p-Hydroxyphenylacetaldehyde, the major product of L-tyrosine oxidation by the myeloperoxidase-H₂O₂-chloride system of phagocytes, covalently modifies ε-amino groups of protein lysine residues

Activated human phagocytes employ the myeloperoxidase-H₂O₂-Cl^- system to convert L-tyrosine to p-hydroxyphenylacetaldehyde (pHA). We have explored the possibility that pHA covalently reacts with proteins to form Schiff base adducts, which may play a role in modifying targets at sites of inflammation. Because Schiff bases are labile to acid hydrolysis, prior to analysis the adducts were rendered stable by reduction with NaCNBH₃. Purified pHA reacted with N(α)-acetyllysine, an analog of protein lysine residues. The reduced reaction product was identified as N(α)-acetyl-N(ε)-(2-(p- hydroxyphenyl)ethyl)lysine by ¹H NMR spectroscopy and mass spectrometry. The compound N(ε)-(2-(p-hydroxyphenyl)ethyl)lysine (pHA-lysine) was likewise identified in acid hydrolysates of bovine serum albumin (BSA) that were first exposed to myeloperoxidase, H₂O₂, L-tyrosine, and Cl^- and then reduced with NaCNBH₃. Other halides (F^-, Br^-, I^-) and the pseudohalide SCN^- could not replace Cl^- as a substrate in the myeloperoxidase-H₂O₂-L- tyrosine system. In the absence of the enzymatic system, pHA-lysine was detected in reduced reaction mixtures of BSA, L-tyrosine, and reagent HOCl. In contrast, pHA-lysine was undetectable when BSA was incubated with L- tyrosine and HOBr, peroxynitrite, hydroxyl radical, or a variety of other peroxidases, indicating that the aldehyde-protein adduct was selectively produced by HOCl. Human neutrophils activated in the presence of tyrosine also modified BSA lysine residues. pHA-lysine formation required L-tyrosine and cell activation; it was inhibited by peroxidase inhibitors and catalase, implicating myeloperoxidase and H₂O₂ in the reaction pathway. pHA-lysine was detected in inflamed human tissues that were reduced, hydrolyzed, and then analyzed by mass spectrometry, indicating that the reaction of pHA with proteins may be of physiological importance. These observations raise the possibility that the identification of pHA-lysine in tissues will pinpoint targets where phagocytes inflict oxidative damage in vivo.