Rat liver-mediated metabolism of hydroxyurea to nitric oxide.

Hydroxyurea is an approved treatment for sickle cell disease. Oxidation of hydroxyurea results in the formation of nitric oxide (NO), which also has drawn considerable interest as a sickle cell disease therapy. Although patients on hydroxyurea demonstrate elevated levels of nitric oxide-derived metabolites, little information regarding the site or mechanism of the in vivo conversion of hydroxyurea to nitric oxide exists. Chemiluminescence detection experiments show the ability of crude rat liver homogenate to convert hydroxyurea to nitrite/nitrate, evidence for NO production. Nitrite/nitrate form at therapeutic concentrations of hydroxyurea in a clinically relevant time frame. Electron paramagnetic resonance (EPR) studies show the formation of iron nitrosyl complexes during this incubation and experiments with labeled hydroxyurea show the NO derives from the drug. Gas chromatography-mass spectrometry measurements indicate the hydrolysis of hydroxyurea to hydroxylamine in this system. Incubation of hydroxylamine with crude rat liver homogenate also generates nitrite/nitrate and iron nitrosyl complexes. A line of evidence including inhibitor studies, EPR spectroscopy, and nitrite/nitrate detection identifies catalase as a possible oxidant for the conversion of hydroxyurea to NO. These results reveal the ability of liver tissue to convert hydroxyurea to nitric oxide and provide insight into the metabolism of this drug.

Synthesis of chemical probes to map sulfenic acid modifications on proteins.

Cysteine sulfenic acids in proteins can be identified by their ability to form adducts with dimedone, but this reagent imparts no spectral or affinity tag for subsequent analyses of such tagged proteins. Given its similar reactivity toward cysteine sulfenic acids, 1,3-cyclohexadione was synthetically modified to an alcohol derivative and linked to fluorophores based on isatoic acid and 7-methoxycoumarin. The resulting compounds retain full reactivity and specificity toward cysteine sulfenic acids in proteins, allowing for incorporation of the fluorescent label into the protein and "tagging" it based on its sulfenic acid redox state. Control experiments using dimedone further show the specificity of the reaction of 1,3-diones with protein sulfenic acids in aqueous media. These new compounds provide the basis for an improved method for the detection of protein sulfenic acids.

Rates of nitric oxide dissociation from hemoglobin.

Nitric oxide (NO) plays a major role in human physiology and in many pathological states. Although oxyhemoglobin is known to destroy NO activity, NO activity can, in principle, be conserved through iron nitrosylation at vacant hemes. In order for this NO activity to be delivered, the NO must dissociate from the heme. Despite its study over the past few decades, our understanding of NO dissociation from hemoglobin is incomplete. In principle, there are at least four NO dissociation rates: kR(alpha), kR(beta), kT(alpha), and kT(beta), where the subscript refers to the quaternary state and the superscript to the hemoglobin chain. In the T-state, a proportion of the proximal histidine bonds break forming pentacoordinate alpha-nitrosyl hemoglobin. In vivo, alpha-nitrosyl hemoglobin predominates over beta-nitrosyl hemoglobin. In this study we have used a fast NO trap, Fe(II)-proline-dithiocarbamate, to measure NO dissociation rates from hemoglobin. We have varied solution conditions so the rate of dissociation from pentacoordinate alpha-nitrosyl hemoglobin could be definitively measured for the first time; kT(alpha) = 4.2 +/- 1.5 x 10(-4) s(-1). We have also found that the fastest NO dissociation rate is on the order of 10(-3) s(-1) and that NO dissociation from sickle cell hemoglobin is the same as that from normal adult hemoglobin.