Binding of the volatile anesthetic chloroform to albumin demonstrated using tryptophan fluorescence quenching
Binding of the volatile anesthetic chloroform to albumin demonstrated using tryptophan fluorescence quenching
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DOI：
10.1074/jbc.272.29.17961
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发表时间：
1997-07-18
影响因子：
4.8
通讯作者：
Johansson, JS
中科院分区：
文献类型：
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作者：
Johansson, JS
挥发性全身麻醉药的作用部位仍有争议，但支持特异性蛋白靶点的证据正在积累。直接测量挥发性麻醉剂与蛋白质结合的技术仍在开发中。进一步的经验与内在的蛋白质荧光猝灭的方法来监测麻醉剂蛋白质络合报告使用氯仿。氯仿淬灭牛血清白蛋白（ESA）的稳态色氨酸荧光的浓度依赖性，饱和的方式与Kd = 2.7 +/- 0.2 mM。色氨酸荧光寿命分析表明，大部分的淬灭是由于一个静态的机制，指示麻醉剂结合。氯仿淬灭BSA色氨酸荧光的能力在50%2，2，2-三氟乙醇的存在下显着降低，这导致BSA中三级结构接触的损失，表明蛋白质构象对麻醉剂结合至关重要。圆二色性光谱显示氯仿对BSA的二级结构没有可测量的影响。结果表明，氯仿结合到亚结构域IB和IIA在ESA，其中每个包含一个单一的色氨酸。早期的研究表明，这些地点也被氟烷占据。因此，本研究提供了实验支持的理论，结构不同的全身麻醉药可能占据相同的结构域的蛋白质目标。
The site(s) of action of the volatile general anesthetics remain(s) controversial, but evidence in favor of specific protein targets is accumulating. The techniques to measure directly volatile anesthetic binding to proteins are still under development. Further experience with the intrinsic protein fluorescence quenching approach to monitor anesthetic-protein complexation is reported using chloroform. Chloroform quenches the steady state tryptophan fluorescence of bovine serum albumin (ESA) in a concentration-dependent, saturable manner with a K-d = 2.7 +/- 0.2 mM. Tryptophan fluorescence lifetime analysis reveals that the majority of the quenching is due to a static mechanism, indicative of anesthetic binding. The ability of chloroform to quench BSA tryptophan fluorescence was decreased markedly in the presence of 50% 2,2,2-trifluoroethanol, which causes loss of tertiary structural contacts in BSA, indicating that protein conformation is crucial for anesthetic binding. Circular dichroism spectroscopy revealed no measurable effect of chloroform on the secondary structure of BSA. The results suggest that chloroform binds to subdomains IB and IIA in ESA, each of which contains a single tryptophan. Earlier work has shown that these sites are also occupied by halothane. The present study therefore provides experimental support for the theory that structurally distinct general anesthetics may occupy the same domains on protein targets.