Although protein degradation by neutrophil-derived
hypochlorous acid (HOCl) and eosinophil-derived
hypobromous acid (
HOBr) can contribute to the inactivation of pathogens, collateral damage to host
proteins can also occur and has been associated with inflammatory diseases ranging from
arthritis to
atherosclerosis. Though previous research suggested halotyrosines as
biomarkers of
protein damage and
lysine as a mediator of the transfer of a
halogen to
tyrosine, these reactions within whole
proteins are poorly understood. Herein, reactions of HOCl and
HOBr with three well-characterized
proteins [
adenylate kinase (ADK),
ribose binding protein, and
bovine serum albumin] were characterized. Three assessments of oxidative modifications were evaluated for each of the
proteins: (1) covalent modification of electron-rich
amino acids (assessed via liquid chromatography and tandem mass spectrometry), (2) attenuation of secondary structure (via circular dichroism), and (3) fragmentation of
protein backbones (via
sodium dodecyl sulfate-
polyacrylamide gel electrophoresis). In addition to forming halotyrosines, HOCl and
HOBr converted
lysine into
lysine nitrile (2-amino-5-cyanopentanoic acid), a relatively stable and largely overlooked product, in yields of up to 80%. At uniform
oxidant levels, fragmentation and loss of secondary structure correlated with
protein size. To further examine the role of
lysine, a
lysine-free ADK variant was rationally designed. The absence of
lysine increased yields of chlorinated tyrosines and decreased yields of brominated tyrosines following treatments with HOCl and
HOBr, respectively, without influencing the susceptibility of ADK to HOX-mediated losses of secondary structure. These findings suggest that
lysine serves predominantly as a sacrificial
antioxidant (via formation of
lysine nitrile) toward HOCl and as a
halogen-transfer mediator [via reactions involving ε-N-(di)haloamines] with
HOBr.