Rate constants for the formation and decay of the intermediates that formed upon mixing (CN)₂-Cbi with HOCl.

<p>(CN)₂-Cbi was rapid-mixed with buffer containing HOCl at various concentrations. Rates of complex formation and decay were determined at three different pHs (6.5, 7.4, and 9.0) by following absorbance change at 613 or 493 nm, at 10°C, and the rate constants determined as described in the text. These data are representative of three independent experiments and the standard error for each individual rate constant has been estimated to be less than 3%.</p

Rate constants of the axial ligands replacement and corrin ring destruction of dicyanocobinamide as a function of HOCl concentration.

<p>Upper panel, the observed rate constants of (OCl)(CN)-Cbi complex formation (open circles) and its conversion to (OCl)₂-Cbi (monitored at 613 nm) (closed circles) observed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110595#pone-0110595-g003" target="_blank">Fig. 3</a> were plotted as a function of HOCl concentration. R² values for the first and second phases were 0.992 and 0.995 respectively. Data represent the mean of triplicate determinations from an experiment performed three times. Lower panel, the rate constants for the corrin ring destruction, for the same reaction, monitored at 493 nm as a function of HOCl concentration. R² value was found to be 0.987. These data are representative of three independent experiments and the standard error for each individual rate constant has been estimated to be less than 8%.</p

Diode array rapid scanning spectra for the intermediates and corrin ring destruction by reacting (CN)₂-Cbi with HOCl at three sequential time frames.

<p>Panel A, spectra traces collected at 0.0, 0.4, 0.8, 1.2, 1.6, and 2.4 s and was attributed to the replacement of the first molecule of CN^- with OCl in (CN)₂-Cbi. Panel B, spectra traces collected at 2.4, 5.0, 7.4, and 12.0 and were attributed to the replacement of the second molecule of CN^- with OCl in (CN)₂-Cbi. Panel C, spectra collected at 12.0, 22.0, 40.0, and 120.0 s and was attributed to corrin ring destruction. Experiments were carried out by rapid mixing a phosphate buffer solution (200 mM, pH 7.0), at 25°C, supplemented with 20 µM (CN)₂-Cbi with a same volume of a buffer solution supplemented with 80-fold excess of HOCl. Arrows indicate the direction of spectral change over time as each intermediate advanced to the next. These data are representative of three independent experiments.</p

The effect of HOCl concentration on the formation, duration of (OCl) (CN)-Cbi and its conversion to (OCl)₂-Cbi.

<p>A solution containing sodium phosphate buffer (200 mM, pH 7.0) supplemented with 5 µm (final) dicyanocobiamide was rapidly mixed with an equal volume of buffer containing increasing concentrations of HOCl (200, 300, 600, 800, and 1200 µM, final) at 25°C. Replacement of the first CN^- molecule by OCl^-, duration, and its decay to (OCl)₂-Cbl were monitored as a function of time by observing spectral changes at 613 nm. The final concentration of HOCl in mixtures is indicated.</p

Structures of cyanocobalamin (Cbl) (left panel) and dicyanocobinamide ((CN)₂-Cbi)) (right panel).

<p>Structures of cyanocobalamin (Cbl) (left panel) and dicyanocobinamide ((CN)₂-Cbi)) (right panel).</p

Cyanocobalamin and (CN)₂-Cbi destruction mediated by HOCl causes the liberation of CNCl.

<p>Equal concentrations of Cbl and (CN)₂-Cbi (110 µM) were treated with 50-fold molar excess of HOCl and CNCl generation were assayed colorimetrically as detailed under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110595#s2" target="_blank">Materials and methods</a>. The data are representative of three independent experiments with the error bars representing the standard error measurements.</p

HPLC analysis shows MLT oxidation thereby preventing MPO heme destruction and generation of free iron.

<p>A) HPLC trace for MLT (elution time 3.98 min) dissolved in DMF (elution time 3.31 min) and phosphate buffer (elution time 2.48 min). B) Addition of MPO and Cl^- causes no significant change in MLT peak intensity and/or retention time. C) Addition of H₂O₂ (sequential addition of 20 μM, total 200 μM) results in a significant shift in MLT retention time elution time (3.71 min) as well as the appearance of a small peak around 3.57 min. D) Increasing levels of H₂O₂ (400 μM) resulted in the domination of the MLT metabolite eluted at 3.57 min showing the retention and absorbance properties of AFMK (elution time 3.57 min) as shown in panel (E).</p

Melatonin prevents MPO heme destruction mediated by self-generated HOCl during steady state catalysis.

<p>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.</p

Melatonin prevents HOCl mediated MPO heme destruction and subsequent free iron release during MPO catalysis.

<p>MPO (1.2 μM) was incubated with 100 mM Cl⁻ in the absence and presence of 400 μM MLT followed by the addition of aliquots of H₂O₂ (in increments of 20 μM) to the reaction mixture. The free iron released was measured using ferrozine assay as detailed under Materials and methods. No free iron was detected before the addition of H₂O₂. The data are the averages of three independent experiments with the error bars representing the standard error of measurement.</p