Nonequivalence of chains in hemoglobin oxidation

Mansouri, Ali; Winterhalter, Kaspar H.

doi:10.1021/bi00748a020

Cited by 113 publications

(82 citation statements)

References 13 publications

(9 reference statements)

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“…Since MetHb was shown to be formed during the oxidation of isolated haemoglobin (Kikuchi et al, 1955;Mansouri & Winterhalter, 1973), it seems to be generally believed that MetHb is also produced in human erythrocytes. However, present results show that half-oxidized haemoglobins such as the (a2+fi3+)2 and (a3+fl2+)2 forms are present rather than MetHb as met-form haemoglobins in the erythrocytes under physiological conditions.…”

Section: Discussionmentioning

confidence: 99%

Analyis of met-form haemoglobins in human erythrocytes of normal adults and of a patient with hereditary methaemoglobinaemia due to deficiency of NADH-cytochrome b5 reductase

Tomoda¹,

Imoto²,

Hirano³

et al. 1979

Biochemical Journal

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Isoelectric-focusing analysis on an Ampholine/polyacrylamide-gel plate revealed that met-form haemoglobins are present as half-oxidized haemoglobins such as the (a2+fl3+)2 and (a3+,f2+)2 forms rather than as methaemoglobin in the erythrocytes of normal human adults and also of a patient with hereditary methaemoglobinaemia due to deficiency of NADH-cytochrome b5 reductase.It seems to be generally accepted that the oxidized haemoglobin, which represents less than 1 % of the total haemoglobins in normal human erythrocytes, is methaemoglobin (Bodansky, 1951). However, analytical investigations into whether the met-form haemoglobin is fully oxidized or partially oxidized have not been reported. Previously we showed that met-form haemoglobin in glucose-depleted erythrocytes is not MetHb, but half-oxidized haemoglobin (Tomoda et al., 1978a). This finding suggests that fully oxidized haemoglobin (methaemoglobin) might be absent in human erythrocytes even under physiological conditions. To study the possibility further, we carried out the identification of the met-form haemoglobins in intact human erythrocytes of normal subjects and also from a patient with hereditary methaemoglobinaemia due to deficiency of NADHcytochrome b5 reductase, by using isoelectricfocusing analysis.As a result, it was found that these contain halfoxidized haemoglobins such as the (a2+,f3+)2 and (a3+fl+)2 forms rather than MetHb as met-form haemoglobins. On the basis of the results, the mechanism of haemoglobin oxidation in human erythrocytes is discussed. ExperimentalBlood samples (5 ml) were freshly obtained without anticoagulant from 20 normal human adults (with their informed consent). Clotting ofthe blood samples was not observed, since silicone-coated injectors and glass tubes were used for collecting the blood and the Abbreviations used: HbA, haemoglobin A; MetHb, methaemoglobin. Throughout this paper the haemoglobins are, for convenience, referred to as though they were the unoxygenated forms.Vol. 181 samples were stood in an ice bath. The samples were centrifuged at 10000 rev./min (ray. 7.2 cm) for 1 min within 30 min. After removal of the plasma, the erythrocytes were suspended in 0.9 % NaCl solution and centrifuged at 10000rev./min for 1 min. The erythrocytes obtained by this procedure were haemolysed by the addition of 5 vol. of ice-cold distilled water. The haemolysates were further centrifuged at 10000rev./min for 20min to remove 'ghosts', and used for the experiments.The heparinized blood of a patient with hereditary methaemoglobinaemia due to deficiency of NADHcytochrome b5 reductase was also treated by the same procedure as stated above within 4h of drawing the sample, and used for the experiments. [The ferricyanide reductase and NADH-cytochrome b5 reductase activities in the haemolysate, which were measured by the methods of Hegesh & Avron (1967) and Sugita et al. (1971) respectively, were extremely low.]Concentrations of met-form haemoglobins were determined as described by Evelyn & Malloy (1938). The contents of met-form haemo...

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Section: Discussionmentioning

confidence: 99%

Analyis of met-form haemoglobins in human erythrocytes of normal adults and of a patient with hereditary methaemoglobinaemia due to deficiency of NADH-cytochrome b5 reductase

Tomoda¹,

Imoto²,

Hirano³

et al. 1979

Biochemical Journal

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“…A number of reports have appeared indicating that both electrolyte in general [4] and chloride ion [6] in particular influence the rate Of methemoglobin formation in blood. In addition there now seems little doubt that superoxide is present in erythrocytes in which normal autoxidation is occurring [7,8].…”

Section: Introductionmentioning

confidence: 99%

A role for chloride in the autoxidation of hemoglobin under conditions similar to those in erythrocytes

1974

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“…The unequal tendency for x and ,9 chains to undergo various oxidations has been noted previously by kinetic and magnetic resonance techniques [17,30,31], although not for carbonyls. The I chains are more easily oxidized in reactions that are dependent on pH, ligand, and amounts of organic phosphate.…”

Section: Discussionmentioning

confidence: 60%

Oxidation of Carbonyl

Lanir¹,

Caughey

Charache

1982

European Journal of Biochemistry

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Oxidation reactions of carbonyl species of hemoglobins A, Zurich, and Osler with ferricyanide to form methemoglobins have been investigated at pH 7.0 and 25 T . The kinetics support a two-step process in which, tirst, CO is lost to give the unliganded reduced species (deoxy), followed by oxidation in a one-electron transfer to ferricyanide ion. On and off rates for CO markedly affect the overall oxidation rate. That 2 subunits oxidize more rapidly than fl subunits was noted by changes in C -0 stretch frequency during the course of reactions with Hb A and Hb Zurich. The substitution of Arg for His at ,963 in Hb Zurich resulted in a slower overall oxidation rate ascribable to greater affinity for CO. MetHb formation from stripped HbCO A was biphasic with the rate for the final = 50 7; of the reaction about 30 :< faster than that for the initial = 50 "/,. Addition of inositol hexakisphosphate increased rates several-fold in both phases and shifted the point of change from slow to fast phase.

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Nonequivalence of chains in hemoglobin oxidation

Cited by 113 publications

References 13 publications

Analyis of met-form haemoglobins in human erythrocytes of normal adults and of a patient with hereditary methaemoglobinaemia due to deficiency of NADH-cytochrome b5 reductase

Analyis of met-form haemoglobins in human erythrocytes of normal adults and of a patient with hereditary methaemoglobinaemia due to deficiency of NADH-cytochrome b5 reductase

A role for chloride in the autoxidation of hemoglobin under conditions similar to those in erythrocytes

Oxidation of Carbonyl

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