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Melatonin prevents myeloperoxidase heme destruction and the generation of free iron mediated by self-generated hypochlorous acid.

Shaeib F, Khan SN, Ali I, Najafi T, Maitra D, Abdulhamid I, Saed GM, Pennathur S, Abu-Soud HM - PLoS ONE (2015)

Bottom Line: Myeloperoxidase (MPO) generated hypochlorous acid (HOCl) formed during catalysis is able to destroy the MPO heme moiety through a feedback mechanism, resulting in the accumulation of free iron.Collectively, in addition to acting as an antioxidant and MPO inhibitor, MLT can exert its protective effect by preventing the release of free iron mediated by self-generated HOCl.Our work may establish a direct mechanistic link by which MLT exerts its antioxidant protective effect in chronic inflammatory diseases with MPO elevation.

View Article: PubMed Central - PubMed

Affiliation: Departments of Obstetrics and Gynecology, the C.S. Mott Center for Human Growth and Development, Wayne State University School of Medicine, Detroit, Michigan, United States of America.

ABSTRACT
Myeloperoxidase (MPO) generated hypochlorous acid (HOCl) formed during catalysis is able to destroy the MPO heme moiety through a feedback mechanism, resulting in the accumulation of free iron. Here we show that the presence of melatonin (MLT) can prevent HOCl-mediated MPO heme destruction using a combination of UV-visible photometry, hydrogen peroxide (H2O2)-specific electrode, and ferrozine assay techniques. High performance liquid chromatography (HPLC) analysis showed that MPO heme protection was at the expense of MLT oxidation. The full protection of the MPO heme requires the presence of a 1:2 MLT to H2O2 ratio. Melatonin prevents HOCl-mediated MPO heme destruction through multiple pathways. These include competition with chloride, the natural co-substrate; switching the MPO activity from a two electron oxidation to a one electron pathway causing the buildup of the inactive Compound II, and its subsequent decay to MPO-Fe(III) instead of generating HOCl; binding to MPO above the heme iron, thereby preventing the access of H2O2 to the catalytic site of the enzyme; and direct scavenging of HOCl. Collectively, in addition to acting as an antioxidant and MPO inhibitor, MLT can exert its protective effect by preventing the release of free iron mediated by self-generated HOCl. Our work may establish a direct mechanistic link by which MLT exerts its antioxidant protective effect in chronic inflammatory diseases with MPO elevation.

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Related in: MedlinePlus

Melatonin prevents MPO heme destruction mediated by self-generated HOCl during steady state catalysis.Fixed amount of MPO (1 μM) was incubated with fixed amount of Cl- (100 mM) and increasing concentration of MLT (12–200 μM), and the reaction mixtures were incrementally received fixed amount of H2O2 (20 μM, total concentration of 180 μM). After reaction completion, the spectra of the reaction mixtures were scanned from 300–700 nm.
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pone.0120737.g002: Melatonin prevents MPO heme destruction mediated by self-generated HOCl during steady state catalysis.Fixed amount of MPO (1 μM) was incubated with fixed amount of Cl- (100 mM) and increasing concentration of MLT (12–200 μM), and the reaction mixtures were incrementally received fixed amount of H2O2 (20 μM, total concentration of 180 μM). After reaction completion, the spectra of the reaction mixtures were scanned from 300–700 nm.

Mentions: We next performed UV-visible photometry to correlate the degree of catalytic inhibition with HOCl-mediated heme destruction. As shown in the Fig. 2 inset; blue trace, MPO-Fe(III) as isolated displays a Soret absorbance peak centered at 430 nm, with three additional peaks at 573, 630, and 694 nm. Since the addition of a high molar ratio of H2O2 to MPO causes the conversion of MPO to Compound (III) (MPO-FeII-O2 complex) [50], the oxidation of the MPO heme moiety mediated by self-generated HOCl was monitored by sequential addition of H2O2 (20 μM; 3 μl) (180 μM H2O2 total) to the MPO-Fe(III)/Cl− mixture. With each incremental addition of H2O2, there was a proportional decrease in the MPO Soret peak, indicating that HOCl-mediated MPO feedback inhibition is associated with MPO heme destruction. After the last addition of H2O2 (180 μM total) solution to enzyme mixture, the spectrum recording showed a flattening in the Soret peak at 430 nm indicating MPO heme destruction (Fig. 2 inset; red trace). This flattening in the Soret peak region occurred solely in the presence of Cl−, signifying HOCl to be the major cause of MPO heme destruction. To confirm that MLT prevents HOCl-mediated MPO heme destruction, a fixed amount of MPO/Cl− mixture was preincubated with increasing concentrations of MLT prior to incremental additions of H2O2 to the reaction mixture. Fig. 2 shows the percentage recovery of MPO heme content, measured at 430 nm after the last addition of the incremental H2O2 to the enzyme solution, as a function of MLT concentration. In the presence of a saturating amount of MLT (>100 μM), spectral analysis indicated no losses in the heme content. Under these conditions, the MPO-H2O2 system utilized MLT as a 1e- substrate for the formation and subsequent decay of Compound II. The accumulation and stability of MPO Compound II (characterized by a Soret absorbance peak at 450 nm) during catalysis depended on the MLT concentration. In the presence of lower MLT levels, addition of limited amounts of H2O2 (10 μM) to the solution mixture caused immediate appearance of MPO Compound II, which then decayed to the ferric form in the next few seconds. In the presence of higher MLT concentrations (e.g. 100–400 μM), no significant change in absorbance was observed upon the addition of an H2O2 solution to the MPO mixture, indicating that the rate of MPO compound II decay exceeded the rate of formation, which was consistent with previous results [51]. In the presence of 50 μM MLT, only 50% recovery was noted in the MPO Soret absorbance peak of the total enzyme. As shown in Fig. 2, the full protection of the MPO heme contents required the presence of a ratio 1:2 MLT: H2O2. Collectively, our results showed that heme destruction did not occur in the presence of MLT, where MPO began reducing H2O2 without generating HOCl, indicating that self-generated HOCl is the major cause of MPO inactivation.


Melatonin prevents myeloperoxidase heme destruction and the generation of free iron mediated by self-generated hypochlorous acid.

Shaeib F, Khan SN, Ali I, Najafi T, Maitra D, Abdulhamid I, Saed GM, Pennathur S, Abu-Soud HM - PLoS ONE (2015)

Melatonin prevents MPO heme destruction mediated by self-generated HOCl during steady state catalysis.Fixed amount of MPO (1 μM) was incubated with fixed amount of Cl- (100 mM) and increasing concentration of MLT (12–200 μM), and the reaction mixtures were incrementally received fixed amount of H2O2 (20 μM, total concentration of 180 μM). After reaction completion, the spectra of the reaction mixtures were scanned from 300–700 nm.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4383586&req=5

pone.0120737.g002: Melatonin prevents MPO heme destruction mediated by self-generated HOCl during steady state catalysis.Fixed amount of MPO (1 μM) was incubated with fixed amount of Cl- (100 mM) and increasing concentration of MLT (12–200 μM), and the reaction mixtures were incrementally received fixed amount of H2O2 (20 μM, total concentration of 180 μM). After reaction completion, the spectra of the reaction mixtures were scanned from 300–700 nm.
Mentions: We next performed UV-visible photometry to correlate the degree of catalytic inhibition with HOCl-mediated heme destruction. As shown in the Fig. 2 inset; blue trace, MPO-Fe(III) as isolated displays a Soret absorbance peak centered at 430 nm, with three additional peaks at 573, 630, and 694 nm. Since the addition of a high molar ratio of H2O2 to MPO causes the conversion of MPO to Compound (III) (MPO-FeII-O2 complex) [50], the oxidation of the MPO heme moiety mediated by self-generated HOCl was monitored by sequential addition of H2O2 (20 μM; 3 μl) (180 μM H2O2 total) to the MPO-Fe(III)/Cl− mixture. With each incremental addition of H2O2, there was a proportional decrease in the MPO Soret peak, indicating that HOCl-mediated MPO feedback inhibition is associated with MPO heme destruction. After the last addition of H2O2 (180 μM total) solution to enzyme mixture, the spectrum recording showed a flattening in the Soret peak at 430 nm indicating MPO heme destruction (Fig. 2 inset; red trace). This flattening in the Soret peak region occurred solely in the presence of Cl−, signifying HOCl to be the major cause of MPO heme destruction. To confirm that MLT prevents HOCl-mediated MPO heme destruction, a fixed amount of MPO/Cl− mixture was preincubated with increasing concentrations of MLT prior to incremental additions of H2O2 to the reaction mixture. Fig. 2 shows the percentage recovery of MPO heme content, measured at 430 nm after the last addition of the incremental H2O2 to the enzyme solution, as a function of MLT concentration. In the presence of a saturating amount of MLT (>100 μM), spectral analysis indicated no losses in the heme content. Under these conditions, the MPO-H2O2 system utilized MLT as a 1e- substrate for the formation and subsequent decay of Compound II. The accumulation and stability of MPO Compound II (characterized by a Soret absorbance peak at 450 nm) during catalysis depended on the MLT concentration. In the presence of lower MLT levels, addition of limited amounts of H2O2 (10 μM) to the solution mixture caused immediate appearance of MPO Compound II, which then decayed to the ferric form in the next few seconds. In the presence of higher MLT concentrations (e.g. 100–400 μM), no significant change in absorbance was observed upon the addition of an H2O2 solution to the MPO mixture, indicating that the rate of MPO compound II decay exceeded the rate of formation, which was consistent with previous results [51]. In the presence of 50 μM MLT, only 50% recovery was noted in the MPO Soret absorbance peak of the total enzyme. As shown in Fig. 2, the full protection of the MPO heme contents required the presence of a ratio 1:2 MLT: H2O2. Collectively, our results showed that heme destruction did not occur in the presence of MLT, where MPO began reducing H2O2 without generating HOCl, indicating that self-generated HOCl is the major cause of MPO inactivation.

Bottom Line: Myeloperoxidase (MPO) generated hypochlorous acid (HOCl) formed during catalysis is able to destroy the MPO heme moiety through a feedback mechanism, resulting in the accumulation of free iron.Collectively, in addition to acting as an antioxidant and MPO inhibitor, MLT can exert its protective effect by preventing the release of free iron mediated by self-generated HOCl.Our work may establish a direct mechanistic link by which MLT exerts its antioxidant protective effect in chronic inflammatory diseases with MPO elevation.

View Article: PubMed Central - PubMed

Affiliation: Departments of Obstetrics and Gynecology, the C.S. Mott Center for Human Growth and Development, Wayne State University School of Medicine, Detroit, Michigan, United States of America.

ABSTRACT
Myeloperoxidase (MPO) generated hypochlorous acid (HOCl) formed during catalysis is able to destroy the MPO heme moiety through a feedback mechanism, resulting in the accumulation of free iron. Here we show that the presence of melatonin (MLT) can prevent HOCl-mediated MPO heme destruction using a combination of UV-visible photometry, hydrogen peroxide (H2O2)-specific electrode, and ferrozine assay techniques. High performance liquid chromatography (HPLC) analysis showed that MPO heme protection was at the expense of MLT oxidation. The full protection of the MPO heme requires the presence of a 1:2 MLT to H2O2 ratio. Melatonin prevents HOCl-mediated MPO heme destruction through multiple pathways. These include competition with chloride, the natural co-substrate; switching the MPO activity from a two electron oxidation to a one electron pathway causing the buildup of the inactive Compound II, and its subsequent decay to MPO-Fe(III) instead of generating HOCl; binding to MPO above the heme iron, thereby preventing the access of H2O2 to the catalytic site of the enzyme; and direct scavenging of HOCl. Collectively, in addition to acting as an antioxidant and MPO inhibitor, MLT can exert its protective effect by preventing the release of free iron mediated by self-generated HOCl. Our work may establish a direct mechanistic link by which MLT exerts its antioxidant protective effect in chronic inflammatory diseases with MPO elevation.

No MeSH data available.


Related in: MedlinePlus