Specific cleavage of histidine‐containing peptides by copper (II)

Abstract

Copper( 11) cleaves with moderate specificity peptides containing Ser-His or Thr-His sequences, at the Nterminal side of the hydroxyaminoacyl residue. The reaction is slow, and is first-order in peptide : Cu" complex, with a half-life of several hours at 62 "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 Cu" 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. 0 Munksgaard 1996.

Fragmentation of monoclonal antibodies

“…In particular for IgG1 molecules, it was demonstrated 29 that the main copper cleavage site is in the hinge region sequence S 219 CDK 222 T 223 HTC between Lys 222 -Thr 223 , presumably supported 30 by the His 224 residue. Iron atoms in the presence of histidine buffer were found to catalyze cleavage of the hinge region of IgG1 molecules containing lambda LCs; no fragmentation catalyzed by iron atoms was observed in the absence of histidine buffer or for IgG1 molecules with kappa LCs.…”

Metal assisted peptide bond hydrolysis: Chemistry, biotechnology and toxicological implications

“…They reported the hydrolysis of a series of heptapeptide substrates by Cu(II) ions [91]. This work was inspired by their discovery of site specific degradation of an antibody upon storage in buffers containing a residual Cu(II) contamination [92].…”

Metal ion interactions with mAbs: Part 1

“…They observed that the rate of the reaction was slow below pH 5 and increased gradually from pH 7 to 9.5. Hydrolysis primarily occurred at the Lys-Thr peptide bond, 31 which is consistent with the trypsin cleavage site. Allen et al deliberately eliminated cysteine residues from the peptide to avoid non-native reactivity with the free cysteine sulfhydryl groups in the presence of Cu 2C .…”