Relative reactivity of lysine and other peptide-bound amino acids to oxidation by hypochlorite

Antibacterial and inflammatory responses of neutrophils and macrophages pro duce hypochlorite as a major oxidant. Numerous side chains of amino acids f ound in extracellular proteins can be modified by hypochlorite, including H is, Arg, Tyr, Lys, Trp, and Met. We studied the relative reactivity of each of these amino acid residues in short N-blocked peptides, where other resi dues in the peptide were highly resistant to hypochlorite attack. Hypochlor ite treatment led to modified peptides in each case, which were detected by changes in retention on reversed-phase HPLC. A distinct single product, co nsuming two equivalents of hypochlorite per equivalent of peptide, was obta ined from the Lys-containing peptides. UV spectroscopy, nuclear magnetic re sonance (NMR), and electrospray/mass spectroscopy identified this product a s the dichloramine at the epsilon-amino group of the Lys side chain. The di chloramine at Lys did not decompose to form a detectable amount of carbonyl reactive with dinitrophenylhydrazine. The dichloramine at Lys did however quantitatively revert back to Lys during HCl digestion of the tetrapeptide for amino acid analysis, with simultaneous modification of the adjacent Phe residue. The formation of the dichloramine at Lys was not blocked by pepti des or acetylated amino acids that contained Tyr, His, or Arg. In contrast, the presence of equimolar Met-containing peptide, or N-Acetyl-Trp, both in hibited the formation of the dichloramine at Lys. Thus, Met and Trp side ch ains of proteins might be able to protect Lys from chloramine formation und er some circumstances, but this interpretation must consider that Met and T rp are typically found in relatively inaccessible hydrophobic sites, wherea s lysine is typically exposed on the protein surface. The hierarchy of amin o acid reactivities examined here will aid in the prediction of residues in biological samples most Likely to be modified by hypochlorite. (C) 2000 El sevier Science Inc.