Inactivation of protease inhibitors and lysozyme by hypochlorous acid: role of side-chain oxidation and protein unfolding in loss of biological function.
Excessive or misplaced activation of leukocytes causes host tissue damage which has been implicated in diseases such as atherosclerosis and chronic inflammation. This may arise via either the generation of oxidants such as hypochlorous acid (HOCl) by the heme enzyme myeloperoxidase, the action of released enzymes including lysozyme and proteases, or a combination of these two activities. Thus, oxidant-mediated inactivation of protease inhibitors that modulate tissue proteolysis by the released enzymes may exacerbate protease-induced degradation of host tissue. The role of myeloperoxidase-derived oxidants, such as HOCl, in the inactivation of Kunitz-type inhibitors and lysozyme is not well-characterized and is the subject of the current study. Exposure of both trypsin inhibitor and lysozyme to low molar excesses of HOCl compared to protein is shown to result in loss of function. With trypsin inhibitor, this loss of activity is associated with the selective oxidation of Trp, Tyr, and His residues, which results in protein unfolding and the disruption of complex formation with active trypsin. Oxidation of Met residues, a major target for HOCl, or the active site Arg, does not appear to play a key role in this loss of activity. In contrast, with lysozyme, oxidation of Met residues to Met sulfoxide appears to be the major process resulting in loss of enzyme activity. With both proteins, inactivation occurs in a time-dependent manner, consistent with both direct oxidation by HOCl and secondary reactions of protein chloramines formed from amine groups (e.g., from Lys and His) playing a role in loss of activity.
Hawkins
Clare L
CL
The Heart Research Institute, 114 Pyrmont Bridge Road, Camperdown, Sydney, NSW 2050, Australia. c.hawkins@hri.org.au
Davies
Michael J
MJ
D000818:Animals; D002700:Chloramines / Q000737:chemistry; D004795:Enzyme Stability; D006997:Hypochlorous Acid / Q000494:pharmacology*; D009113:Muramidase / Q000737:chemistry / Q000378:metabolism*; D016877:Oxidants / Q000494:pharmacology*; D010084:Oxidation-Reduction; D011480:Protease Inhibitors / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013025:Glycine max; D013997:Time Factors; D014361:Trypsin Inhibitors / Q000737:chemistry / Q000378:metabolism
D002700:Chloramines; D016877:Oxidants; D011480:Protease Inhibitors; D014361:Trypsin Inhibitors; D006997:Hypochlorous Acid; D009113:Muramidase