磷光寿命法研究变性剂对大肠杆菌碱性磷酸酶构象的影响

色氨酸残基光寿命监测了大肠杆菌碱性磷酸酶在不同变性剂中展开过程的构象 变化．结果表明：不同变性剂加人蛋白质溶液中，色氨酸残基的微环境发生了较大 的变化，磷光发射减弱，寿命缩短，预示了色氨酸残基从刚性的疏水内芯转移到蛋 白质表面；通过Arrthenius关系式获得的热动力学参数如活化能（E_a）、活化熵 （△S°）、活开过程中间态的形成．     

不同介质中大肠杆菌碱性磷酸酶构象变化的研究

利用色氨酸残基作为内源荧光探针的膜模拟剂（各种表面活性剂）和不同变性剂中对大肠杆菌碱性磷酸酶（ＡＰ）的构象变化进行了系统的研究。通过测定在不同变性时间下盐酸胍浓度对荧光强度的影响以及荧光强度随pＨ有规律的变化,进一步证实了该蛋白质变性过程中形成较稳定中间态的结论。     

Lin光探针法研究不同变性剂对大肠杆菌石破天惊生磷酸酶构象的影响

通过监测大肠杆菌碱性磷酸酶（AP）的109位色氨酸（Trp-109）室温Lin光（RTP）的强度与寿命的变化，探讨了变性剂酸、盐酸及EDTA对AP构象变化的影响。结果表明：Lin光强度、Lin寿命与Trp-109所处的微环境刚性化程度有密切的关系。尤其是盐酸胍的加入，AP经历了曲型的3种变化状态：从稳定折叠态到中间态，最后形成展开态。极少量的EDTA加入，导致蛋白质变性、Lin光减弱和Lin光寿命缩短。     

用Lin光寿命变化分析血中游离色氨酸

全血用三氯醋酸去蛋白的上清液中加入不同量色氨酸，测Ｌｉｎ光寿命，以加入色氨酸与Ｌｉｎ光寿命变化回归方程与应用推导的公式ｘ０＝（τｉ－ｂｘｉ）／ｂ，计算血中游离色氨酸为１１．１μｇ／ｍＬ。与文献报道的一致。     

基于时间分辨方法的LicT蛋白荧光动力学特性

使用时间分辨荧光方法,结合紫外吸收光谱和稳态荧光光谱技术,测量了LicT蛋白中色氨酸残基的荧光动力学特性,进而对LicT蛋白质激活前后的局部微环境和结构变化进行了研究。LicT蛋白质的激活态使得有关糖类利用的基因转录过程继续进行,促进机体新陈代谢。通过色氨酸残基的荧光发射和寿命的差异判断出激活型蛋白AC 141和野生型蛋白Q 22不同的结构性质和微环境差异。在此基础上,通过衰减相关光谱(DAS)和时间分辨发射光谱(TRES)阐释了两种蛋白色氨酸残基和溶剂的相互作用,说明了激活型AC 141的比野生型Q 22的结构更加紧密。此外,TRES还说明了蛋白中的色氨酸残基存在连续光谱弛豫过程。各向异性结果则对残基和整个蛋白的构象运动进行了阐述,说明了色氨酸残基在蛋白质体系内有独立的局部运动,且在激活型蛋白中该运动更加强烈。     

人纤维蛋白溶酶原中色氨酸残基的化学修饰

以Ｎ－溴代琥珀酰亚胺为修饰剂，对人纤维蛋白溶酶原（ＨＰｇ）中色氨酸（Ｔｒｐ）残基的分布及其与酶活力的关系进行了研究，发现每个ＨＰｇ分子有１９个Ｔｒｐ残基，５个位于分子表面：有２个是快反应残基，其中１个是活性必需的氨基酸，酶被修饰后其荧光光谱及圆二色谱发生了变化。     

磷光寿命的研究及生物体内色氨酸的测定

研究了磷光寿命的特性及ｐＨ、溶剂极性、碘、铅、钡、铊等重原子对磷光寿命的影响。应用磷光寿命变化法，分析生物样品中色氨酸含量。小鼠体内，血、脑、心、肺、肝、脾、肾中游离色氨酸分别为８．９μｇ／ｍＬ，１７．３，５１．８，２９．１，２９．９，４６，６８．８μｇ／ｇ。其中，组织血脑中色氨酸含量与文献报道一致。     

家蝇幼虫抗菌肽MDL-1的荧光特性分析

本文研究了家蝇幼虫抗菌肽MDL-1的荧光光谱和淬灭剂对内源性荧光的影响。家蝇幼虫抗菌肽MDL-1在激发波长280 nm时,其荧光光谱为酪氨酸（Tyr）残基和色氨酸（Trp）残基共同提供。结果表明,KI不能淬灭抗菌肽MDL-1的Trp残基的荧光,而丙烯酰胺（Acr）能淬灭几乎所有的Trp残基的荧光（f-0．92）;这说明,Trp残基不是位于抗菌肽分子的表面,而是位于分子的内部。     

溶液中溶菌酶的荧光光谱研究

本文采用荧光分析法(荧光光谱、荧光偏振度)结合内源荧光探针色氨酸残基对溶菌酶及其在不同条件下的构象变化进行了研究。结果表明,利用荧光光谱可对溶菌酶溶液构象进行有效的分析,能够较直观地表征色氨酸残基在溶菌酶分子中的微环境及其在不同条件下构象的变化,得出了一些较有价值的结果。     

用燐光寿命变化测定碘离子与燐光体反应动力学参数

利用I-促进系间窜越，加快发射过程，缩短磷光寿命的特点，研究I-量改变与色氨酸，色胺燐光寿命关系，分别为：τ=3．98e－0.0658x,与τ=3．5e－0.0899x,应用静态分析，推导Stern－Volmer式，计算平衡常数Ka与Ki，分别为：0．109与0．200mmol；0．027与0．051smmol－1。利用本法分析了酪氨酸，苯丙氨酸，L-色氨酸，牛血清白蛋白，葡糖苷酸酶，链霉蛋白酶，硫酸酯酶，顺-雄（甾）酮．吲满氨酯，催醒安，阿托品等的动力学参数。     

Study on Conformation of Escherichia Coli Alkaline Phosphatase by Intrinsic tryptophan Room Temperature Phosphorescence Probe

Room temperature phosphorescence(RTP) probe is a powerful tool for studying the kinetics and relationship between the conformation change and physiological function of protein because some slow kinetic processes of protein in solution just occur the same time scales as phosphorescence lifetime and it susceptibly indicates microenvironment properties. RTP of Escherichia coli alkaline phosphatase(AP) comes mainly from tryptophan residues (Trp-109). It is well known that the deeper Trp-109 is embedded, The stronger phosphorescence emitting is, and the longer its lifetime is. The current work is a preliminary study of the conformation changes of AP, the local microenvironment of Trp-109 and the quenching kinetic natures in the unfolded processes of AP in the presence of different denaturants.     

Study on Escherichia coli alkaline phosphatase conformation by phosphorimetry in the presence of denaturant

The influence of different denaturants on the phosphorescence spectrum and lifetime decay of Escherichia coli alkaline phosphatase (AP) was investigated. Phosphorescence intensity and lifetime of tryptophan residue (Trp-109) decrease upon addition of guanidine hydrochloride, ethylene diamine tetraacetic acid, and urea or decreasing acidity. The experiments show that AP undergoes different pathways with different denaturants and that the activation energy data, DeltaS degrees (not equal) and deltaH degrees (not equal) further confirm that there is a stable intermediate state between the folded and unfolded AP states in solution.     

Differentiation of the local structure around tryptophan 51 and 64 in recombinant human erythropoietin by tryptophan phosphorescence

Abstract Recombinant human erythropoietin is a 4-helix bundle, glycosylated cytokine containing three tryptophan residues at positions 51, 64 and 88 whose phosphorescence emission may represent a sensitive probe of the structure at multiple sites near or at the protein surface. This report characterizes the phosphorescence properties (spectral energy, thermal spectral relaxation and phosphorescence lifetime), from low temperature glasses to ambient temperature, of the native protein plus that of three single point mutation analogs where each Trp was replaced by Phe. The structural information inferred from the phosphorescence parameters was essentially in good agreement with the structure of the Escherichia coli-produced nonglycosylated protein determined by nuclear magnetic resonance (Cheetham et al., Nat Struct Biol [1998] 5:861). The results showed that the fluorescence and phosphorescence spectra of the native protein were entirely due to independent contributions of Trp51 and Trp64 and that Trp88 was quenched under all conditions. The phosphorescence emissions of Trp51 and Trp64 were differentiated by their unique spectra at 77 K with Trp64 exhibiting an unusually blueshifted spectrum likely due to the attractive interaction of Arg110 and Lys116 with the ground state dipole of Trp64. In the native protein the room temperature phosphorescence lifetime of Trp64 was relatively short with a time of 1.62 ms whereas the lifetime of Trp51 was five-fold longer. Characterization of the single point mutation analogs showed that each lifetime was composed of multiple components revealing the presence of multiple stable conformations of the protein at these surface sites.     

Temperature dependence of tryptophan phosphorescence in proteins

The phosphorescence yield and decay kinetics of tryptophan (Trp) in apoazurin from Pseudomonas aeruginosa, subtilisin Carlsberg, Staphylococcal nuclease and liver alcohol dehydrogenase were determined as a function of temperature from 150 K (glassy matrix) to 300 K (fluid solution). The constancy of the lifetime-normalized phosphorescence yield with apoazurin and with Trp-314 in alcohol dehydrogenase establishes that the intersystem crossing quantum yield is practically unaffected across the temperature range. Consequently, any decrease in phosphorescence intensity not accounted for by lifetime-shortening is a signal either of the selective quenching of specific Trp residues in the same macromolecule or that the protein sample is heterogeneous in its emission properties. From an analysis of the thermal profile it is concluded that subtilisin Carlsberg and S. nuclease, as opposed to apoazurin, are not phosphorescent at ambient temperature, their residual emission probably arising from protein impurities. Criteria for distinguishing conformer emission from a contribution by protein impurities are discussed.     

PHOSPHORESCENCE LIFETIME STUDIES OF INTERACTIONS BETWEEN SERUM ALBUMINS AND SODIUM DODECYL SULFATE

Binding of sodium dodecyl sulfate (SDS) to bovine serum albumin (BSA) and human serum albumin (HSA) in aqueous solutions at room temperature induces significant changes in the phosphorescence lifetime of tryptophan (Trp) residues. A steep rise of the phosphorescence lifetime from 1.9 ms to 10.0 ms for BSA and from 1.9 ms to 5.5 ms for HSA is observed when the total SDS concentration increased from 0.0 mM to 0.22 mM at 1 mg/mL protein concentration. As the total SDS concentrationis further inccreased to 2.2 mM, a slower increase in the phosphorescence lifetime is observed, from 10.0 ms to 19.5 ms for BSA and from 5.5 ms to 7.2 ms for HSA. It appears that the phosphorescence lifetime modifications are mainly due to an increase of protein matrix rigidity around Trp residues. The observed differences (between HSA and BSA) allow us to distinguish the contribution of the two Trp residues to the BSA phosphorescence.     

盐酸胍诱导的淀粉液化芽孢杆菌α-淀粉酶去折叠过程的研究

分别用内源荧光光谱法、荧光相图法、荧光探针法、荧光猝灭法、蛋白质电泳法以及体积排阻色谱法研究了盐酸胍诱导的淀粉液化芽孢杆菌a-淀粉酶的去折叠过程. 内源荧光光谱和荧光相图结果表明, 当变性液中盐酸胍浓度约为1.0 mol/L时, 芽孢杆菌a-淀粉酶的去折叠过程中出现一个部分折叠中间体, 其去折叠过程符合“三态模型”; 荧光探针结果表明, 在溶液中盐酸胍浓度约为1.0 mol/L时, 中间态芽孢杆菌a-淀粉酶分子中存在着能够与探针分子1-苯胺 基-8-萘磺酸(ANS)结合的稳定的疏水区域; 荧光猝灭研究给出了不同程度变性的淀粉液化芽孢杆菌a-淀粉酶中的Trp的分布情况, 结果表明中间态芽孢杆菌a-淀粉酶分子中能够被碘化钾猝灭的位于分子表面的色氨酸残基数目达到最大的8个; 蛋白电泳和体积排阻色谱结果表明, 在盐酸胍诱导的芽孢杆菌a-淀粉酶分子的整个去折叠过程中, 不会以共价键或非共价键形式形成芽孢杆菌a-淀粉酶分子之间的集聚体或集聚体沉淀. 在此基础上, 对盐酸胍诱导的淀粉液化芽孢杆菌a-淀粉酶的去折叠过程进行了描述.     

Stability of proteins with multi-state unfolding behavior

A new model used to calculate the free energy change of protein unfolding is presented.In this model,proteins are considered to be composed of structural elements.The unfolding of a structural element obeys a two-state mechanism and the free energy change of the element can be obtained by a linear extrapolation method.If a protein consists of the same structural elements,its unfolding will displays a two-state process,and only the average structural element free energy change < △G 0 element(H2O)> can be measured.If protein consists of completely different structural elements,its unfolding will show a multi-state behavior.When a protein consists of n structural elements its unfolding will shows a(n+1)-state behavior.A least-squares fitting can be used to analyze the contribution of each structural element to the protein and the free energy change of each structural element can be obtained by using linear extrapolation to zero denaturant concentration,not to the start of each transition.The measured △G0 protein(H2 O) is the sum of the free energy change for each structural element.Using this new model,we can not only analyze the stability of various proteins with similar structure and similar molecular weight,which undergo multi-state unfolding processes,but also compare the stability of proteins with different structures and molecular weights using the average structural element free energy change < △G0 element(H2O)>.Although this method cannot completely provide the exact free energy of proteins,it is better than current methods.     

Distribution and Transition of Native and Completely Unfolded Conformations in the Unfolding of Bovine Heart Cytochrome c Induced by Urea and Guanidine Hydrochloride

The unfolding of bovine heart cytochrome c induced by urea and guanidine hydrochloride was first studied through intrinsic fluorescence emission spectra and fluorescence phase diagram and the results showed that both of them separately followed a two‐state model. As the simplest sample of the unfolding of protein molecules induced by denaturants, an equation was presented to show the effect of the denaturant concentrations in denaturation solution on the residual activity ratios of bovine heart cytochrome c in their two‐state unfolding. There are two characteristic unfolding parameters K and m in this equation. The former is the thermodynamic equilibrium constant of the unfolding of bovine heart cytochrome c induced by denaturants, the latter is the number of denaturant molecules associated with a bovine heart cytochrome c molecule during the unfolding procedure, and through them the distribution and transition of native and completely unfolded bovine heart cytochrome c conformations under different concentrations of urea or guanidine hydrochloride in denaturation solution can be accurately described.     

Interactions between hydrophobic and ionic solutes in aqueous guanidinium chloride and urea solutions: lessons for protein denaturation mechanism

In order to clarify the mechanism of denaturant-induced unfolding of proteins we have calculated the interactions between hydrophobic and ionic species in aqueous guanidinium chloride and urea solutions using molecular dynamics simulations. Hydrophobic association is not significantly changed in urea or guanidinium chloride solutions. The strength of interaction between ion pairs is greatly diminished by the guanidinium ion. Although the changes in electrostatic interactions in urea are small, examination of structures, using appropriate pair functions, of urea and water around the solutes show strong hydrogen bonding between urea's carbonyl oxygen and the positively charged solute. Our results strongly suggest protein denaturation occurs by the direct interaction model according to which the most commonly used denaturants unfold proteins by altering electrostatic interactions either by solvating the charged residues or by engaging in hydrogen bonds with the protein backbone. To further validate the direct interaction model we show that, in urea and guanidinium chloride solutions, unfolding of an unusually stable helix (H1) from mouse PrPC (residues 144-153) occurs by hydrogen bonding of denaturants to charged side chains and backbone carbonyl groups.     

Characterization of the tryptophan residues of human placental ribonuclease inhibitor and its complex with bovine pancreatic ribonuclease A by steady-state and time-resolved emission spectroscopy

Human placental ribonuclease inhibitor (hRI) containing six tryptophan (Trp) residues located at positions 19, 261, 263, 318, 375, and 438 and its complex with RNase A have been studied using steady-state and time-resolved fluorescence (298 K) as well as low-temperature phosphorescence (77 K). Two Trp residues in wild-type hRI and also in the protein-protein complex with RNase A are resolved optically. The accessible surface area values of Trp residues in the wild-type hRI and its complex and consideration of inter-Trp energy transfer in the wild-type hRI reveal that one of the Trp residues is Trp19, which is located in a hydrophobic buried region. The other Trp residue is tentatively assigned as Trp375 based on experimental results on wild-type hRI and its complex. This residue in the wild-type hRI is more or less solvent exposed. Both the Trp residues are perturbed slightly on complex formation. Trp19 moves slightly toward a more hydrophobic region, and the environment of Trp375 becomes less solvent exposed. The complex formation also results in a more heterogeneous environment for both the optically resolved Trp residues.