Steady - state fluorescence anisotropy binding titrations were performed on a PTI Model QM-4 spectrofluorimeter at 25 C following the intrinsic fluorescence of the single tryptophan residue ( exc 295 nm and em 324 nm ) . To measure the affinity of the protein toward rrm1 , the THAP zinc finger was diluted to 0.5 mM in a volume of 4 ml and the 16 - bp rrm1 DNA duplex ( 100 mM ) was prepared in a buffer consisting of 50 mM Tris , 30 mM NaCl , pH 6.8 . The rrm1 solution was progressively added to the protein sample with protein : DNA ratios ranging from 1:0 to 1:6 . To study the influence of the ionic strength on the non - specific binding , samples with different protein : DNA ratios ranging from 1:0 to 1:6 were initially prepared in 250 mM NaCl ( 100 ml of THAP zinc finger at 3 mM ) and were then exchanged in buffer containing suitable NaCl concentrations ( 30 or 150 mM ) . Fluorescence anisotropy was calculated including a correction factor as previously described ( 27 ) and the data were fitted from a previously described equation ( 28 ) using a non - linear fit with GOSA software ( 22 ) .