Methemoglobin Research Articles

Formation of methemoglobin and metmyoglobin using 8‐aminoquinoline derivatives or sodium nitrite and subsequent reaction with cyanide

The kinetics of the oxidation of hemoglobin (Hb) and myoglobin (Mb) by sodium nitrite, 8-[(4-amino-1-methylbutyl)amino]-6-methoxy-quinoline diphosphate (primaquine), 6-methoxy-8-(6-diethylaminohexylamino)-4-methyl-quinoline dihydrochloride (WR6026) and 8-[(4-amino-1-methylbutyl)amino]-2,6-dimethoxy-4-methyl- 5-[(3-trifluoromethyl)phenoxy]quinoline succinate (WR238,605) were studied at pH values ranging from 7.4 to 7.6 and at 37 +/- 1 degrees C. The reaction between Hb and primaquine, WR6026 and WR238,605 resulted in precipitation, as did the reaction between Mb and WR238,605. The reaction between nitrite ion (NO2-) and Hb showed a lag period followed by an autocatalytic phase. The data in this study are consistent with and substantiate the proposed mechanism for the Hb-NO2- oxidation reaction. The reaction between Mb and NO2- at higher NO2- concentrations also showed a lag period followed by an autocatalytic period, while at lower NO2- concentrations no lag period was seen. The data suggest a shift in rate constant at these lower NO2- concentrations. The reaction between Mb and both WR6026 and primaquine followed a two-term rate law with oxidant-dependent and -independent terms. Concentration-effect curve data, along with these results, suggest the presence of a catalytic pathway. The rates of formation of cyanomethemoglobin and cyanometmyoglobin complexes from cyanide ion and methemoglobin (MHb) and metmyoglobin (MMb), respectively, were followed in the presence of the heme oxidants. The rate constants were all within a narrow range and suggest that complexation of cyanide by MHb and MMb is not affected by the presence of oxidants.

Effect of inositol hexakisphosphate on the spectroscopic properties of the nitric oxide derivative of ferrous horse and bovine hemoglobin

The effect of inositol hexakisphosphate (IHP) on the spectroscopic (EPR and absorbance) properties of the nitric oxide derivative of ferrous horse and bovine hemoglobin (Hb) has been investigated. In the absence of IHP, the nitric oxide derivative of ferrous horse Hb shows spectroscopic properties similar to those of the corresponding derivative of ferrous human Hb that are generally taken as typical of the high affinity state of tetrametric hemoproteins. Similar to human Hb, the addition of IHP to the nitric oxide derivative of ferrous horse Hb induces a transition toward a species characterized by spectral properties typical of the low affinity state of hemoglobins. Nevertheless, the equilibrium constant for IHP binding to the nitric oxide derivative of ferrous horse Hb (= 1.5 x 10(2) M-1) is much lower than that reported for the association of the polyphosphate to the same derivative of ferrous human Hb (greater than 3 x 10(5) M-1). Conversely, the spectroscopic properties of the nitric oxide derivative of ferrous bovine Hb are characteristic of the low affinity state of tetrameric hemoproteins, both in the absence and in the presence of IHP. These results, taken together with the behavior of the nitric oxide derivative of ferrous human Hb, provide further evidence for the peculiar oxygen binding properties of horse and bovine Hb.

Hemoglobins of the Lucina pectinata/bacteria symbiosis. II. An electron paramagnetic resonance and optical spectral study of the ferric proteins.

We report an optical and EPR spectral study of three hemoglobins, Hb I, II, and III, from the gill of the clam Lucina pectinata. Hemoglobin I reacts much more avidly with hydrogen sulfide than do Hbs II and III. The proximal ligand to the heme iron of each hemoglobin is histidyl imidazole. The acid/alkaline transition of ferric Hb I occurs with pK 9.6; those of ferric Hbs II and III with pK 6.6 and 5.9, respectively. At their acid limits each ferric hemoglobin exists as aquoferric hemoglobin. Broadening of the g = 6 resonance suggests that the bound water enjoys great positional freedom. Ferric Hb I, at the alkaline limit (pH 11), exists as ferric hemoglobin hydroxide. Ferric Hbs II and III, at their alkaline limit (pH 7.5), each exist as equal mixtures of two species. The low spin species with optical maxima near 541 and 576 nm and g values of 2.61, 2.20, and 1.82, are identified as ferric hemoglobin hydroxide. The high spin species, with optical maxima near 486 and 603 nm and g values of 6.71, 5.87, and 5.06, resemble Dicrocoelium hemoglobin and hemoglobin MSaskatoon. Here we show that Hbs II and III resemble hemoglobin MSaskatoon in which a distal tyrosinate oxygen ligated to the ferric heme iron at alkaline pH is displaced by water at acid pH. The H2S product of ferric Hb I is identified as ferric hemoglobin sulfide.

Hemoglobins of the Lucina pectinata/bacteria symbiosis. I. Molecular properties, kinetics and equilibria of reactions with ligands.

Three hemoglobins have been isolated from the symbiont-harboring gill of the bivalve mollusc Lucina pectinata. Oxyhemoglobin I (Hb I), which may be called sulfide-reactive hemoglobin, reacts with hydrogen sulfide to form ferric hemoglobin sulfide in a reaction that may proceed by nucleophilic displacement of bound superoxide anion by hydrosulfide anion. Hemoglobins II and II, called oxygen-reactive hemoglobins, remain oxygenated in the presence of hydrogen sulfide. Hemoglobin I is monomeric; Hb II and Hb III self-associate in a concentration-dependent manner and form a tetramer when mixed. Oxygen binding is not cooperative. Oxygen affinities are all nearly the same, P50 = 0.1 to 0.2 Torr, and are independent of pH. Combination of Hb I with oxygen is fast; k'on = (estimated) 100-200 x 10(6) M-1 s-1. Combination of Hb II and Hb III with oxygen is slow: k'on = 0.4 and 0.3 x 10(6) M-1 s-1, respectively. Dissociation of oxygen from Hb I is fast relative to myoglobin: koff = 61 s-1. Dissociation from Hb II and Hb III is slow: koff = 0.11 and 0.08 s-1, respectively. These large differences in rates of reaction together with differences in the reactions of carbon monoxide suggest differences in configuration of the distal heme pocket. The fast reactions of Hb I are comparable to those of hemoglobins that lack distal histidine residues. Slow dissociation of oxygen from Hb II and Hb III suggest that a distal residue may interact strongly with the bound ligand. We infer that Hb I may facilitate delivery of hydrogen sulfide to the chemoautotrophic bacterial symbiont and Hb II and Hb III may facilitate delivery of oxygen. The midpoint oxidation-reduction potential of the ferrous/ferric couple of Hb I, 103 +/- 8 mV, was independent of pH. Potentials of Hb II and Hb III were pH-dependent. At neutral pH all three hemoglobins have similar midpoint potentials. The rate constant for combination of ferric Hb I with hydrogen sulfide increases 3000-fold from pH 10.5 to 5.5, with apparent pK 7.0, suggesting that undissociated hydrogen sulfide is the attacking ligand. At the acid limit combination of ferric Hb I with hydrogen sulfide, k'on = 2.3 x 10(5) M-1 s-1, is 40-fold faster than combination with ferric Hb II or myoglobin.(ABSTRACT TRUNCATED AT 400 WORDS)

Oxygen binding to partially oxidized hemoglobin

We report on oxygen binding to partially oxidized (aquomet) hemoglobin. The fractional saturation with oxygen is evaluated by deconvoluting the optical absorption spectra, in the 500-700 nm wavelength region, in terms of oxyhemoglobin, deoxyhemoglobin and methemoglobin spectral components. Experiments have been performed with auto-oxidized samples and with samples obtained by mixing ferrous hemoglobin with fully oxidized hemoglobin (mixed samples). An increase in oxygen affinity and a decrease in cooperativity are observed on increasing the amount of ferric hemoglobin in the sample. A high cooperativity (nH approximately 2) is maintained even in the presence of 50-60% ferric hemes. Moreover, for equal amounts of methemoglobin the oxygen affinity is lower and the cooperativity higher for mixed samples than for those auto-oxidized. The results are analyzed within the framework of a modified Monod-Wyman-Changeux allosteric model taking into account the effects brought about by the presence of oxidized hemes and of alpha betta dimers. The distribution of ferric subunits within the tetramers in fully deoxygenated and fully oxygenated samples, as derived from the model, provides details on the cooperative behavior of partially oxidized hemoglobin.

On the pathogenesis of abortion in acute nitrite toxicosis of pregnant dairy cows

Six cows, 220 to 260 d pregnant, were used to study possible abortifacient effects of absorbed nitrite. Maternal and fetal bloodvessels were catheterized. Nitrite was infused intravenously (i.v.) for 30 minutes on Days 1, 3 and 5, commencing at least 5 d postcatheteration. The dose used was 7, 9.5 and 12 mg NO 2 −/kg bodyweight, respectively. Nitrite caused a dose-related (P < 0.05) conversion of maternal hemoglobin (Hb) into methemoglobin (MHb); a 30 to 50% decrease of mean arterial bloodpressure; an increase in heart rate, with dose-related recovery periods; and a decrease in partial oxygen tension (PO 2) of maternal blood. Maternal partial carbon dioxide tension (PCO 2), pH and electromyographic activity of the uterus were unchanged. Fetal changes included a small increase in MHb content, variable changes in heart rate (tachycardia and bradycardia), and decreases in fetal PO 2, with considerable differences between animals. All calves were born alive. Three cows calved early, 2 to 3 d after the highest nitrite dose. The hematological and cardiovascular data suggest that these three fetuses experienced a more serious hypoxemic stress than the other fetuses.

Temporal Changes in the Oxidation State in In Vitro Blood

The rates at which the paramagnetic compounds deoxyhemoglobin (Hb) and methemoglobin (MHb) form in vivo within an area of hemorrhage are unknown. The present experiment establishes the baseline concentrations and rates of change in paramagnetic hemoglobin concentrations, as well as the pH in normal heparinized and clotted human blood maintained in vitro at 37 degrees C under anaerobic conditions over 30 hours. There was a moderate increase in Hb concentration in normal heparinized blood (average increase was 15.5%, rate = 0.50%/hour) and a slight increase in MHb concentration in the heparinized blood and clots (average increase was 1.4%, rate = 0.044%/hour). A second experiment was done to verify the activity of the RBC systems responsible for maintaining the hemoglobin molecule in the reduced state. Conversion of MHb to Hb in these samples proceeded at a rate of 5.6%/hour. In a third experiment, blood from 11 normal subjects maintained at 4 degrees C 25 degrees C was analyzed for MHb concentration over the course of 28 days. The level of MHb formation remained in the range of normal for at least 11 days in all subjects. The authors conclude that at basal conditions created in vitro, the blood levels of both Hb and MHb remain at relatively low levels. Therefore, if the accumulation of Hb and/or MHb occurs in acute in vivo hematomas it must be driven by intrinsic tissue factors.

Optimization of Gradient-Echo Imaging Parameters for Intracaval Filters and Trapped Thromboemboli

Flow-phantom magnetic resonance (MR) gradient-echo (GRE) imaging at 1.5 T was performed on a titanium Greenfield filter containing trapped blood clots with a high concentration of either deoxyhemoglobin (DHb) or methemoglobin (MHb), simulating acute and older thromboemboli, respectively. Flip angle, repetition time (TR), and echo time (TE) were varied, and a contrast-to-noise ratio between trapped clots and flowing fluid (clot-flow contrast) was determined for each set of imaging parameters. Use of very low flip angles (less than or equal to 10 degrees) rendered MHb clots indistinguishable from flowing fluid. In general, DHb clots displayed greater clot-flow contrast than MHb clots regardless of flip angle. With increasing TE values, T2* effect was observed with MHb clots, and magnetic susceptibility artifacts increased. Overall, optimum clot-flow contrast for imaging of both DHb and MHb clots was achieved with a flip angle of 45 degrees-60 degrees, a TR of 50 msec, and the shortest TE possible. Using GRE parameters similar to the optimum parameters determined in vitro, the authors imaged four patients with nickel-titanium Simon filters and one dog with a titanium Greenfield filter. MR imaging was successful in demonstrating filter location, caval patency, and the presence and extent of intraluminal thrombus.

An antioxidant role for hemoglobin

Hemoglobin readily undergoes one-electron oxidations and reductions, and it can act as a source or sink of free radicals. An autoxidation of the heme groups produces O2– and, indirectly, H2O2. Hemoglobin also interacts with redox-active xenobiotics and metabolites, forming the xenobiotic radical and initiating a series of reactions that generate other radicals and oxidant species and often result in oxidative denaturation of the hemoglobin. This chapter focuses on the measurement of oxidant-mediated changes to hemoglobin. It defines the different oxidation products of hemoglobin and briefly describes general mechanisms for their production. The oxidation of oxyhemoglobin (oxyHb)4 gives O2– and methemoglobin (metHb). If the globin structure is destabilized, metHb can convert to hemichrome in which either the distal histidine or an external ligand occupies the sixth coordination position of the ferric heme. Hemichromes precipitate readily and are the main constituent of Heinz bodies. Choleglobin is a term used to describe denatured hemoglobin in which the porphyrin ring has been hydroxylated or broken open. Ferrylhemoglobin (ferrylHb or Hb2+H2O2) is an Fe(IV) complex formed from ferrous hemoglobin and H2O2. The reaction of metHb with H2O2 produces short-lived ferryl radicals, which are Fe (IV) species with the additional oxidizing equivalent localized on the globin.

The use of a superconducting magnetometer to measure spin equilibria in hemoglobin

Abstract We are very glad to have the opportunity to outline the outcome of so many years of happy collaboration in this volume to honour Prof. Giorgia Careri. Indeed it is quite appropriate to do so. Even if the reader will not find referenced any paper in collaboration with him, still his role in this work has been just as great and relevant. It is thanks to Prof. Careri that one of us (M.C.) was at first introduced to the world of superfluidity and superconductivity and then to biophysics. Superconducting electronics, with SQUIDs etc., was offering good opportunities for the developement of susceptometers sensitive and accurate enough for biological studies. The first such efforts were carried out in Jim Mercereau's laboratory in Caltech by E. Hoenig and Run Han Wang. One of the authors, M.C., following the style of Careri's laboratory, had the opportunity to spend one year doing research abroad in an outstanding laboratory. As he had been using SQUIDs for high resolution magnetic studies in solid state physics and wantend to expand his studies to biological matter, he chose to go to Caltech, where he also developed his interest in hemoglobin. Afterwards, in Careri's laboratories in Rome and Monterotondo, SQUID susceptometers were developed, with which the authors carried out the first experiment on ferric hemoglobin with Max Perutz. These studies have been pursued up to the present, even after M.C.'s laboratory moved to Trento. Therefore, we would like to thank Prof. Giorgio Careri for creating the opportunity by which we have been able to enjoy this collaboration for all these years.

XANES spectroscopy sensitivity to small electronic changes Case of carp azidomethemoglobin

Spin states equilibrium of hemoglobin-iron varies with external conditions: pH, allosteric effectors, temperature. The small electronic reorganization of the iron caused by the spin state changes has been detected by X-ray absorption near edge structure (XANES) spectroscopy at room temperature. The iron K-edge region which is sensitive to spin state is located in 7110–7130 eV. Here are presented the 100% high spin and 100% low spin XANES spectra of carp azido ferric hemoglobin.

Gill Hemoglobin May Deliver Sulfide to Bacterial Symbionts ofSolemya velum(Bivalvia, Mollusca)

Two different hemoglobins occur in nearly equal concentrations in the gill of the bivalve mollusc, Solemya velum (total hemoglobin concentration is 200 SM/kg wet weight gill). A spectrophotometric study of intact gill filaments demonstrates that in the absence of sulfide, the gill hemoglobin may be oxygenated and de oxygenated, with part (5â€20%) in the aquoferric form. In the presence ofsulfide, about halfofthe gill hemoglobin is rapidly and reversibly converted to ferric hemoglobin, which then binds sulfide to form ferric hemoglobin sul fide (ferric hemoglobin with sulfide ligated to the heme iron in the distal ligand position); the balance continues to bind oxygen as oxyhemoglobin. S. velum inhabits re duced marine sediments where oxygen and hydrogen sulfide meet, and houses a dense population of intracel lular chemoautotrophic sulfur-oxidizing symbiotic bac teria in its gill. We suggest that gill hemoglobins may me diate sulfide and oxygen delivery to the bacterial symbi ont. Because sulfide is the dominant electron donor to fuel the Solemya/bacteria symbiosis, a cytoplasmic sul fide-binding protein that prevents the spontaneous reac tion ofsulfide with oxygen may be ofutility in the nutri tion ofthe animal.

Susceptibility of glucose-6-phosphate dehydrogenase deficient red cells to primaquine enantiomers and two putative metabolites—I: Effect on reduced glutathione, methemoglobin content and release of hemoglobin

The effects of the primaquine (PQ) enantiomers, (+)PQ and (−)PQ, and two putative metabolites [5-hydroxyprimaquine (5HPQ) and 6-desmethyl-5-hydroxyprimaquine (6D5HPQ)] on methemoglobin (Met Hb) and glutathione content and release of hemoglobin into plasma from glucose-6-phosphate dehydrogenase (G-6-PD) deficient red cells were studied in vitro. The results show that a 1.5 mM concentration of (−)PQ produced a significantly greater increase in Met Hb content and decrease in reduced glutathione (GSH) level than did (+)PQ. However, the release of plasma hemoglobin was greater with (+)PQ than with (−)PQ. The hydroxy derivatives of primaquine, 5HPQ and 6D5HPQ, were significantly more active than PQ. Their individual effects differed; whereas 5HPQ produced significantly greater reduction in GSH compared to 6D5HPQ, the effect of 6D5HPQ on Met Hb content and release of plasma hemoglobin was greater than that of 5HPQ. The qualitative effects of these compounds on normal, heterozygous and hemizygous G-6-PD deficient red cells were similar, but quantitatively the effects were greatest on hemizygous G-6-PD deficient cells and intermediate on heterozygous cells.

Hemoglobin-mediated oxidation of the carcinogen 1,2-dimethylhydrazine to methyl radicals

Oxidation of 1,2-dimethylhydrazine (SDMH) catalyzed by hemoglobin is investigated by oxygen consumption studies, ESR spin-trapping experiments, and gas chromatography. Kinetic analysis and the study of the effects of superoxide dismutase, catalase, and azide on reaction rates indicate that SDMH oxidation is primarily dependent on ferric hemoglobin and autoxidatively formed H 2O 2. SDMH oxidation generates both methyl and hydroxyl radicals as ascertained by spin-trapping experiments with α-(4-pyridyl-1-oxide)- N-tert-butylnitrone, 5,5-dimethyl-1-pyrroline- N-oxide, and tert-nitrosobutane. Quantitative estimates indicate that the yield of the methyl radical trapped by α-(4-pyridyl-1-oxide)- N-tert-butylnitrone is about 8% of the consumed oxygen. Analysis of the gaseous products by gas chromatography shows methane formation at a yield 10 times lower than that obtained for the spin-trap methyl adduct. These results are discussed within the context of the spin-trapping technique. The relative efficiencies of oxyhemoglobin in catalyzing SDMH, 2-phenylethylhydrazine, and phenylhydrazine oxidation, defined as V max K M , are estimated as 1, 13, and 386, respectively. The higher efficiency obtained for the monosubstituted derivatives leads us to suggest that hemoglobin-catalyzed oxidation could be a detoxification route for these compounds. By contrast, SDMH oxidation requires a peroxidase-like activity, a fact that may be related to the efficacy and specificity of this carcinogen.

The effects of ethanol on the hematotoxicity of twelve pharmaceutical and environmental agents

Abstract The ability of ethanol (5%) to potentiate the oxidant stressor effects of twelve well‐known hematoxic agents was investigated in vitro using human erythrocytes. Human whole blood was incubated with one of the following agents with and without ethanol for one hour at 37°C: o‐aminophenol (0.05 mM); p‐benzoquinone (4.0 mM); butyl nitrite (1.0 mM); p‐hydroxyacetophenone (3.0 mM); hydroxylamine (0.5 mM); 0,N‐dimethylhydroxylamine (7.0 mM); 1,2‐naphthoquinone (0.4 mM); 1,4‐naphthoquinone (0.5 mM); p‐phenylenediamine (5.0 mM); phenylhydrazine (1.0 mM); potassium nitrite (1.0 mM) and primaquine (8.0 mM). Methemoglobin (METHB) and reduced glutathione (GSH) levels were subsequently measured. Synergistic increases in METHB levels occurred for primaquine, 1,2‐naphthoquinone and p‐phenylenediamine incubated with ethanol (p <.05).

The effect of ethanol on nitrite‐ and 1‐naphthol‐induced oxidant stress in human and sheep erythrocytes

Abstract The enhancement by ethanol of nitrite‐ and 1‐naphthol‐Induced oxidant stress was assessed in vitro in human and Dorset sheep erythrocytes as measured by changes in methemoglobin (MetHB) and glutathtone (GSH) levels. The human and sheep erythrocytes treated with nitrite (0.5, 1.0 and 2.0 mM), 1‐naphthol (1.0, 2.0 and 3.0 mM) or ethanol (0.1, 0.5, 1.0 and 5.0%) alone revealed significant increases in MetHb (P .05) except for sheep erythrocytes exposed to 1‐naphthol (P < .01) and ethanol (P < .05). The combined nitrite‐ethanol treatment resulted in greater than additive increases in MetHb leveis in both species; however, a protective effect occurred in sheep erythrocytes at the lowest combined treatment levels. The Joint naphthol‐ethanol treatment also resulted In synergtstic increases in MetHb levels in both species. No synergistic decreases in GSH levels were detected for either of the combined treatments. These results suggest that ethanol combined ...

The effect of BCNU on the responses of human erythrocytes to six oxidant stressors

Abstract It was hypothesized that the antineoplastic drug (1, 3‐bis(2‐chloroethyl)‐i‐nitrosourea (BCNU), a known and potent Inhibitor of erythrocyte glutathlone reductase (GSSG‐R), may enhance the oxidant stress of known oxidizing agents commonly found in the environment. Consequently, this paper assessed the effects of BCNU‐induced decreases (i.e. down to approximately 20% of normal) In human erythrocyte GSSG‐R on the toxicity of six known oxldant stressor agents (i.e. chlorite, copper, nitrite, p‐chloroaniline, paraquat and carbaryl) as measured by changes in methemoglobin (METHB) and reduced glutathlone (GSH). The results Indicated that a BCNU‐induced deficiency of GSSG‐R produced no enhanced formation of METHB or decrease in GSH under exposure to the above stressors.

Methemoglobin formation resulting from administration of candidate 8-aminoquinoline antiparasitic drugs in the dog

In vivo methemoglobin (MHb) formation caused by five 8-aminoquinoline compounds was tested in beagle dogs. Male beagle dogs were dosed orally once per day at 0.0116 mmol/kg for 4 consecutive days with primaquine (8-[4-amino-1-methylbutyl)amino]-6-methoxyquinoline, diphosphate), three candidate 8-aminoquinoline antimalarial drugs (WR 225,448 5-(3-trifluoromethyl)phenoxy-4-methyl primaquine, succinate); WR 238,605 2,6-dimethoxy-5-(3-trifluoromethyl)phenoxy-4-methyl primaquine, succinate; or WR 242,511 5-hexoxy-4-methyl primaquine, diphosphate dihydrate), or a candidate 8-aminoquinoline antileishmanial drug WR 6026 (8-[(6-diethylamino)amino]-6-methoxy-4-methyl quinoline, dihydrochloride). MHb and total hemoglobin levels were determined daily prior to dosing and for 29 days after drug administration. All compounds caused prolonged levels of MHb that peaked at Days 4 to 5 with disappearance half-lives of 5 to 9 days. Peak percentage MHb of primaquine, WR 6026, WR 238,605, WR 225,448, and WR 242,511 was 6.3, 20.7, 16.0, 25.3, and 48.1%, respectively. Total MHb as measured by area under the time-concentration curve was highest for WR 242,511, followed by WR 225,448, WR 238,605, WR 6026, and primaquine, respectively. The results of this study, in conjunction with other toxicity and efficacy studies, have been utilized to select one of these compounds for development as a replacement for the antimalarial drug primaquine, and also to characterize the MHb-forming properties of WR 6026.

Multiplicity of Different Stable Quaternary Conformations in Ferric Hemoglobin fromCamelus dromedarius.Modulation by Small Anions

The effect of small anions in modulating polyphosphate-induced conformational changes in ferric dromedary hemoglobin (Camelus dromedarius) was investigated. The results obtained indicate that: i) in solution, the simultaneous presence of polyphosphate and small anions is essential to obtain quaternary changes in the protein; ii) the charge, rather than the size or sterical configuration, mainly affects the quaternary structural equilibrium of the macromolecule; iii) three distinct, stable quaternary conformers of ferric dromedary hemoglobin were detected spectroscopically as a function of charge and concentration of small anion.

1H-NMR heme resonance assignments by selective deuterations in low-spin complexes of ferric hemoglobin A

The heme methyl and vinyl α-proton signals have been assigned in low-spin ferric cyanide and azide ligated derivatives of the intact tetramer of hemoglobin A, as well as the isolated chains, by reconstituting the proteins with selectively deuterated hemins. For the hemoglobin cyanide tetramer, assignment to individual subunits was effected by forming hybrid hemoglobins possessing isotope-labeled hemins in only one type of subunit. The heme methyl contact shift pattern has 1-methyl and 5-methyl shifts furthest downfield in both chains and the individual subunits of the intact hemoglobin in both the cyanide- and azide-ligated species, which is consistent with a dominant rhombic perturbation due to the proximal His-F8 imidazole π bonding in the known structure for human adult hemoglobin. The individual chain and subunit assignments confirm that the detailed electronic/magnetic properties of the heme pocket are essentially unaltered upon assembling the R-state tetramer from the isolated subunits.