Togashi Denisio M, Szczupak Boguslaw, Ryder Alan G, Calvet Amandine, O'Loughlin Muireann
Nanoscale Biophotonics Laboratory, School of Chemistry and National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Galway, Ireland.
J Phys Chem A. 2009 Mar 26;113(12):2757-67. doi: 10.1021/jp808121y.
Fluorescein is one of most used fluorescent labels for characterizing biological systems, such as proteins, and is used in fluorescence microscopy. However, if fluorescein is to be used for quantitative measurements involving proteins then one must account for the fact that the fluorescence of fluorescein-labeled protein can be affected by the presence of intrinsic amino acids residues, such as tryptophan (Trp). There is a lack of quantitative information to explain in detail the specific processes that are involved, and this makes it difficult to evaluate quantitatively the photophysics of fluorescein-labeled proteins. To address this, we have explored the fluorescence of fluorescein in buffered solutions, in different acidic and basic conditions, and at varied concentrations of tryptophan derivatives, using steady-state absorption and fluorescence spectroscopy, combined with fluorescence lifetime measurements. Stern-Volmer analyses show the presence of static and dynamic quenching processes between fluorescein and tryptophan derivatives. Nonfluorescent complexes with low association constants (5.0-24.1 M(-1)) are observed at all pH values studied. At low pH values, however, an additional static quenching contribution by a sphere-of-action (SOA) mechanism was found. The possibility of a proton transfer mechanism being involved in the SOA static quenching, at low pH, is discussed based on the presence of the different fluorescein prototropic species. For the dynamic quenching process, the bimolecular rate constants obtained (2.5-5.3 x 10(9) M(-1)s(-1)) were close to the Debye-Smoluchowski diffusion rate constants. In the encounter controlled reaction mechanism, a photoinduced electron transfer process was applied using the reduction potentials and charges of the fluorophore and quencher, in addition to the ionic strength of the environment. The electron transfer rate constants (2.3-6.7 x 10(9) s(-1)) and the electronic coupling values (5.7-25.1 cm(-1)) for fluorescein fluorescence quenching by tryptophan derivatives in the encounter complex were then obtained and analyzed.
荧光素是用于表征生物系统（如蛋白质）的最常用荧光标记之一，并且用于荧光显微镜检查。然而，如果要将荧光素用于涉及蛋白质的定量测量，那么必须考虑到荧光素标记的蛋白质的荧光会受到诸如色氨酸（Trp）等内在氨基酸残基的存在的影响。缺乏详细解释所涉及的具体过程的定量信息，这使得难以对荧光素标记的蛋白质的光物理性质进行定量评估。为了解决这个问题，我们使用稳态吸收和荧光光谱结合荧光寿命测量，研究了荧光素在缓冲溶液中、不同的酸性和碱性条件下以及不同浓度的色氨酸衍生物存在下的荧光。斯特恩-沃尔默分析表明荧光素和色氨酸衍生物之间存在静态和动态猝灭过程。在所有研究的pH值下都观察到了具有低缔合常数（5.0 - 24.1 M⁻¹）的非荧光复合物。然而，在低pH值下，发现了一种由作用球（SOA）机制引起的额外静态猝灭贡献。基于不同荧光素质子异构体的存在，讨论了在低pH值下质子转移机制参与SOA静态猝灭的可能性。对于动态猝灭过程，获得的双分子速率常数（2.5 - 5.3×10⁹ M⁻¹s⁻¹）接近德拜-斯莫卢霍夫斯基扩散速率常数。在遭遇控制反应机制中，除了环境的离子强度外，还利用荧光团和猝灭剂的还原电位和电荷应用了光诱导电子转移过程。然后获得并分析了色氨酸衍生物在遭遇复合物中猝灭荧光素荧光的电子转移速率常数（2.3 - 6.7×10⁹ s⁻¹）和电子耦合值（5.7 -
25.1 cm⁻¹）。
