SYNTHESIS OF DANSYL RIBONUCLEOTIDES AND THEIR USE IN STEADY-STATE FLUORESCENCE ANISOTROPY STUDIES OF NUCLEOTIDE-BINDING BY INITIATION FACTOR-II (EIF-2) AND HISTONE H1

1. Fluorescent analogs of GDP and ATP were prepared with DANSYL-beta-a lanine (DbetaA) coupled to the (2')3' hydroxyl of the ribose. 2. Obser vation of changes in both total fluorescence and anisotropy accompanyi ng the binding of DbetaA-GDP to eIF-2 allowed determination of K(d) (3 3 nM). 3. When DbetaA-ATP bound to H1 histone, the fluorescence quantu m yield increased and the emission was blue shifted. Analysis yielded a K(d) of 3.4 muM and 20 binding sites per histone. At high levels of ATP, fluorescence anisotropy values and fight scattering intensities p ointed to significant aggregation of H1 that is strongly dependent on ATP concentration.