Binding of ATP to FGF2 and nucleotide-induced conformational change. (A) The binding of ATP to FGF2 was determined by equilibrium microdialysis. FGF2 (10 μM) was incubated with different concentrations of ATP in rapid equilibrium dialysis inserts in a Teflon Base Plate for 16 h at 20°C. ATP in the dialysis chambers was determined using ATP Kit SL and a LKB Wallac luminometer. (B) The data were analyzed in a Scatchard plot using a calculated amount of bound and free ATP to determine KD of FGF2/ATP-complex. The equation was used to calculate the number of binding sites of 1.2 (y-value is set = 0) and the KD value of 59.8 μM (the reciprocal of the negative gradient). (C) CD spectra of FGF2 (25 μM) measured in Tris-HCl (pH 7.5). Effects of different nucleotides (100 μM, respectively) are shown. The ordinate axis presents the molecular ellipticity.