Time-resolved fluorescence measurements of cyanine dyes in biomimetic systems

摘要

In the present study the photophysical properties of DY-635B, a cyanine dye, bound to streptavidin were characterized in detail. Special emphasis was given to I) the alterations in the intrinsic photophysical characteristics of the dye due to (un)specific interactions with streptavidin and ii) the evaluation of interaction between the fluorescence probe and streptavidin in the presence of unlabeled biotin. Fluorescence correlation spectroscopy (FCS) and time-resolved anisotropy experiments were carried out in the presence of excess biotin to monitor also a possible cooperative effect on the fluorescence behavior of DY-635B.rnBased on the evaluation of FCS and time-resolved anisotropy data it is shown that due to binding to streptavidin the rotational freedom of DY-635B is restricted. This restriction is further increased by additional biotin indicating that the biotin binding is altering the tertiary structure of streptavidin. The intrinsic photophysical deactivation processes of DY-635B are changed as well. From FCS measurements it is concluded that due to the specific interaction of DY-635B and streptavidin, the deactivation via a "dark state" becomes less effective, shown as an increase of the corresponding decay time τ_R.