Specific cleavage of histidine-containing peptides by copper(II).

Abstract

Copper(II) cleaves with moderate specificity peptides containing Ser-His or Thr-His sequences, at the N-terminal side of the hydroxyaminoacyl residue. The reaction is slow, and is first-order in peptide: CuII complex, with a half-life of several hours at 62 degrees C in sodium bicarbonate buffer, pH 8. Cleavage of other histidine-containing peptides also occurs, at a rate around 10-100-fold less. EDTA completely quenches the cleavage. The reaction is stoichiometric in CuII and is inhibited by amine-containing buffer components; Tris at 19 mM inhibits cleavage by 50%. The reaction has a complex pH-dependence, being very slow below pH 5, and with rates increasing with pH from pH 7 to pH 9.5. Slower degradative side reactions do occur, with destruction of tyrosine residues, particularly in the presence of high concentrations of chloride ion, but the specific cleavage appears to be a hydrolysis, as determined by amino-acid analysis and mass spectrometry of the products. The cleavage is clearly different from the previously described oxidative degradation of proteins catalysed by copper ions. Cleavage of denatured IgG protein occurs with sufficient specificity to reveal distinct bands on SDS-polyacrylamide gel electrophoresis under reducing conditions.