Figure 1.
Structures of cyanocobalamin (Cbl) (left panel) and dicyanocobinamide ((CN)2-Cbi)) (right panel).
Figure 2.
Stability of (CN)2-Cbi as a function of pH.
UV-visible difference measurements recorded after dissolving equal amounts of (CN)2-Cbi (14 µM, final) at different pH values as described under “Materials and Methods.”
Figure 3.
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.
Figure 4.
The effect of HOCl concentration on the formation, duration of (OCl) (CN)-Cbi and its conversion to (OCl)2-Cbi.
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.
Figure 5.
Rate constants of the axial ligands replacement and corrin ring destruction of dicyanocobinamide as a function of HOCl concentration.
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 Fig. 3 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%.
Figure 6.
Cyanocobalamin and (CN)2-Cbi destruction mediated by HOCl causes the liberation of CNCl.
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 Materials and methods. The data are representative of three independent experiments with the error bars representing the standard error measurements.
Figure 7.
Rate constants for the formation and decay of the intermediates that formed upon mixing (CN)2-Cbi with HOCl.
(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%.