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Biomedical subjects

C C Winterbourn

Publications and source records attributed to C C Winterbourn.

At least 181 records · Page 10Linked to original sources
PubMed1978

Mechanism of oxyhaemoglobin breakdown on reaction with acetylphenylhydrazine.

The reaction of oxyhaemoglobin and acetylphenylhydrazine, which results in haemoglobin denaturation and precipitation, was found to be influenced by H202 and superoxide (O2-.) generated during the reaction. By analysing the different haemoglobin oxidation products, it was found that by influencing the rate at which oxyhaemoglobin was oxidized, H2O2 accelerated the overall haemoglobin breakdown, and O2-. inhibited it. By adding GSH (reduced glutathione) or ascorbate, it was possible to slow down the rates of both oxyhaemoglobin oxidation and O2-. production, and the overall rate of haemoglobin breakdown. These results are compatible with a mechanism involving production of the acetylphenylhydrazyl free radical, and with GSH, ascorbate and O2-. acting as radical scavengers and preventing its further reactions. The reaction produced choleglobin, as well as acetylphenyldiazine and methaemoglobin, which combined to form a haemichrome. The haemichrome was less stable and precipitated first. It was also less stable than the haemichrome formed by direct reaction of acetylphenyldiazine with methaemoglobin, and it is proposed that this is because the methaemoglobin produced from oxyhaemoglobin and acetylphenylhydrazine was modified by the free radicals and H2O2 produced in the reaction.

Azo Compounds↗
PubMed1977

Haemoglobin North Shore, beta134 Val replaced by Glu, a new unstable haemoglobin.

A new mildly unstable haemoglobin, Hb North Shore (beta134 Val replaced by Glu) is described. It was found in a 4th generation Australian of Anglo-Celtic origin with a mild microcytic anaemia. The charge change involved is partially obscured as evidenced by the electrophoretic mobility of the native haemoglobin. The structural studies illustrate the usefulness of Staphylococcus aureus strain V8 protease as a specific cleavage enzyme at glutamyl residues.

Adult↗
PubMed1977

Oxidation of human haemoglobin by copper. Mechanism and suggested role of the thiol group of residue beta-93.

Addition of Cu(II) ions to human oxyhaemoglobin caused the rapid oxidation of the haem groups of the beta-chain. Oxidation required binding of Cu(II) to sites involving the thiol group of beta-93 residues and was prevented when these groups were blocked with iodoacetamide or N-ethylmaleimide. Equilibrium-dialysis studies showed three pairs of binding sites, two pairs with high affinity for Cu(II) and one pair with lower affinity. It was the second pair of high-affinity sites that were blocked with iodoacetamide and were involved in haem oxidation. Cu(II) oxidized deoxyhaemoglobin at least ten times as fast as oxyhaemoglobin, and analysis of rates suggested that binding rather than electron transfer was the rate-determining step. No thiol-group oxidation to disulphides occurred during the period of haem oxidation, although it did occur subsequently in the presence of oxygen, or when Cu(II) was added to methaemoglobin. It is proposed that thiol oxidation did not occur because there exists a pathway of electron transfer between the haem group and copper bound to the beta-93 thiol groups. The route for this electron transfer is discussed, as well as the implications as to the function of the beta-93 cysteine in the haemoglobin molecule.

Binding Sites↗
PubMed1977

Haemoglobin--a frustrated oxidase? Implications for red cell metabolism.

The haem proteins can be considered, in one aspect of their function, as machines for activating oxygen. In the case of oxygen-carriers such as haemoglobin, the globin has evolved so that its conformation limits access to the haem group, with resultant reversible release of oxygen. However, distortion of the globin may allow either the discharge of oxygen as the activated product superoxide or, more threateningly, allow direct function of the haemoglobin as an oxidative enzyme. Support for this is shown by the reaction with acetylphenylhydrazine where haemoglobin functions as both an oxidase and oxygenase. An implication of oxidase activity is the potential to initiate free radical formation particularly with unsaturated lipids. Observations of the acetylphenylhydrazine reaction emphasize the role of glutathione as a free radical scavenger.

Ascorbic Acid↗
PubMed1977

Isolation of high oxygen affinity hemoglobins.

This paper describes a method for isolating high oxygen affinity hemoglobins. It involves the selective derivatisation of the cysteine residue at position 93 in the beta chain. The iodoacetamide derivative of the high oxygen affinity hemoglobin was separated from the more negatively charged iodoacetic acid derivative of HbA on DEAE-Sephadex. The method was used to isolate two high oxygen affinity hemoglobins, one of which was subsequently identified as Hb Heathrow.

Amino Acid Sequence↗
PubMed1976

Reactions involving superoxide and normal and unstable haemoglobins.

Superoxide ions (O2-) oxidized oxyhaemoglobin to methaemoglobin and reduced methaemoglobin to oxyhaemoglobin. The reactions of superoxide and H2O2 with oxyhaemoglobin or methaemoglobin and their inhibition by superoxide dismutase or catalase were used to detect the formation of superoxide or H2O2 on autoxidation of oxyhaemoglobin. The rate of autoxidation was decreased at about 35% in the presence of both enzymes. The copper-catalysed autoxidation of Hb (haemoglobin) was also shown to involve superoxide production. Superoxide was released on autoxidation of three unstable haemoglobins and isolated alpha and beta chains, at rates faster than with Hb A. Reactions of superoxide with Hb Christchurch and Hb Belfast were identical with those with Hb A, and occurred at the same rate. Hb Koln contrasted with the other haemoglobins in that the thiol groups of residue beta-93 as well as the haem groups reacted with superoxide. Haemichrome formation from methaemoglobin occurred very rapidly with Hb Christchurch and Hb Belfast, as well as the isolated chains, compared with Hb A. The process did not involve superoxide production or utilization. The relative importance of autoxidation and superoxide production compared with haemichrome formation in the haemolytic process associated with these abnormal haemoglobins and thalassaemia is considered.

Copper↗
PubMed1976

The rate of reaction of superoxide radical ion with oxyhaemoglobin and methaemoglobin.

Superoxide radical ions (O2-) produced by the radiolytic reduction of oxygenated formate solutions and by the xanthine oxidase-catalysed oxidation of xanthine were shown to oxidize the haem groups in oxyhaemoglobin and reduce those in methaemoglobin as in reactions (1) and (2): (see articles) Reaction (1) is suppressed by reaction (8) when [O2-]exceeds 10 muM, but consumes all the O2- generated in oxyhaemoglobin solutions when [oxyhaemoglobin] greater than 160 muM and [O2-]less than 1 nM at pH 7. The yield of reaction (2) is also maximal in methaemoglobin solutions under similar conditions, but less than one haem group is reduced per O2- radical. From studies of (a) the yield of reactions (1) and (2) at variable [haemoglobin] and rates of production of O2-, (b) their suppression by superoxide dismutase, and (c) equilibria observed with mixtures of oxyhaemoglobin and methaemoglobin, it is shown that k1/k2=0.7 +/- 0.2 and k1 = (4 +/- 1) X 10(3) M-1-S-1 At pH7, and k1 and k2 decrease with increasing pH. Concentrations and rate constants are expressed in terms of haem-group concentrations. Concentrations of superoxide dismutase observed in normal erythrocytes are sufficient to suppress reactions (1) and (2), and hence prevent the formation of excessive methaemoglobin.

Heme↗
PubMed1975

The estimation of red cell superoxide dismutase activity.

A method is described for the estimation of red cell superoxide dismutase (erythrocuprein) and a normal range of activity established. It is likely that this enzyme is essential to the red cell for the detoxification of superoxide radicals, and plays a protective role similar to that of the glutathione-glutathione peroxidase system. It is suggested that superoxide dismutase deficiency may be an unrecognized cause of Heinz body hemolytic anemia. The separation of superoxide dismutase from hemoglobin by polyacrylamide gel electrophoresis is also described. Normal superoxide dismutase activity was measured in one case of Wilson's disease.

Adolescent↗
PubMed1974

Studies of hemoglobin denaturation and Heinz body formation in the unstable hemoglobins.

The sequential changes that occur during the precipitation on mild heating of the unstable hemoglobins, Hb Christchurch, Hb Sydney, Hb Köln, and Hb A, were examined with particular attention to the possibility of an accompanying oxidative process. Hb Christchurch, Hb Sydney, and Hb A precipitated with equal amounts of alpha- and beta-chains and full heme complement. Hb Köln, however, was one-half hemedepleted and showed a slight excess of precipitated beta-chains. In all cases the spectrum of the precipitated material was typical of a hemichrome. There was no evidence that sulfhydryl oxidation contributed to the precipitation process. Reduced glutathione was unable to protect the hemoglobin against precipitation, and mixed disulfide formation between the precipitating hemoglobin and glutathione was insignificant, even in the presence of excess glutathione. No blockade of beta93 cysteines could be demonstrated in the unstable hemoglobins. Precipitation of oxyhemoglobin and carboxyhemoglobin in all cases gave nonspecific oxidation of approximately two of the six hemoglobin sulfhydryl groups to give intra- and intermolecular disulfide bonds. Single alpha- and beta-chains, plus polymers of up to five or six chains linked by disulfide bridges, were demonstrated by polyacrylamide gel electrophoresis. This disulfide oxidation was not observed with deoxy- or methemoglobin and did not appear to influence the rate of precipitation. These findings fit the theoretical prediction that autoxidation of oxy- and carboxyhemoglobin is accompanied by formation of a free radical, with the reactions of this free radical being confined intramolecularly.Together, these results are in keeping with predictions based on the known structural abnormalities of the unstable hemoglobins, all of which result in greater molecular flexibility. Our findings support the conclusion that the usual precipitating event is altered bonding at the heme to give the formation of hemichromes. There is no evidence of an accompanying oxidative process that could pose a threat to the integrity of the red cell.

Anemia, Hemolytic, Congenital↗