Department of Physical Chemistry and Research Institute of Theoretical and Computational Chemistry of University of Barcelona, Barcelona, Spain.
J Phys Chem B. 2010 Mar 25;114(11):4028-34. doi: 10.1021/jp910811j.
Fluorescence recovery after photobleaching (FRAP) is one of the most powerful and used techniques to study diffusion processes of macromolecules in membranes or in bulk. Here, we study the diffusion of alpha-chymotrypsin in different crowded (Dextran) in vitro solutions using a confocal laser scanning microscope. In the considered experimental conditions, confocal FRAP images could be analyzed applying the uniform circular disk approximation described for a nonscanning microscope generalized to take into account anomalous diffusion. Considering the slow diffusion of macromolecules in crowded media, we compare the fitting of confocal FRAP curves analyzed with the equations provided by the Gaussian and the uniform circular disk profile models for nonscanning microscopes. As the fitted parameter variation with the size and concentration of crowders is qualitatively similar for both models, the use of the uniform circular disk or the Gaussian model is justified for these experiments. Moreover, in our experimental conditions, alpha-chymotrypsin shows anomalous diffusion (alpha < 1), depending on the size and concentration of Dextran molecules, until a high concentration and high size of crowding agent are achieved. This result indicates a range of validity of the idealized fitting expressions used, beyond which other physical phenomena must be considered.
荧光漂白后恢复（FRAP）是研究膜或体相中大分子扩散过程的最强大和最常用的技术之一。在这里，我们使用共焦激光扫描显微镜研究了在不同拥挤（葡聚糖）体外溶液中α-糜蛋白酶的扩散。在考虑的实验条件下，可以应用针对非扫描显微镜描述的均匀圆形盘近似来分析共焦 FRAP 图像，该近似被推广以考虑异常扩散。考虑到大分子在拥挤介质中的扩散缓慢，我们比较了用高斯和非扫描显微镜的均匀圆形盘轮廓模型提供的方程拟合共焦 FRAP 曲线。由于两种模型的拟合参数随拥挤物的大小和浓度的变化具有定性相似性，因此对于这些实验，使用均匀圆形盘或高斯模型是合理的。此外，在我们的实验条件下，α-糜蛋白酶显示异常扩散（α<1），这取决于葡聚糖分子的大小和浓度，直到达到高浓度和高拥挤剂大小时才会出现。该结果表明，所使用的理想化拟合表达式的有效范围超出了必须考虑其他物理现象的范围。
