Department of Clinical Microbiology, Mahidol University, Bangkok-Noi, Bangkok, Thailand.
Biol Trace Elem Res. 2010 Jun;134(3):352-63. doi: 10.1007/s12011-009-8476-9. Epub 2009 Aug 1.
In the present study, spectroscopic determinations of copper ions using chimeric metal-binding green fluorescent protein (His6GFP) as an active indicator have been explored. Supplementation of copper ions to the GFP solution led to a remarkable decrease of fluorescent intensity corresponding to metal concentrations. For circumstances, rapid declining of fluorescence up to 60% was detected in the presence of 500 microM copper. This is in contrast to those observed in the case of zinc and calcium ions, in which approximately 10-20% of fluorescence was affected. Recovery of its original fluorescence up to 80% was mediated by the addition of ethylenediamine tetraacetic acid. More importantly, in the presence of metal ions, the emission wavelength maximum remains unchanged while reduction of the optical density of the absorption spectrum has been observed. This indicates that the chromophore's ground state was possibly affected by the static quenching process. Results from circular dichroism measurements revealed that the overall patterns of circular dichroism spectra after exposure to copper ions were not significantly different from that of the control, where the majority of sharp positive band around 195-196 nm in combination with a broad negative deflection around 215-216 nm was obtained. Taken together, it can be presumed that copper ions exerted their static quenching on the fluorescence rather than structural or conformational alteration. However, notification has to be made that some peptide rearrangements may also occur in the presence of metal ions. Further studies were conducted to investigate the feasibility of using the His6GFP as a sensing unit for copper ions. The His6GFP was encapsulated in Sol-gel and immobilized onto the optical fiber connected with a fluorescence detecting device. The Sol-gel was doped into the metal solution where the quenching of fluorescence could be monitored in real time. The sensing unit provided a high sensitivity of detection in the range of 0.5 microM to 50 mM with high selectivity for copper ions. All these findings open up a high potential to apply the fluorescent protein-based bioanalytical tool for copper determination in the future.
在本研究中，我们探索了使用嵌合金属结合绿色荧光蛋白（His6GFP）作为活性指示剂测定铜离子的光谱方法。向 GFP 溶液中添加铜离子会导致荧光强度显著降低，与金属浓度相对应。例如，在存在 500 μM 铜的情况下，检测到荧光迅速下降 60%。这与锌和钙离子的情况形成对比，在这些情况下，约有 10-20%的荧光受到影响。通过添加乙二胺四乙酸，可将其原始荧光恢复至 80%。更重要的是，在存在金属离子的情况下，发射波长最大值保持不变，同时观察到吸收光谱的光密度降低。这表明生色团的基态可能受到静态猝灭过程的影响。圆二色性测量结果表明，暴露于铜离子后圆二色性光谱的整体模式与对照没有显著差异，其中在 195-196nm 左右获得了大多数尖锐的正带，同时在 215-216nm 左右获得了宽的负偏转。综上所述，可以推测铜离子对荧光产生了静态猝灭作用，而不是结构或构象改变。然而，必须注意的是，在存在金属离子的情况下，可能会发生一些肽重排。进一步的研究旨在探讨将 His6GFP 用作铜离子传感单元的可行性。His6GFP 被包埋在溶胶-凝胶中，并固定在与荧光检测装置相连的光纤上。将溶胶-凝胶掺杂到金属溶液中，可以实时监测荧光的猝灭情况。传感单元在 0.5 μM 至 50mM 的范围内提供了高灵敏度的检测，对铜离子具有高选择性。所有这些发现为将来应用荧光蛋白基生物分析工具进行铜测定开辟了广阔的前景。
