Covalent Modifications of Hemoglobin by Nitrite Anion: Formation Kinetics and Properties of Nitrihemoglobin
Department of Chemistry, Carnegie Mellon University, Mellon Institute, Pittsburgh, Pennsylvania 15213, USA. Chemical Research in Toxicology
(Impact Factor: 3.53).
10/2010; 23(11):1786-95. DOI: 10.1021/tx100242w
The green nitrihemoglobin (α(2)β(2) tetramer, NHb) was prepared by the aerobic reaction of excess nitrite with human hemoglobin A(0) under mildly acidic conditions. A rate equation was determined and found to depend on nitrite, hydrogen ion, and oxygen concentrations: -d[HbNO(2)]/dt = [k(1) + k(2)(K(a)[HNO(2)])[O(2)](1/2)][HbNO(2)], where k(1) = (2.4 ± 0.9) × 10(-4) s(-1), k(2) = (1 ± 0.2) × 10(5) M(-5/2) s(-1), and K(a) is the acid dissociation constant for nitrous acid (4.5 × 10(-4) M). Also, the chemical properties of NHb are compared to those of the normal hemoglobin (including the addition products of common oxidation states with exogenous ligands, the alkaline transitions of the ferric forms, and the oxygen binding characteristics of the ferrous forms) and were found to be nearly indistinguishable. Therefore, the replacement of a single vinyl hydrogen with a nitro group on the periphery of each macrocycle in hemoglobin does not significantly perturb the interaction between the hemes and the heme pockets. Because nonphotochemical reaction chemistry must necessarily be most dependent on electronic ground states, it follows that the clearly visible difference in color between hemoglobin A(0) and NHb must be associated primarily with the respective electronic excited states. The possibility of NHb formation in vivo and its likely consequences are considered.
Available from: Prashanth Jayanna
- "Earlier studies were focusing on the methemoglobin-forming effect of nitrite that act as a scavenger by forming a relative stable complex of cyanomethemoglobin  . Very recent studies are focusing on the mitochondria-linked mechanism of nitrite as a nitric oxide donor   . Extensive researches are also focusing on developing effective sulfur-containing compounds serving as sulfur donors for reacting with CN with or without Rh. "
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ABSTRACT: Present studies have focused on a novel cyanide antidotal system, on the coencapsulation of a new sulfur donor DTO with rhodanese within sterically stabilized liposomes. The optimal lipid composition for coencapsulation of DTO with rhodanese is the combination of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, cholesterol, cationic lipid (DOTAP), and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] ammonium salt (with molar ratios of 82.7 : 9.2 : 3.0 : 5.1). With the optimized compositions, prophylactic and therapeutic in vivo efficacy studies were carried out in a mice model. When DTO was coencapsulated with rhodanese and thiosulfate the prophylactic antidotal protection was 4.9 × LD(50). Maximum antidotal protection against cyanide intoxication (15 × LD(50)) was achieved with coencapsulated rhodanese and DTO/thiosulfate in combination with sodium nitrite. When applied therapeutically, 100% survival rate (6/6) was achieved at 20 mg/kg cyanide doses with the encapsulated DTO-rhodanese-thiosulfate antidotal systems with and without sodium nitrite. These data are indicating that the appropriately formulated DTO is a promising sulfur donor for cyanide antagonism.
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ABSTRACT: Sodium nitrite alone is shown to ameliorate sublethal cyanide toxicity in mice when given from ∼1 h before until 20 min after the toxic dose as demonstrated by the recovery of righting ability. An optimum dose (12 mg/kg) was determined to significantly relieve cyanide toxicity (5.0 mg/kg) when administered to mice intraperitoneally. Nitrite so administered was shown to rapidly produce NO in the bloodsteam as judged by the dose-dependent appearance of EPR signals attributable to nitrosylhemoglobin and methemoglobin. It is argued that antagonism of cyanide inhibition of cytochrome c oxidase by NO is the crucial antidotal activity rather than the methemoglobin-forming action of nitrite. Concomitant addition of sodium thiosulfate to nitrite-treated blood resulted in the detection of sulfidomethemoblobin by EPR spectroscopy. Sulfide is a product of thiosulfate hydrolysis and, like cyanide, is known to be a potent inhibitor of cytochrome c oxidase, the effects of the two inhibitors being essentially additive under standard assay conditions rather than dominated by either one. The findings afford a plausible explanation for an observed detrimental effect in mice associated with the use of the standard nitrite-thiosulfate combination therapy at sublethal levels of cyanide intoxication.
Available from: George Richter-Addo
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ABSTRACT: Heme is an important cofactor in a large number of essential proteins and is often involved in small molecule binding and activation. Loss of heme from proteins thus negatively affects the function of these proteins but is also an important component of iron recycling. The characterization of intermediates that form during the loss of heme from proteins has been problematic, in a large part, because of the instability of such intermediates. We have characterized, by X-ray crystallography, three compounds that form during the nitrite-induced degradation of human α(2)β(2) hemoglobin (Hb). The first is an unprecedented complex that exhibits a large β heme displacement of 4.8 Å toward the protein exterior; the heme displacement is stabilized by the binding of the distal His residue to the heme Fe, which in turn allows for the unusual binding of an exogenous ligand on the proximal face of the heme. We have also structurally characterized complexes that display regiospecific nitration of the heme at the 2-vinyl position; we show that heme nitration is not a prerequisite for heme loss. Our results provide structural insight into a possible pathway for nitrite-induced loss of heme from human Hb.
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