NICKEL-DEPENDENT OXIDATIVE CROSS-LINKING OF A PROTEIN

A model protein, ribonuclease A (bovine pancreas), was examined for it s ability to coordinate Ni2+ and promote selective oxidation. In the p resence of a peracid such as monopersulfate, HSO5-, nickel induced the monomeric RNase A to form dimers, trimers, tetramers, and higher olig omers without producing fragmentation of the polypeptide backbone. Co2 + and to a lesser extent Cu2+ exhibited similar activity. The nickel-d ependent reaction appeared to result from a specific association betwe en the protein and Ni2+ that allowed for transient and in situ oxidati on of the bound nickel to yield intermolecular tyrosine-tyrosine cross -links. Macrocylic nickel complexes that had previously been shown to promote guanine oxidation were unable to mimic the activity of the fre e metal salt. Amino acid analysis of the protein dimer confirmed the e xpected consumption of one tyrosine per polypeptide and formation of d ityrosine. The presence of excess tyrosine efficiently inhibited forma tion of the protein dimer and produced instead a ribonuclease-tyrosine cross-link. In contrast, high concentrations of the hydroxyl radical quenching agent mannitol only partially inhibited ribonuclease dimeriz ation. The polypeptide-mediated activation of nickel and its subsequen t reactivity mimic a process that could contribute to the adverse effe cts of nickel in vivo.