Ca 2+-induced spectral changes in human prothrombin

Abstract

Ca 2+ binding to human prothrombin as well as to the N-terminal region of the molecule, prothrombin fragment 1, is a highly cooperative process. This suggests that the filling of these Ca 2+ binding sites is related to a conformational change in the protein. We have attempted to characterize such a change by difference spectroscopy in the ultraviolet region and by solvent perturbation studies. Ca 2+ binding to human prothrobin is associated with a red-shift tryptophan difference spectrum, the contribution of tyrosine residues being minimal. An identical spectrum is observed upon the binding of Ca 2+ to prothrombin fragment 1. Conversely, no spectral change can be obtained with the fragment 2 region. The fact that such spectral change is not decreased in an apolar solvent (20% glycerol) supports the idea that it is due mainly to a local charge effect of Ca 2+ on chromophore. Primary structure data and comparison with similar results already performed on Factor Xa suggest that the tryptophan residue located in the vicinity of Ca 2+ binding sites and, therefore, involved in the spectral changes might be tryptophan 41. In the absence of Ca 2+, solvent perturbations of prothrombin with 20% glycerol result in a large difference spectrum involving tryptophan and tyrosine residues. This spectral change is markedly reduced in the presence of 2 mM Ca 2+, suggesting that, besides a local vicinity charge effect, the binding of Ca 2+ to prothrombin leads to an overall structural change in the protein corresponding to the burial of an average of 1.7 tryptophan and 6 tyrosine.