The thermodynamic binding parameters for intracellular heparin-acridine orange (AO) complexes were determined for Furth murine mastocytoma cells and were found to agree with 1) results from binding studies on heparin-AO complexes in solution, and 2) with biochemical analyses of the cells. The cells exhibited cooperative binding with a binding constant of 1.18 x 10(6) M-1. The cooperative binding constant of heparin-AO in 1 mM buffer was found to be 1.13 x 10(6) M-1. The addition of 1 mM NaCl to heparin-AO system in vitro detectably decreased the cooperative binding constant. Low ionic strength is the only condition in solution under which the cell and solution binding constants are equal. The cells have an average of 1.2 x 10(-14) mol of AO binding sites per cell. Using the biochemically measured heparin content per cell and the amount of AO bound by heparin in solution, 8 x 10(15) mol of sites/cell can be attributed to heparin. The remaining cellular binding sites (4 x 10(-15) mol of sites per cell) are essentially all accounted for by AO binding to DNA, the amount of which is calculated from its previously determined thermodynamic binding parameters. A theoretical isotherm, calculated from the binding parameters of both heparin-AO in solution and DNA-AO complexes in situ, agreed closely with the isotherm experimentally determined for the Furth mastocytoma cells. Ligand-binding analysis yields a binding constant, which may aid in identification of cellular bipolymers, and the number of ligand binding sites per cell. The latter is a measure of the amount of a given intracellular biopolymer present.
Determination of the Kinetics and Thermodynamics of Ligand Binding to a Specific Inactive Conformation in Protein Kinases
