Synthesis of dansyl ribonucleotides and their use in steady-state fluorescence anisotropy studies of nucleotide binding by initiation factor-2 (eIF-2) and histone H1.
1. Fluorescent analogs of GDP and ATP were prepared with DANSYL-beta-alanine (D beta A) coupled to the (2')3' hydroxyl of the ribose. 2. Observation of changes in both total fluorescence and anisotropy accompanying the binding of D beta A-GDP to eIF-2 allowed determination of Kd (33 nM). 3. When D beta A-ATP bound to H1 histone, the fluorescence quantum yield increased and the emission was blue shifted. Analysis yielded a Kd of 3.4 microM 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.
Mueser
T C
TC
Department of Chemistry, University of Nebraska-Lincoln 68588-0304.
Parkhurst
L J
LJ
D000255:Adenosine Triphosphate / Q000138:chemical synthesis* / Q000378:metabolism; D003619:Dansyl Compounds / Q000737:chemistry*; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism*; D005454:Fluorescence Polarization; D006160:Guanosine Triphosphate / Q000138:chemical synthesis* / Q000378:metabolism; D006657:Histones / Q000378:metabolism*
D003619:Dansyl Compounds; D015852:Eukaryotic Initiation Factor-2; D006657:Histones; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate