SURFACE-INDEPENDENT ACCELERATION OF FACTOR-XII ACTIVATION BY ZINC IONS .2. DIRECT BINDING AND FLUORESCENCE STUDIES

To determine the role of Zn(II)-factor XII interactions in the rate-en hancing effect of Zn(II) on factor XII activation demonstrated in the preceding paper, equilibrium binding of zinc ions to factor XII, and t he spectroscopic changes accompanying this binding were investigated. Equilibrium dialysis provided direct evidence for the binding of Zn(II ) to factor XII. The binding data were consistent with 7.8 +/- 0.3 zin c ions binding with an indistinguishable K(d) of 91 +/- 6 muM. Binding of Zn(II) was accompanied by a 10% quenching of the intrinsic protein fluorescence and a 2-nm red shift of the wavelength of maximum emissi on. These spectroscopic changes were specific for factor XII and were not observed with factor XIIa. The Zn(II) concentration dependence of factor XII fluorescence quenching was sigmoid and paralleled the Zn(II )-accelerating effect on factor XII activation by kallikrein and facto r XIIa, indicating that the spectral change was reporting Zn(II)-facto r XII interactions responsible for the enhanced activation rate. The a pparent cooperativity of Zn(II) effects on factor XII fluorescence que nching and activation kinetics, and the apparent noncooperativity in Z n(II) binding to factor XII measured by equilibrium dialysis could be explained by a two-state model in which Zn(II) binding is linked to a conformational change in the protein. The Zn(II)-induced quenching of factor XII fluorescence exhibited a pH dependence consistent with the involvement of histidine residues in the binding of Zn(II). Dynamic qu enching of factor XII protein fluorescence by iodide or acrylamide, in the absence and presence of Zn(II), revealed heterogeneity in the env ironment of the 13 tryptophan residues of factor XII that was markedly reduced by metal ion binding. Together, these results indicate that c ooperative interactions of Zn(II) with factor XII induce structural ch anges in the zymogen that facilitate its proteolytic cleavage and acti vation.