Preparation and Fluorescence Anisotropy Study of a Ribonuclease-Lucifer Yellow Conjugate

We have prepared a chemical derivative of ribonuclease A (RNase) with lucifer yellow (LY). The rotational dynamics of the LY-RNase conjugate were characterized by steady state and time resolved fluorescence techniques. Steady state anisotropy measurements were performed at varying viscosities at 10C and 20C, and the rotational correlation time of both RNase and the covalently linked LY probe were determined by time resolved frequency domain measurements. Our data suggest that the fluorophore is rigidly bound at 10C.