Fluorescence correlation spectroscopy and photon correlation spectroscopy of tryptophan-containing proteins in sugar solutions.

摘要

Sugars are common ingredients for many commercial cryopreserving agents used in the long term preservation of biological tissues and proteins. However, their mechanism in this role is not resolved yet. Some believe that sugars preferentially bind with a protein thereby replacing ice-forming water. Others argue that aqueous sugars are easily vitrified into non-crystalline solid at sufficiently high concentrations, which avoids the formation of ice crystals. In an attempt to test the former idea, we have undertaken studies of the diffusion of L-tryptophan and streptavidin-coated polystyrene spheres (340 tryptophan residues on each) using fluorescence correlation spectroscopy in an effort to determine whether the hydrodynamic radius is influenced by the addition of sugar. Some novelty of our approach lies in exploiting the native fluorescence of tryptophan by use of two-photon excitation. Photon correlation spectroscopy was also utilized to check the results of fluorescence correlation spectroscopy. However, the two techniques showed different results.