Generation of the Superoxide Radical during Autoxidation of Oxymyoglobin

Gotoh, Toshio; Shikama, Keiji

doi:10.1093/oxfordjournals.jbchem.a131289

Cited by 100 publications

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“…Bovine Mb02 rs oxidized easily to metMb by the generation of superoxide with a primary chemical reaction (Brown & Mebine, 1969;Gotoh & Shikama, 1976).…”

Section: Resultsmentioning

confidence: 99%

Development of a Spectrophotometric Assay System for Evaluating Reducibility of Water-Soluble Substances

Ashida

Noguchi

Satō

2004

FSTR

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A spectrophotometric assay system for evaluating reducibility of water-soluble substances using native bovine oxymyoglobin (Mb02) was developed. With incubation of Mb02 solution at fixed temperature and pH, the autoxidation rate to metmyoglobin (metMb) was measured. The reductive effects of test samples on metMb could be estimated as numerical values based on the changes in the rate constants from Mb02 to metMb. The reductive effects of four kinds of water-soluble compounds were examined: L-ascorbic acid (AsA) strongly reduced the autoxidation rate of Mb02, glutathione and kojic acid showed reductive effects inferior to that of ASA, while gallic acid tended to promote autoxidation at high concentrations. The results of test compounds obtained by the present Mb02 assay were compared with data by ferricyanide assay and XTT assay, which are known methods for evaluating reducibility. The Mb02 assay is useful because of its superior features of sensitivity, application flexibility and capability of evaluating pro-oxidant activity of reducing agents.

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“…Bovine Mb02 rs oxidized easily to metMb by the generation of superoxide with a primary chemical reaction (Brown & Mebine, 1969;Gotoh & Shikama, 1976).…”

Section: Resultsmentioning

confidence: 99%

Development of a Spectrophotometric Assay System for Evaluating Reducibility of Water-Soluble Substances

Ashida

Noguchi

Satō

2004

FSTR

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“…According to our standard procedure, the rate of autoxidation of oxyhemoglobin was measured in 0.1 M buffer at 25°C over a wide pH range (4)(5)(6)(7)(8)(9)(10)(11)(12) and in the presence of 1 mM EDTA. For example, a 1-mL solution containing 0.2 M appropriate buffer and 2 mM EDTA was placed in a test tube and incubated in a water bath maintained at 25 (± 0.1)°C.…”

Section: Autoxidation Rate Measurementsmentioning

confidence: 99%

“…where k obs represents the observed first-order rate constant at a given pH value [9]. Therefore, the rate of the autoxidation reaction is written by:…”

Section: Stopped-flow Measurements For the Distal Histidine Swinging mentioning

confidence: 99%

The swinging movement of the distal histidine residue and the autoxidation reaction for midge larval hemoglobins

Kamimura

Matsuoka

Imai

et al. 2003

European Journal of Biochemistry

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Some insects have a globin exclusively in their fast-growing larval stage. This is the case in the 4th-instar larva of Tokunagayusurika akamusi, a common midge found in Japan. In the polymorphic hemoglobin comprised of 11 separable components, hemoglobin VII (Ta-VII Hb) was of particular interest. When its ferric met-form was exposed to pH 5.0 from 7.2, the distal histidine was found to swing away from the E7 position. As a result, the iron(III) was converted from a hexacoordinate to a pentacoordinate form by a concomitant loss of the axial water ligand. The corresponding spectral changes in the Soret band were therefore followed by stopped-flow and rapid-scan techniques, and the observed first-order rate constants of k out ¼ 25 s )1 and k in ¼ 128 s )1 were obtained for the outward and inward movements, respectively, of the distal histidine residue in 0.1 M buffer at 25°C. For O 2 affinity, Ta-VII Hb showed a value of P 50 ¼ 1.7 Torr at pH 7.4, accompanied with a remarkable Bohr effect (dH + ¼ )0.58) almost equal to that of mammalian hemoglobins. We have also investigated the stability property of Ta-VII HbO 2 in terms of the autoxidation rate over a wide range of pH from 4 to 11. The resulting pH-dependence curve was compared with those of another component Ta-V HbO 2 and sperm whale MbO 2 , and described based on a nucleophilic displacement mechanism. In light of the O 2 binding affinity, Bohr effect and considerable stability of the bound O 2 against acidic autoxidation, we conclude that T. akamusi Hb VII can play an important role in O 2 transport and storage as the major component in the larval hemolymph.Keywords: Insect (midge) Hb; distal (E7) histidine; pH jump; swinging movement; heme oxidation.In previous papers, we reported that the hemoglobin from the 4th-instar larva of Tokunagayusurika akamusi, a common midge (Diptera) found in eutrophic lakes in Japan, is comprised of as many as 11 separable components (IA, IB, II, III, IV, V, VIA, VIB, VII, VIII and IX) on a DEAEcellulose column, and found that these can be classified into two groups on the basis of their presence or absence of the distal (E7) histidine residue. For instance, hemoglobin VII consists of 150 amino-acid residues and contains the usual distal histidine at position 64, whereas component V replaces it by an isoleucine at position 66 [1]. This is certainly one of the unique characters of T. akamusi hemoglobins, as all the Chironomus hemoglobins have a distal histidine residue. Among the T. akamusi hemoglobins, component VII was of particular interest. When its ferric met-form was placed in acidic pH range, its Soret peak was considerably blue-shifted and accompanied by a marked decrease in intensity, indicative of the hemoglobin being converted into a structure quite similar to that of Aplysia (sea hare) myoglobin lacking the usual distal histidine residue. The pH-dependent Soret magnetic circular dichroism (CD) spectra also revealed that hemoglobin VII is in an equilibrium between a hexacoordinate and a pentacoordinate structure for it...

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“…Under air-saturated conditions, oxymyoglobin is oxidized easily to metmyoglobin with generation of the superoxide anion as M b 0 2 * metMb + 0; (1) where kobs represents the first-order rate constant observed at a given pH [13]. Therefore, the rate of autoxidation is given by Here it seemed of interest to examine the autoxidation rate of Paramecium MbOz compared with those of other oxymyoglobins from different sources.…”

Section: Stability Properties Of Paramecium Oxymyoglobinmentioning

confidence: 99%

Protozoan myoglobin from Paramecium caudatum

Tsubamoto

Matsuoka

et al. 1990

European Journal of Biochemistry

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Native oxymyoglobin (Mb02) was isolated directly from the cells of Paramecium caudatum with complete separation from metmyoglobin (metMb) on a DEAE-cellulose column. It was examined for its spectral and stability properties. When compared with sperm whale MbOz used as a reference, Paramecium MbO, was found to be much more susceptible to autoxidation over a wide range of pH (4-11) in 0.1 M buffer at 25°C. Kinetic analysis has revealed that a proton-catalyzed displacement of 0; from M b 0 2 by an entering water molecule can play a dominant role in the autoxidation reaction of Paramecium Mb02 to metMb, as in the case of sperm whale MbOz involving the distal histidine as its catalytic residue. At pH values higher than 9.5, however, Paramecium MbOz was found to be oxidized to yield a hemichrome. The spontaneous formation of hemichromes is at variance with the other known myoglobins and is therefore discussed in relation to the unusual amino acid sequence of Paramecium myoglobin having a large number of deletion [Iwaasa, H. et al. (1989) J . Mol. Biol. 208,.The occurrence of a hemoglobin-like protein in protozoa was first reported by Sat0 and Tamiya on the basis of spectroscopic observations on cell suspensions of Paramecium caudatum [I]. This was later confirmed by Keilin and Ryley with estimations of its content of 1.12 -1.74% [2]. Isolation and characterization of the so-called Paramecium hemoglobin, which is functionally referred to rather as myoglobin [3], have thus been carried out from Paramecium aurelia [4], P. tetraurelia [3], and P. caudatum and P. primaurelia [5].Very recently, we have determined the complete amino acid sequence of a myoglobin isolated from P. caudatum [6]. It consists of 116 amino acid residues with a molecular mass of 12565 Da including the heme moiety, this being much smaller than mammalian myoglobins by 37 residues. The protein contains two histidine residues at positions 68 and 84, but we failed to find any notable degree of sequence similarity with other known hemoglobins. In relation to these structural studies, the stability properties of the oxygenated form of Paramecium myoglobin should be of most importance and of primary concern, but these are little understood.In this paper, we describe procedures for isolating oxymyoglobin directly from the ciliated cells of P. caudatum and examine for the first time the autoxidation rate of oxymyoglobin (MbO,) to metmyoglobin (metMb) over a wide range of pH values. The resulting pH dependence was subjected to some mechanistic analysis in order to unravel the elementary processes involved in the stability properties of Paramecium MbOz. This kinetic analysis seems to be of great interest when compared with that of sperm whale MbOz as a reference, and should deepen our insight into the myoglobinhemoglobin chemistry and the evolution of these molecules from protozoa to higher animals, since the kinetics of the autoxidation should reflect differences on the distal side of the heme iron [7, 81. Correspondence to K. Shikama, Biological Institute,...

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Generation of the Superoxide Radical during Autoxidation of Oxymyoglobin

Cited by 100 publications

References 0 publications

Development of a Spectrophotometric Assay System for Evaluating Reducibility of Water-Soluble Substances

Development of a Spectrophotometric Assay System for Evaluating Reducibility of Water-Soluble Substances

The swinging movement of the distal histidine residue and the autoxidation reaction for midge larval hemoglobins

Protozoan myoglobin from Paramecium caudatum

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