Synthesis of dansyl ribonucleotides and their use in steady-state fluorescence anisotropy studies of nucleotide binding by initiation factor-2 (eif-2) and histone hi

1. 1. Fluorescent analogs of GDP and ATP were prepared with DANSYL-β-alanine (DβA) coupled to the (2′)3′ hydroxyl of the ribose. 2. 2. Observation of changes in both total fluorescence and anisotropy accompanying the binding of DβA-GDP to eIF-2 allowed determination of K_d (33 nM). 3. 3. When DβA-ATP bound to HI histone, the fluorescence quantum yield increased and the emission was blue shifted. Analysis yielded a K_d of 3.4 μM and 20 binding sites per histone. At high levels of ATP, fluorescence anisotropy values and light scattering intensities pointed to significant aggregation of H1 that is strongly dependent on ATP concentration.