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首页> 外文学位 >Fluorescence lifetime studies of ligand-protein binding using the impulse response technique with refined data analysis methods.
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Fluorescence lifetime studies of ligand-protein binding using the impulse response technique with refined data analysis methods.

机译：使用脉冲响应技术和完善的数据分析方法研究配体-蛋白质结合的荧光寿命。

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摘要

An instrument for the measurement of fluorescence decay lifetimes was constructed and used for studies of ligand-protein binding. The light source was a nitrogen-pumped dye laser with a subnanosecond pulse width. Detection was by the impulse response method using a boxcar averager to poll and digitize the output of a photomultiplier tube mounted on a laboratory-made base. Digitized signal information was sent from the boxcar to a dedicated microcomputer from which it was uploaded over a network to a workstation.;Data acquired in this way contained significant instrumental distortions. In order to resolve biexponential decay laws from such data, an analysis method was developed which took advantage of the computing power of the workstation. Lifetime parameters were roughly determined by the simulated annealing method and these estimates were used to provide starting guesses and upper and lower bounds for each parameter for analysis by simplex searching. The combination of these two techniques enabled accurate estimation of decay law parameters in the presence of instrumental distortion and noise.;One of the systems examined was an aqueous solution of quinine. The fluorescence from this system followed a biexponential decay law whose parameters were dependent upon emission wavelength.;The ultimate goal of this project was to estimate binding parameters of fluorescent ligands to proteins. The system chosen for this study was the binding of dansylated amino acids to bovine serum albumin. In this case, the bound amino acid derivative has a much longer fluorescence lifetime than the free form. Hence, it was possible to follow, for example, the increase in fluorescence from the longer lifetime component (the bound form) as the ratio of protein to ligand was increased. It was also possible to obtain evidence of the displacement of the fluorescent dansylated amino acid ligand from its binding site on albumin by nonfluorescent competitor ligands. Unfortunately, because the quantity proportional to concentration calculable in these experiments is dependent upon the spectral quantum yield of the fluorescent species, which is different for the free and the bound forms of the ligands, it was not possible to quantitatively estimate binding parameters from these experiments.
机译：构造了用于测量荧光衰减寿命的仪器，并将其用于研究配体-蛋白质结合。光源是具有亚纳秒脉冲宽度的氮泵浦染料激光器。检测是通过脉冲响应方法，使用棚车平均器对安装在实验室基座上的光电倍增管的输出进行轮询和数字化。数字化的信号信息从棚车发送到专用的微型计算机，然后通过网络从网络上传到工作站。以这种方式获取的数据包含严重的仪器失真。为了从此类数据中解决双指数衰减定律，开发了一种利用工作站的计算能力的分析方法。寿命参数是通过模拟退火方法粗略确定的，这些估计值用于为每个参数提供开始猜测以及上下限，以便通过单纯形搜索进行分析。这两种技术的结合使得在仪器失真和噪声存在的情况下能够精确估计衰减定律参数。所研究的系统之一是奎宁水溶液。该系统发出的荧光遵循双指数衰减定律，其参数取决于发射波长。该项目的最终目标是估计荧光配体与蛋白质的结合参数。本研究选择的系统是丹磺酰化氨基酸与牛血清白蛋白的结合。在这种情况下，结合的氨基酸衍生物具有比游离形式更长的荧光寿命。因此，例如随着蛋白质与配体之比的增加，来自更长寿命组分（结合形式）的荧光增加是可能的。也有可能获得荧光丹磺化氨基酸配体从其在白蛋白上的结合位点被非荧光竞争配体置换的证据。不幸的是，由于与这些实验中可计算的浓度成比例的量取决于荧光物质的光谱量子产率，这对于配体的游离形式和结合形式而言是不同的，因此无法从这些实验中定量估计结合参数。

著录项

  • 作者

    Shew, Sanford Len.;

  • 作者单位

    The Ohio State University.;

  • 授予单位 The Ohio State University.;
  • 学科 Chemistry Analytical.
  • 学位 Ph.D.
  • 年度 1990
  • 页码 216 p.
  • 总页数 216
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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