

Allogeneic and autogenous transplantations of MSCs in treatment of the physeal bone bridge in rabbits

Background

Serum and high ionic strength solutions constitute important barriers to cationic lipid-mediated intravenous gene transfer. Preparation or incubation of lipoplexes in these media results in alteration of their biophysical properties, generally leading to a decrease in transfection efficiency. Accurate quantification of these changes is of paramount importance for the success of lipoplex-mediated gene transfer in vivo.

Results

In this work, a novel time-resolved fluorescence resonance energy transfer (FRET) methodology was used to monitor lipoplex structural changes in the presence of phosphate-buffered saline solution (PBS) and fetal bovine serum. 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/pDNA lipoplexes, prepared in high and low ionic strength solutions, are compared in terms of complexation efficiency. Lipoplexes prepared in PBS show lower complexation efficiencies when compared to lipoplexes prepared in low ionic strength buffer followed by addition of PBS. Moreover, when serum is added to the referred formulation no significant effect on the complexation efficiency was observed. In physiological saline solutions and serum, a multilamellar arrangement of the lipoplexes is maintained, with reduced spacing distances between the FRET probes, relative to those in low ionic strength medium.

Conclusion

The time-resolved FRET methodology described in this work allowed us to monitor stability and characterize quantitatively the structural changes (variations in interchromophore spacing distances and complexation efficiencies) undergone by DOTAP/DNA complexes in high ionic strength solutions and in presence of serum, as well as to determine the minimum amount of potentially cytotoxic cationic lipid necessary for complete coverage of DNA. This constitutes essential information regarding thoughtful design of future in vivo applications.     

Histone H1: Ultracentrifugation studies of the effects of ionic strength and denaturants on the solution conformation

The conformation of histone H1 has been examined under native and denaturing conditions in the absence of DNA or chromatin. Sedimentation coefficients were determined for Histone H1 in 0.1 m KCl and in 6 m guanidine hydrochloride solutions at pH 7.4. The influence of ionic strength on the conformation of histone H1 has been determined by measurement of the sedimentation coefficient in tetramethylammonium chloride solutions of up to 2.5 m and extrapolated to infinite ionic strength. Results from these experiments suggest that the native conformation of histone H1 is very asymmetric in shape. The molecule is best described as a prolate ellipsoid with axes of 312 Å (2a) and 16 Å (2b) in low ionic strength media and also as a prolate ellipsoid with axes of 202 Å (2a) and 20 Å (2b) at high ionic strength or when associated with polyanions, e.g., DNA. Denaturation of histone H1 by guanidine hydrochloride was found to be completely reversible. In 6 m guanidine hydrochloride, the H1 molecule collapses to a sphere but the original extended conformation of the protein is readily restored on dialysis. This suggests rigid conformational requirements for the H1 molecule as incorporated into chromatin. The shape and dimensions for the H1 molecule at high ionic strength are not sufficiently conclusive to locate H1 in the chromatin structure. It is proposed, however, that viable models for chromatin architecture must be consistent with the histone H1 solution dimensions obtained here.     

Fluorescence resonance energy transfer analysis of the structure of the four-way DNA junction.

We have carried out fluorescence resonance energy transfer (FRET) measurements on four-way DNA junctions in order to analyze the global structure and its dependence on the concentration of several types of ions. A knowledge of the structure and its sensitivity to the solution environment is important for a full understanding of recombination events in DNA. The stereochemical arrangement of the four DNA helices that make up the four-way junction was established by a global comparison of the efficiency of FRET between donor and acceptor molecules attached pairwise in all possible permutations to the 5' termini of the duplex arms of the four-way structure. The conclusions are based upon a comparison between a series of many identical DNA molecules which have been labeled on different positions, rather than a determination of a few absolute distances. Details of the FRET analysis are presented; features of the analysis with particular relevance to DNA structures are emphasized. Three methods were employed to determine the efficiency of FRET: (1) enhancement of the acceptor fluorescence, (2) decrease of the donor quantum yield, and (3) shortening of the donor fluorescence lifetime. The FRET results indicate that the arms of the four-way junction are arranged in an antiparallel stacked X-structure when salt is added to the solution. The ion-related conformational change upon addition of salt to a solution originally at low ionic strength progresses in a continuous noncooperative manner as the ionic strength of the solution increases. The mode of ion interaction at the strand exchange site of the junction is discussed.