The reaction of HOCl and cyanocobalamin: corrin destruction and the liberation of cyanogen chloride

Abstract

Overproduction of hypochlorous acid (HOCl) has been associated with the development of a variety of disorders such as inflammation, heart disease, pulmonary fibrosis, and cancer through its ability to modify various biomolecules. HOCl is a potent oxidant generated by the myeloperoxidase-hydrogen peroxide-chloride system. Recently, we have provided evidence to support the important link between higher levels of HOCl and heme destruction and free iron release from hemoglobin and RBCs. Our current findings extend this work and show the ability of HOCl to mediate the destruction of metal-ion derivatives of tetrapyrrole macrocyclic rings, such as cyanocobalamin (Cobl), a common pharmacological form of vitamin B12. Cyanocobalamin is a water-soluble vitamin that plays an essential role as an enzyme cofactor and antioxidant, modulating nucleic acid metabolism and gene regulation. It is widely used as a therapeutic agent and supplement, because of its efficacy and stability. In this report, we demonstrate that although Cobl can be an excellent antioxidant, exposure to high levels of HOCl can overcome the beneficial effects of Cobl and generate proinflammatory reaction products. Our rapid kinetic, HPLC, and mass spectrometric analyses showed that HOCl can mediate corrin ring destruction and liberate cyanogen chloride (CNCl) through a mechanism that initially involves α-axial ligand replacement in Cobl to form a chlorinated derivative, hydrolysis, and cleavage of the phosphonucleotide moiety. Additionally, it can liberate free Co, which can perpetuate metal-ion-induced oxidant stress. Taken together, these results are the first report of the generation of toxic molecular products through the interaction of Cobl with HOCl.

Figure 1

Structure of cyanocobalamin.

Figure 2

UV/Vis spectral changes and kinetic analysis of Cobl-HOCl interaction. Panel A shows the spectral changes for concentration dependence of HOCl-mediated corrin ring destruction. The dashed line represents spectral traces of Cobl (11 μM) recorded in phosphate buffer (200 mM, pH 7), at 25°C. Spectral traces (solid lines, from top to bottom) were recorded after 2 h of incubation of a fixed amount Cobl with increasing concentration of HOCl (100, 200, 300, 400, 600 and 1000 μM), at 25°C. Arrows in the panel indicate the direction of spectral change as a function of increasing concentration of HOCl. Panel B shows the stopped-flow trace monitored at 363 nm when a buffer solution containing Cobl (11 μM, final) was rapidly mixed with an equal volume of buffer solution supplemented with HOCl (1000 μM, final), at 25 °C. The red line represents the theoretical fit generated by the software when the raw data (blue line) was fitted to a two exponential function (Eq. 2). Panel C contains the kinetic traces for the reaction monitored at 590 nm, when Cobl (11 μM) was mixed with increasing concentrations of HOCl (250, 750, 1250 and 1500 μM). These data are representative of three independent experiments.

Figure 3

Rate constant of α-axial ligand replacement and corrin ring destruction of Cobl as a function of HOCl concentration. The observed rate constants for the formation of Cobl intermediate upon reacting Cobl with HOCl (panel A) and subsequent Cobl destruction (panel B) monitored at 363 nm observed in Fig. 3 were plotted as a function of HOCl concentration. A solution containing 11 μM Cobl was rapidly mixed with an equal volume of sodium phosphate buffer (200 mM, pH 7) supplemented with varying concentration of HOCl at 25 °C. The high concentration of the phosphate buffer is to keep the pH of the solution unaltered after the addition of HOCl. These data are representative of three independent experiments and the standard error for each individual rate constant has been estimated to be less than 5%.

Figure 4

Cobl destruction mediated by HOCl causes the liberation of CNCl. Cobl was treated with increasing molar ratios of HOCl:Cobl and CNCl generation was assayed colorimetrically as detailed in the Materials and Methods section. The data are a representative of three independent experiments with the error bars representing the standard error measurements.

Figure 5

Mass spectrometric detection of Cobl-HOCl reaction products. The extracted ion chromatogram (A) and MS spectrum (B) of unreacted Cobl as detected from the Cobl-HOCl reaction mixture. The molecular ion was detected in the [M+H]⁺ form and had a m/z of 1355. Disruption of coordination in Cobl when reacted with HOCl formed a ‘base-off’ intermediate. Examination of the MS spectrum revealed that the [M+H]⁺ ion had a m/z 1354. (C) The extracted ion chromatogram, (D) The MS spectrum of the peak and (E) the assigned structure of m/z 1354. Formation of a chlorinated derivative of Cobl on reacting with HOCl with a [M + H]⁺ ion having a m/z 1389. (F) The extracted ion chromatogram, (G) the MS spectrum of the peak. Note the presence of one chlorine atom as the ion intensity of the [M+H+2]⁺ ion is approximately 40% of [M+H]⁺, indicating a chlorine isotope pattern. (H) The assigned structure of m/z 1389.

Figure 6

Oxidative modification of the phosphonucleotide moiety and the corrin ring of Cobl. Extracted ion chromatogram (A & B) and MS spectrum (C) of oxidatively modified phosphonucleotide moiety and corrin degradation product, detected from the Cobl-HOCl reaction mixture. The molecular ion was detected in the [M+H]⁺ form and had a m/z of 279 and 579. The assigned structures of m/z 279 and 579 are shown in D & E.

Scheme 1

Kinetic model depicting the reaction between Cobl and HOCl leading to ligand replacement and corrin ring destruction.

Scheme 2

A general kinetic model explains the interaction of Cobl with HOCl leading to the axial ligand replacement, corrin ring degradation.

Scheme 2

A general kinetic model explains the interaction of Cobl with HOCl leading to the axial ligand replacement, corrin ring degradation.