Binding of zinc to alpha-2-macroglobulin and its role in enzyme binding activity.

Physicochemical studies performed on alpha-2-macroglobulin were correlated with the biological activities of this protein. Equilibrium dialysis of the binding of 65Zn by alpha-2-macroglobulin at pH 7.9 showed heterogeneous binding which could be attributed to two classes of binding sites. The site of greatest affinity for zinc had an apparent stoichiometry (n1 in gatoms/mol of alpha-2-macroglobulin monomer) of 12 and an apparent association constant (K1) of 3.06.10(7). The second binding site had an n2 of 60 and K2 of 1.32.10(5). The trypsin binding activity of alpha-2-macroglobulin did not depend on the presence of zinc in this protein since all but traces of this metal could be removed by EDTA without loss of trypsin binding activity. Saturation of site 1 with zinc did not affect the trypsin binding activity of alpha-2-macroglobulin, but binding of the metal by site 2 progressively decreased the trypsin binding activity by causing an irreversable association of the alpha-2-macroglobulin molecules. Removal of excess zinc from alpha-2-macroglobulin did not restore its trypsin binding activity. Our results also indicate that the high zinc content of alpha-2-macroglobulin (320--770 microgram/g protein) reported in the literature is an artifact and that native alpha-2-macroglobulin contains approximately 150--180 microgram Zn/g protein.