Diffusion of alpha-Chymotrypsin in solution-crowded media. A fluorescence recovery after photobleaching study

Abstract

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 disc 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 disc 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 disc or the Gaussian model is justified for these experiments. Moreover, in our experimental conditions, alpha-chymotrypsin shows anomalous diffusion (a < 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 of which other physical phenomena must be considered.