Limits...
Kinetic studies on the reaction between dicyanocobinamide and hypochlorous acid.

Maitra D, Ali I, Abdulridha RM, Shaeib F, Khan SN, Saed GM, Pennathur S, Abu-Soud HM - PLoS ONE (2014)

Bottom Line: The formation of (OCl)(CN)-Cbi and its conversion to (OCl)2-Cbi was fitted to a first order rate equation with second order rate constants of 0.002 and 0.0002 µM(-1) s(-1), respectively.Plots of the apparent rate constants as a function of HOCl concentration for all the three steps were linear with Y-intercepts close to zero, indicating that HOCl binds in an irreversible one-step mechanism.Collectively, these results illustrate functional differences in the corrin ring environments toward binding of diatomic ligands.

View Article: PubMed Central - PubMed

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

ABSTRACT
Hypochlorous acid (HOCl) is a potent oxidant generated by myeloperoxidase (MPO), which is an abundant enzyme used for defense against microbes. We examined the potential role of HOCl in corrin ring destruction and subsequent formation of cyanogen chloride (CNCl) from dicyanocobinamide ((CN)2-Cbi). Stopped-flow analysis revealed that the reaction consists of at least three observable steps, including at least two sequential transient intermediates prior to corrin ring destruction. The first two steps were attributed to sequential replacement of the two cyanide ligands with hypochlorite, while the third step was the destruction of the corrin ring. The formation of (OCl)(CN)-Cbi and its conversion to (OCl)2-Cbi was fitted to a first order rate equation with second order rate constants of 0.002 and 0.0002 µM(-1) s(-1), respectively. The significantly lower rate of the second step compared to the first suggests that the replacement of the first cyanide molecule by hypochlorite causes an alteration in the ligand trans effects changing the affinity and/or accessibility of Co toward hypochlorite. Plots of the apparent rate constants as a function of HOCl concentration for all the three steps were linear with Y-intercepts close to zero, indicating that HOCl binds in an irreversible one-step mechanism. Collectively, these results illustrate functional differences in the corrin ring environments toward binding of diatomic ligands.

Show MeSH
Diode array rapid scanning spectra for the intermediates and corrin ring destruction by reacting (CN)2-Cbi with HOCl at three sequential time frames.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.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4222763&req=5

pone-0110595-g003: Diode array rapid scanning spectra for the intermediates and corrin ring destruction by reacting (CN)2-Cbi with HOCl at three sequential time frames.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.

Mentions: We next utilized a diode array stopped-flow spectrophotometer to investigate the kinetics of the reaction of (CN)2-Cbi with increasing concentrations of HOCl. Reactions were run in the dark (vitamin B12 derivatives have been considered light sensitive analytes) under aerobic conditions, at 25°C, pH 6.4–9.0. At physiologic pH, rapid mixing of a solution of 20 µM (CN)2-Cbi with 80-fold molar excess of HOCl resulted in the rapid formation of a transient intermediate that displayed a decrease in the corrin absorbance peak at 366 nm and broad visible bands centered at 493 and 613 nm, typical of a six-coordinate complex Figure 3 (upper Panel). This spectrum differs from that of (CN)2-Cbi, whose corrin maxima are centered at 366 with two resolved visible peaks at 538 and 578 nm [37]. This spectrum also differs from that of both α-cyano, β-aqua-cobinamide (UV/Vis 353, 496, and 525 nm) and α-aqua, β-cyano-cobinamide (UV/Vis 354, 497, and 527 nm) [37]. The spectrum for (CN)2-Cbi reported by Zhou and Zelder that was also supported by 1H NMR [37] matches our spectrum indicating that (CN)2-Cbi is the starting compound. Furthermore, there is no alteration in the (CN)2-Cbi upon the addition of excess CN- to the reaction mixture solidifying the evidence in favor of (CN)2-Cbi as the starting compound. The spectrum of the intermediate that initially formed following addition of HOCl/OCl- to (CN)2-Cbi is consistent with the replacement of one of the CN- groups with hypochlorite ion (OCl-). This Cbi intermediate formed within 2.4 s after mixing, but was unstable and rapidly converted into a more stable intermediate within 12 s, as judged by further decrease in the absorbance at 366 nm and a time-dependent shift in visible absorbance peak from 613 to 493 nm. These spectral changes were attributed to the replacement of the second CN- group with OCl-, as shown in Figure 3 (middle Panel). The second transient intermediate that formed within 12 s of initiating the reaction was also unstable and this was followed by spontaneous corrin ring destruction, within minutes of initiating the reaction (Figure 3, lower Panel). This conclusion was made by the decrease and flattening in the absorbance spectra indicating oxidative destruction of the corrin ring. Spectral transitions between each intermediate formed revealed distinct and well-defined isosbestic points (Figure 3). Thus sequential formation and decay of Cbi intermediates occur at sufficiently different rates to enable each process to be studied by conventional (i.e., single mixing) stopped-flow methods. Similar intermediates were observed when the experiments were repeated at pH 6.4 and 9.0 (where HOCl and OCl-, respectively, predominated); however the rate constants of the transition from one intermediate to another were slower (see below). Collectively, these results indicate that HOCl binds (CN)2-Cbi generating two unstable transient intermediates before corrin ring fragmentation. The generation of the transient octahedral intermediate, (OCl)(CN)-Cbi, and its conversion to (OCl)2-Cbi was characterized by one set of isosbestic points at 520 and 602 nm, as well as differing responses to varying HOCl concentrations.


Kinetic studies on the reaction between dicyanocobinamide and hypochlorous acid.

Maitra D, Ali I, Abdulridha RM, Shaeib F, Khan SN, Saed GM, Pennathur S, Abu-Soud HM - PLoS ONE (2014)

Diode array rapid scanning spectra for the intermediates and corrin ring destruction by reacting (CN)2-Cbi with HOCl at three sequential time frames.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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4222763&req=5

pone-0110595-g003: Diode array rapid scanning spectra for the intermediates and corrin ring destruction by reacting (CN)2-Cbi with HOCl at three sequential time frames.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.
Mentions: We next utilized a diode array stopped-flow spectrophotometer to investigate the kinetics of the reaction of (CN)2-Cbi with increasing concentrations of HOCl. Reactions were run in the dark (vitamin B12 derivatives have been considered light sensitive analytes) under aerobic conditions, at 25°C, pH 6.4–9.0. At physiologic pH, rapid mixing of a solution of 20 µM (CN)2-Cbi with 80-fold molar excess of HOCl resulted in the rapid formation of a transient intermediate that displayed a decrease in the corrin absorbance peak at 366 nm and broad visible bands centered at 493 and 613 nm, typical of a six-coordinate complex Figure 3 (upper Panel). This spectrum differs from that of (CN)2-Cbi, whose corrin maxima are centered at 366 with two resolved visible peaks at 538 and 578 nm [37]. This spectrum also differs from that of both α-cyano, β-aqua-cobinamide (UV/Vis 353, 496, and 525 nm) and α-aqua, β-cyano-cobinamide (UV/Vis 354, 497, and 527 nm) [37]. The spectrum for (CN)2-Cbi reported by Zhou and Zelder that was also supported by 1H NMR [37] matches our spectrum indicating that (CN)2-Cbi is the starting compound. Furthermore, there is no alteration in the (CN)2-Cbi upon the addition of excess CN- to the reaction mixture solidifying the evidence in favor of (CN)2-Cbi as the starting compound. The spectrum of the intermediate that initially formed following addition of HOCl/OCl- to (CN)2-Cbi is consistent with the replacement of one of the CN- groups with hypochlorite ion (OCl-). This Cbi intermediate formed within 2.4 s after mixing, but was unstable and rapidly converted into a more stable intermediate within 12 s, as judged by further decrease in the absorbance at 366 nm and a time-dependent shift in visible absorbance peak from 613 to 493 nm. These spectral changes were attributed to the replacement of the second CN- group with OCl-, as shown in Figure 3 (middle Panel). The second transient intermediate that formed within 12 s of initiating the reaction was also unstable and this was followed by spontaneous corrin ring destruction, within minutes of initiating the reaction (Figure 3, lower Panel). This conclusion was made by the decrease and flattening in the absorbance spectra indicating oxidative destruction of the corrin ring. Spectral transitions between each intermediate formed revealed distinct and well-defined isosbestic points (Figure 3). Thus sequential formation and decay of Cbi intermediates occur at sufficiently different rates to enable each process to be studied by conventional (i.e., single mixing) stopped-flow methods. Similar intermediates were observed when the experiments were repeated at pH 6.4 and 9.0 (where HOCl and OCl-, respectively, predominated); however the rate constants of the transition from one intermediate to another were slower (see below). Collectively, these results indicate that HOCl binds (CN)2-Cbi generating two unstable transient intermediates before corrin ring fragmentation. The generation of the transient octahedral intermediate, (OCl)(CN)-Cbi, and its conversion to (OCl)2-Cbi was characterized by one set of isosbestic points at 520 and 602 nm, as well as differing responses to varying HOCl concentrations.

Bottom Line: The formation of (OCl)(CN)-Cbi and its conversion to (OCl)2-Cbi was fitted to a first order rate equation with second order rate constants of 0.002 and 0.0002 µM(-1) s(-1), respectively.Plots of the apparent rate constants as a function of HOCl concentration for all the three steps were linear with Y-intercepts close to zero, indicating that HOCl binds in an irreversible one-step mechanism.Collectively, these results illustrate functional differences in the corrin ring environments toward binding of diatomic ligands.

View Article: PubMed Central - PubMed

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

ABSTRACT
Hypochlorous acid (HOCl) is a potent oxidant generated by myeloperoxidase (MPO), which is an abundant enzyme used for defense against microbes. We examined the potential role of HOCl in corrin ring destruction and subsequent formation of cyanogen chloride (CNCl) from dicyanocobinamide ((CN)2-Cbi). Stopped-flow analysis revealed that the reaction consists of at least three observable steps, including at least two sequential transient intermediates prior to corrin ring destruction. The first two steps were attributed to sequential replacement of the two cyanide ligands with hypochlorite, while the third step was the destruction of the corrin ring. The formation of (OCl)(CN)-Cbi and its conversion to (OCl)2-Cbi was fitted to a first order rate equation with second order rate constants of 0.002 and 0.0002 µM(-1) s(-1), respectively. The significantly lower rate of the second step compared to the first suggests that the replacement of the first cyanide molecule by hypochlorite causes an alteration in the ligand trans effects changing the affinity and/or accessibility of Co toward hypochlorite. Plots of the apparent rate constants as a function of HOCl concentration for all the three steps were linear with Y-intercepts close to zero, indicating that HOCl binds in an irreversible one-step mechanism. Collectively, these results illustrate functional differences in the corrin ring environments toward binding of diatomic ligands.

Show MeSH