The binding of cupric ions to bovine pancreatic ribonuclease studies with diligand metal-ion buffers.

A procedure has been developed for the use of metal-ion buffers that depends on the formation of 2:1 complexes between suitable chelators and metal ions. beta-Alanine has been used as the chelator for Cu(2+) ions in a study of Cu(2+) binding by bovine pancreatic ribonuclease by the equilibrium-dialysis technique at pH7.0, 6.1 and 5.2. The results indicated the presence of two avid binding sites, the more avid group being implicated in the inhibition of enzyme activity by Cu(2+) ions. The binding constants of the more avid site were 2.97x10(7), 7.97x10(5) and 1.25x10(4) at pH7.0, 6.1 and 5.2 respectively, and the binding constants of the less avid site were 5.27x10(6) and 1.71x10(5) at pH7.0 and 6.1 respectively. The data show that the Cu(2+) is chelated to the protein through at least two ligand groups on the ribonuclease molecule.