STUDY OF RIBONUCLEASE PRECRYSTALLIZATION BY FLUORESCENCE ANISOTROPY

The purpose of this work is to study protein interactions under crystallization conditions. Fluorescence appears a suitable method to follow the prenucleation phase since it is sensitive to any factor affecting the rate of rotational diffusion. We present a study using bovine pancreatic Ribonuclease A. The fluorescent signal is obtained from a label covalently attached to one of the histidines of the active site, the fluorescence anisotropy of which reflects the rotational diffusion of the whole protein. It is found that the apparent fluorescence anisotropy of the probe is sensitive to protein-protein interactions. The effects of two kinds of precipitating agent are compared: ethanol and ammonium sulfate. The second virial coefficient for rotational diffusion exhibits a characteristic behavior for each of them. The results demonstrate that fluorescence anisotropy can be used for monitoring prenucleation.