van den Berg P A, Widengren J, Hink M A, Rigler R, Visser A J
MicroSpectroscopy Centre, Department of Biomolecular Sciences, Wageningen University, The Netherlands.
Spectrochim Acta A Mol Biomol Spectrosc. 2001 Sep 14;57(11):2135-44. doi: 10.1016/s1386-1425(01)00494-2.
Fluorescence Correlation Spectroscopy (FCS) was used to investigate the excited-state properties of flavins and flavoproteins in solution at the single molecule level. Flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD) and lipoamide dehydrogenase served as model systems in which the flavin cofactor is either free in solution (FMN, FAD) or enclosed in a protein environment as prosthetic group (lipoamide dehydrogenase). Parameters such as excitation light intensity, detection time and chromophore concentration were varied in order to optimize the autocorrelation traces. Only in experiments with very low light intensity ( < 10 kW/cm2), FMN and FAD displayed fluorescence properties equivalent to those found with conventional fluorescence detection methods. Due to the high triplet quantum yield of FMN, the system very soon starts to build up a population of non-fluorescent molecules, which is reflected in an apparent particle number far too low for the concentration used. Intramolecular photoreduction and subsequent photobleaching may well explain these observations. The effect of photoreduction was clearly shown by titration of FMN with ascorbic acid. While titration of FMN with the quenching agent potassium iodide at higher concentrations ( > 50 mM of I-) resulted in quenched flavin fluorescence as expected, low concentrations of potassium iodide led to a net enhancement of the de-excitation rate from the triplet state, thereby improving the fluorescence signal. FCS experiments on FAD exhibited an improved photostability of FAD as compared to FMN: As a result of stacking of the adenine and flavin moieties, FAD has a considerably lower triplet quantum yield. Correlation curves of lipoamide dehydrogenase yielded correct values for the diffusion time and number of molecules at low excitation intensities. However, experiments at higher light intensities revealed a process which can be explained by photophysical relaxation or photochemical destruction of the enzyme. As the time constant of the process induced at higher light intensities resembles the diffusion time constant of free flavin, photodestruction with the concomitant release of the cofactor offers a reasonable explanation.
荧光相关光谱法（FCS）被用于在单分子水平上研究溶液中黄素和黄素蛋白的激发态性质。黄素单核苷酸（FMN）、黄素腺嘌呤二核苷酸（FAD）和硫辛酰胺脱氢酶作为模型系统，其中黄素辅因子在溶液中是游离的（FMN、FAD），或者作为辅基包裹在蛋白质环境中（硫辛酰胺脱氢酶）。为了优化自相关曲线，改变了诸如激发光强度、检测时间和发色团浓度等参数。只有在非常低的光强度（<10 kW/cm²）实验中，FMN和FAD才表现出与传统荧光检测方法相当的荧光性质。由于FMN的三重态量子产率很高，该系统很快就开始积累非荧光分子群体，这反映在表观粒子数远低于所用浓度应有的数量。分子内光还原和随后的光漂白很可能解释了这些现象。用抗坏血酸滴定FMN清楚地显示了光还原的效果。虽然用较高浓度（>50 mM的I⁻）的猝灭剂碘化钾滴定FMN会导致黄素荧光猝灭，但低浓度的碘化钾会导致三重态去激发速率净增加，从而改善荧光信号。与FMN相比，对FAD进行的FCS实验显示FAD具有更好的光稳定性：由于腺嘌呤和黄素部分的堆积，FAD的三重态量子产率相当低。硫辛酰胺脱氢酶的相关曲线在低激发强度下给出了正确的扩散时间和分子数。然而，在较高光强度下的实验揭示了一个可以用光物理弛豫或酶的光化学破坏来解释的过程。由于在较高光强度下诱导的过程的时间常数类似于游离黄素的扩散时间常数，光破坏伴随着辅因子的释放提供了一个合理的解释。
