TY - JOUR

T1 - Hypochlorous acid oxidizes methionine and tryptophan residues in myoglobin

AU - Szuchman-Sapir, Andrea J

AU - Pattison, David I

AU - Ellis, Natasha A

AU - Hawkins, Clare Louise

AU - Davies, Michael Jonathan

AU - Witting, Paul K

PY - 2008/9/15

Y1 - 2008/9/15

N2 - After acute myocardial infarction (AMI), infiltrating proinflammatory cells generate two-electron oxidants such as hypochlorous acid (HOCl). Myoglobin (Mb) is present at approximately 0.3 mM in cardiomyocytes and, therefore, represents a significant target for oxidation. Exposure of horse Mb (50 microM) to reagent HOCl (0-500 microM) or activated human neutrophils (4-40x10(6) cells/ml) yielded oxidized Mb (Mb(ox)) as judged by amino acid analysis and peptide mass mapping. HOCl/Mb ratios of 1-5 mol/mol gave Mb(ox) with up to four additional oxygen atoms. Hydrolysis of Mb(ox) followed by amino acid analysis indicated that methionine (Met) and tryptophan (Trp) residues were modified by HOCl. Peptide mass mapping revealed that Met55 was oxidized at a lower HOCl/Mb ratio than Met131 and this preceded Trp7/14 modification (susceptibility Met55>Met131>Trp7>Trp14). Incubation of Mb with activated neutrophils and physiological chloride anion yielded Mb(ox) with a composition similar to that determined with HOCl/Mb ratios <2 mol/mol, with oxidation of Met, but not Trp, detected. These data indicate that Mb undergoes site-specific oxidation depending on the HOCl/protein ratio. As Mb is released from necrotic cardiomyocytes into the vasculature after AMI, HOCl-modified Mb may be a useful surrogate marker to gauge the extent of myocardial inflammation.

AB - After acute myocardial infarction (AMI), infiltrating proinflammatory cells generate two-electron oxidants such as hypochlorous acid (HOCl). Myoglobin (Mb) is present at approximately 0.3 mM in cardiomyocytes and, therefore, represents a significant target for oxidation. Exposure of horse Mb (50 microM) to reagent HOCl (0-500 microM) or activated human neutrophils (4-40x10(6) cells/ml) yielded oxidized Mb (Mb(ox)) as judged by amino acid analysis and peptide mass mapping. HOCl/Mb ratios of 1-5 mol/mol gave Mb(ox) with up to four additional oxygen atoms. Hydrolysis of Mb(ox) followed by amino acid analysis indicated that methionine (Met) and tryptophan (Trp) residues were modified by HOCl. Peptide mass mapping revealed that Met55 was oxidized at a lower HOCl/Mb ratio than Met131 and this preceded Trp7/14 modification (susceptibility Met55>Met131>Trp7>Trp14). Incubation of Mb with activated neutrophils and physiological chloride anion yielded Mb(ox) with a composition similar to that determined with HOCl/Mb ratios <2 mol/mol, with oxidation of Met, but not Trp, detected. These data indicate that Mb undergoes site-specific oxidation depending on the HOCl/protein ratio. As Mb is released from necrotic cardiomyocytes into the vasculature after AMI, HOCl-modified Mb may be a useful surrogate marker to gauge the extent of myocardial inflammation.

KW - Amino Acid Sequence

KW - Chloramines

KW - Chromatography, High Pressure Liquid

KW - Humans

KW - Hypochlorous Acid

KW - In Vitro Techniques

KW - Methionine

KW - Molecular Sequence Data

KW - Myoglobin

KW - Neutrophil Activation

KW - Neutrophils

KW - Spectrometry, Mass, Electrospray Ionization

KW - Tryptophan

U2 - 10.1016/j.freeradbiomed.2008.06.010

DO - 10.1016/j.freeradbiomed.2008.06.010

M3 - Journal article

C2 - 18625300

VL - 45

SP - 789

EP - 798

JO - Free Radical Biology & Medicine

JF - Free Radical Biology & Medicine

SN - 0891-5849

IS - 6

ER -