Nitric Oxide Scavenging by the Cobalamin Precursor Cobinamide

Abstract

Nitric oxide (NO) is an important signaling molecule, and a number of NO synthesis inhibitors and scavengers have been developed to allow study of NO functions and to reduce excess NO levels in disease states. We showed previously that cobinamide, a cobalamin (vitamin B 12 ) precursor, binds NO with high affinity, and we now evaluated the potential of cobinamide as a NO scavenger in biologic systems. We found that cobinamide reversed NO-stimulated fluid secretion in Drosophila Malpighian tubules, both when applied in the form of a NO donor and when produced intracellularly by nitricoxide synthase. Moreover, feeding flies cobinamide markedly attenuated subsequent NO-induced increases in tubular fluid secretion. Cobinamide was taken up efficiently by cultured rodent cells and prevented NOinduced phosphorylation of the vasodilator-stimulated phosphoprotein VASP both when NO was provided to the cells and when NO was generated intracellularly. Cobinamide appeared to act via scavenging NO because it reduced nitrite and nitrate concentrations in both the fly and mammalian cell systems, and it did not interfere with cGMP-induced phosphorylation of VASP. In rodent and human cells, cobinamide exhibited toxicity at concentrations >50 M with toxicity completely prevented by providing equimolar amounts of cobalamin. Combining cobalamin with cobinamide had no effect on the ability of cobinamide to scavenge NO. Cobinamide did not inhibit the in vitro activity of either of the two mammalian cobalamin-dependent enzymes, methionine synthase or methylmalonyl-coenzyme A mutase; however, it did inhibit the in vivo activities of the enzymes in the absence, but not presence, of cobalamin, suggesting that cobinamide toxicity was secondary to interference with cobalamin metabolism. As part of these studies, we developed a facile method for producing and purifying cobinamide. We conclude that cobinamide is an effective intra-and extracellular NO scavenger whose modest toxicity can be eliminated by cobalamin.

Cobinamides Are Novel Coactivators of Nitric Oxide Receptor That Target Soluble Guanylyl Cyclase Catalytic Domain

“…Because ligand-free cobinamides may also act as NO scavengers (Broderick et al, 2005), we focused our studies on dicyanocobinamide, which does not bind NO.…”

“…2C). However, in the absence of a bound ligand, the cobalt(II) ion scavenges NO (Broderick et al, 2005), limiting its application as an effective sGC regulator. To avoid the possible artifacts associated with NO scavenging, we focused our work on dicyanocobinamide, which binds NO poorly because all ligating positions of the cobalt(II) ion are occupied.…”

Reactions of Cobinamide with Glucose and Fructose

“…Aquahydroxocobinamide was prepared by acid hydrolysis of hydroxocobalamin and purified by reported procedure. [22] Purity of product was controlled by UV-vis spectrometry [23] and chromatography methods.…”

Nitrosyl-cobinamide (NO-Cbi), a new nitric oxide donor, improves wound healing through cGMP/cGMP-dependent protein kinase

“…This imparts several chemical differences between cobinamide and cobalamin, most notably that cobinamide has higher affinity for ligands, including NO. Thus, cobinamide can be used as an NO scavenger, while NO-Cbi can be used as an NO donor [20][21][22][23]. In this paper we report results of NO-Cbi in a cellular based wound-healing model using a robotic system for generating uniform scratch wounds and a software developed specifically for assessing the results.…”