Highly efficient fluorescent label unquenched by protein interaction to probe the avidin rotational motion

Fluorescent dye alexa-fluor-594 was fixed to biotin with a spacer chain of four atoms. The static, dynamic fluorescence and fluorescence polarization properties of the avidin/labeled-biotin conjugate were investigated and compared with those of free fluorescent dye and directly labeled avidin. With 0.8 label on the average per avidin, one single fluorescence lifetime and one single correlation time related to the actual protein motion were recorded. Moreover, the excited state fluorescent probe is stabilized in the conjugate, with a quantum yield of 0.87, by slowing down the non-radiative processes, while fluorescence lifetime and fluorescence quantum yield increase by 10%, with respect to free alexa-fluor-594.