Hypochlorous acid damages histone proteins forming 3-chlorotyrosine and 3,5-dichlorotyrosine

While the last 30 years chronicles an extensive effort to understand the damage to DNA caused by reactive oxygen species (ROS), little research has examined the chemical damage to the histone proteins found in chromatin. Hypochlorous acid (HOCl), the primary product of activated neutrophils, is known to damage both DNA and proteins. This article describes the use of mass spectrometry to quantitate the formation of 3-chlorotyrosine and 3,5-dichlorotyrosine, stable and unique markers of protein damage caused by HOCl, in the core histone proteins. Our results indicate that up to 25% of the tyrosine in histone proteins become chlorinated by excess HOCl We also observe significant formation of 3-chlorotyrosine and 3,5-dichlorotyrosine at low HOCl concentrations and short reaction times. We use mass spectrometry to identify the tyrosine residues on each histone protein that are chlorinated based on the observation of chlorine-containing peptides following protease digestion of histone proteins exposed to HOCl The tyrosine residues preferentially chlorinated by HOCl are generally within three residues of a lysine or histidine residue, further implicating the initial formation of chloramines in the efficient chlorination of tyrosine residues. The methods and results described here should further our understanding of how HOCl produced at sites of inflammation might damage chromatin.