TY - JOUR
T1 - Reevaluation of the rate constants for the reaction of hypochlorous acid (HOCl) with cysteine, methionine, and peptide derivatives using a new competition kinetic approach
AU - Storkey, Corin
AU - Davies, Michael Jonathan
AU - Pattison, David I
N1 - Copyright © 2014 Elsevier Inc. All rights reserved.
PY - 2014/8
Y1 - 2014/8
N2 - Activated white cells use oxidants generated by the heme enzyme myeloperoxidase to kill invading pathogens. This enzyme utilizes H2O2 and Cl(-), Br(-), or SCN(-) to generate the oxidants HOCl, HOBr, and HOSCN, respectively. Whereas controlled production of these species is vital in maintaining good health, their uncontrolled or inappropriate formation (as occurs at sites of inflammation) can cause host tissue damage that has been associated with multiple inflammatory pathologies including cardiovascular diseases and cancer. Previous studies have reported that sulfur-containing species are major targets for HOCl but as the reactions are fast the only physiologically relevant kinetic data available have been extrapolated from data measured at high pH (>10). In this study these values have been determined at pH 7.4 using a newly developed competition kinetic approach that employs a fluorescently tagged methionine derivative as the competitive substrate (k(HOCl + Fmoc-Met), 1.5 × 10(8)M(-1)s(-1)). This assay was validated using the known k(HOCl + NADH) value and has allowed revised k values for the reactions of HOCl with Cys, N-acetylcysteine, and glutathione to be determined as 3.6 × 10(8), 2.9 × 10(7), and 1.24 × 10(8)M(-1)s(-1), respectively. Similar experiments with methionine derivatives yielded k values of 3.4 × 10(7)M(-1)s(-1) for Met and 1.7 × 10(8)M(-1)s(-1) for N-acetylmethionine. The k values determined here for the reaction of HOCl with thiols are up to 10-fold higher than those previously determined and further emphasize the critical importance of reactions of HOCl with thiol targets in biological systems.
AB - Activated white cells use oxidants generated by the heme enzyme myeloperoxidase to kill invading pathogens. This enzyme utilizes H2O2 and Cl(-), Br(-), or SCN(-) to generate the oxidants HOCl, HOBr, and HOSCN, respectively. Whereas controlled production of these species is vital in maintaining good health, their uncontrolled or inappropriate formation (as occurs at sites of inflammation) can cause host tissue damage that has been associated with multiple inflammatory pathologies including cardiovascular diseases and cancer. Previous studies have reported that sulfur-containing species are major targets for HOCl but as the reactions are fast the only physiologically relevant kinetic data available have been extrapolated from data measured at high pH (>10). In this study these values have been determined at pH 7.4 using a newly developed competition kinetic approach that employs a fluorescently tagged methionine derivative as the competitive substrate (k(HOCl + Fmoc-Met), 1.5 × 10(8)M(-1)s(-1)). This assay was validated using the known k(HOCl + NADH) value and has allowed revised k values for the reactions of HOCl with Cys, N-acetylcysteine, and glutathione to be determined as 3.6 × 10(8), 2.9 × 10(7), and 1.24 × 10(8)M(-1)s(-1), respectively. Similar experiments with methionine derivatives yielded k values of 3.4 × 10(7)M(-1)s(-1) for Met and 1.7 × 10(8)M(-1)s(-1) for N-acetylmethionine. The k values determined here for the reaction of HOCl with thiols are up to 10-fold higher than those previously determined and further emphasize the critical importance of reactions of HOCl with thiol targets in biological systems.
U2 - 10.1016/j.freeradbiomed.2014.04.024
DO - 10.1016/j.freeradbiomed.2014.04.024
M3 - Journal article
C2 - 24794410
SN - 0891-5849
VL - 73
SP - 60
EP - 66
JO - Free Radical Biology & Medicine
JF - Free Radical Biology & Medicine
ER -
