Dye binding probes of lipid-binding structures. An investigation of 2-p-toluidinylnaphthylene-6-sulfonate binding to human and bovine prothrombin and fragment 1 in the presence and absence of calcium and magnesium ions.

TNS (2-p-toluidinylnaphthylene-6-sulfonate) binds to human and bovine prothrombin and Fragment 1 in the absence and presence of added Ca2+. The stoichiometry of TNS binding is 1:1 for human and bovine prothrombin and Fragment 1. The Ca2+-dependence of the fluorescence of TNS bound to bovine prothrombin Fragment 1 yields a modified Hill plot slope of 2.7, which is consistent with the slope obtained by monitoring the Ca2+ dependence of protein fluorescence quenching, CD changes and phospholipid binding. Mg2+ has have no effect on the fluorescence of TNS-prothrombin fluorescence. TNS binding to the amino-terminal region of prothrombin is the first relatively simple probe of the subtle and complex relationship which exists between protein structure and phospholipid binding.

A kinetic model describing the interaction of bovine prothrombin fragment 1 with calcium ions.

A kinetic model is derived for the interaction of bovine prothrombin fragment 1 with calcium ions. The model requires binding of a minimum of two calcium ions for induction of the observed biphasic fluorescence decrease as a function of time. The model is shown to be consistent with experimental kinetic and equilibrium data by fitting theoretical curves for the biphasic fluorescence change to the data through exact solution of the nonlinear differential rate equations derived from the model. The rate constants for the binding of these two required calcium ions are calculated from the solutions as best fit parameters. The thermodynamic equilibrium constants, K1 and K2, for the binding of these two calcium ions are calculated from ratios of the forward and reverse rate constants as 0.6 X 10(4) and 5.4 X 10(4), respectively. Thus, the model correctly predicts positively cooperative calcium ion binding for at least the two calcium ions required to induce fluorescence quenching.