Distinct chromatographic forms of human hemi-myeloperoxidase obtained by reductive cleavage of the dimeric enzyme. Evidence for subunit heterogeneity.

Taylor, K L; Guzman, GS; Pohl, Jan; Kinkade, Jr Jm

doi:10.1016/s0021-9258(18)55488-0

Cited by 25 publications

(4 citation statements)

References 43 publications

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“…However, addition of methionine, which scavenges hypohalous acids, had no effect on the burst phase (not shown). The burst phase also occurred to the same extent with hemi-myeloperoxidase, which is a reductively cleaved and alkylated form of the enzyme that contains only one heme group per molecule of protein ( , ) (not shown). This result rules out the possibility that the catalase activity was due to an intramolecular reaction between the two hemes of myeloperoxidase as proposed by Agner ().…”

Section: Resultsmentioning

confidence: 86%

A Kinetic Analysis of the Catalase Activity of Myeloperoxidase

Kettle

Winterbourn

2001

Biochemistry

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The predominant physiological activity of myeloperoxidase is to convert hydrogen peroxide and chloride to hypochlorous acid. However, this neutrophil enzyme also degrades hydrogen peroxide to oxygen and water. We have undertaken a kinetic analysis of this reaction to clarify its mechanism. When myeloperoxidase was added to hydrogen peroxide in the absence of reducing substrates, there was an initial burst phase of hydrogen peroxide consumption followed by a slow steady state loss. The kinetics of hydrogen peroxide loss were precisely mirrored by the kinetics of oxygen production. Two mols of hydrogen peroxide gave rise to 1 mol of oxygen. With 100 microM hydrogen peroxide and 6 mM chloride, half of the hydrogen peroxide was converted to hypochlorous acid and the remainder to oxygen. Superoxide and tyrosine enhanced the steady-state loss of hydrogen peroxide in the absence of chloride. We propose that hydrogen peroxide reacts with the ferric enzyme to form compound I, which in turn reacts with another molecule of hydrogen peroxide to regenerate the native enzyme and liberate oxygen. The rate constant for the two-electron reduction of compound I by hydrogen peroxide was determined to be 2 x 10(6) M(-1) s(-1). The burst phase occurs because hydrogen peroxide and endogenous donors are able to slowly reduce compound I to compound II, which accumulates and retards the loss of hydrogen peroxide. Superoxide and tyrosine drive the catalase activity because they reduce compound II back to the native enzyme. The two-electron oxidation of hydrogen peroxide by compound I should be considered when interpreting mechanistic studies of myeloperoxidase and may influence the physiological activity of the enzyme.

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Section: Resultsmentioning

confidence: 86%

A Kinetic Analysis of the Catalase Activity of Myeloperoxidase

Kettle

Winterbourn

2001

Biochemistry

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“…A weakly stained band also appeared at 40 kD and could be attributed to the heavy subunit without a prosthetic group, as postulated by several authors. 30,35 A sandwich ELISA for quantification of equine MPO in blood was developed following the classical sandwich ELISA method with 2 different polyclonal antibodies obtained against equine MPO. The sandwich complex (rabbit primary antibody-MPO-guinea pig secondary antibody) was recognized by a third antibody conjugated with alkaline phosphatase.…”

Section: Discussionmentioning

confidence: 99%

Development of an Enzyme-Linked Immunosorbent Assay for Specific Equine Neutrophil Myeloperoxidase Measurement in Blood

et al. 2005

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Equine inflammatory disease is accompanied by a neutrophil activation resulting in the release of granulocytic enzyme myeloperoxidase (MPO). To measure MPO in horse plasma as marker of neutrophil activation, the authors purified equine neutrophil MPO and developed a specific enzyme immunoassay using 2 specific polyclonal antibodies obtained from rabbit (primary antibody) and guinea pig (secondary antibody). The sandwich complex "primary antibody-MPO-secondary antibody" was detected using a goat anti-guinea pig immunoglobulin antibody conjugated to alkaline phosphatase. The enzyme-linked immunosorbent assay (ELISA) showed good precision and accuracy, with intra- and interassay coefficients of variation below 10% for MPO concentrations ranging from 0.78 to 50 ng/ml. A stable plasma MPO value, unaffected by time elapsed between blood collection and centrifugation, was obtained with plasma from EDTA anticoagulated blood. The mean MPO value measured in 38 healthy horses was 181.80 +/- 64.74 ng/ml. In 20 horses suffering from obstruction of the large or small intestine, MPO concentrations measured at the time of arrival at the intensive care unit were significantly higher than mean normal value, ranging from 477.88 to 2,748.13 ng/ml. Work is in progress to apply this MPO ELISA technique to other biological fluids and other equine diseases.

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“…The biphasic time courses could have been caused by a different reactivity of the two hemes, or it could be that the hemes exert a certain degree of negative cooperativity, such that when one heme is bound the rate of binding to the other is reduced. This possibility was considered in view of a recent finding that distinct chromatographic forms of human hemi-myeloperoxidase have been obtained by cleaving the dimeric enzyme (Taylor et al, 1990).…”

Section: Time (Min)mentioning

confidence: 99%

Spectral and Kinetic Studies on the Formation of Myeloperoxidase Compounds I and II: Roles of Hydrogen Peroxide and Superoxide

Marquez

Huang²,

Dunford³

1994

Biochemistry

141

145

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The conversion of myeloperoxidase to compounds I and II in the presence of H2O2 has been reinvestigated in order to explain the abnormal stoichiometry of compound I formation and the fast spontaneous decay of compound I to compound II. Rapid-scan studies show that at least a 20-fold excess of H2O2 is required to obtain a good spectrum of relatively pure compound I; a further increase in H2O2 concentration causes compound I to be reduced to compound II, which is a very stable intermediate. Compound I formation is reversible, with an apparent second-order forward rate constant of (1.8 +/- 0.1) x 10(7) M-1 s-1 and a reverse rate constant of 58 +/- 4 s-1, giving a constant of 3.2 microM for the dissociation of compound I to native enzyme and H2O. This reversibility is one factor that can explain the large excess of H2O2 required to form compound I. The apparent second-order rate constant for compound II formation from compound I and H2O2 is (8.2 +/- 0.2) x 10(4) M-1 s-1. We confirm pH dependence studies, which suggest that the formation of compounds I and II is controlled by a residue in the enzyme with a pKa of about 4.0. Excess H2O2 is also converted to O2 via catalase activity of the enzyme. However, we do not consider this a dominant pathway because it fails to account for the fast spontaneous reduction of compound I to compound II. The time courses for both the decay of compound I and the formation of compound II are biphasic.(ABSTRACT TRUNCATED AT 250 WORDS)

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Distinct chromatographic forms of human hemi-myeloperoxidase obtained by reductive cleavage of the dimeric enzyme. Evidence for subunit heterogeneity.

Cited by 25 publications

References 43 publications

A Kinetic Analysis of the Catalase Activity of Myeloperoxidase

A Kinetic Analysis of the Catalase Activity of Myeloperoxidase

Development of an Enzyme-Linked Immunosorbent Assay for Specific Equine Neutrophil Myeloperoxidase Measurement in Blood

Spectral and Kinetic Studies on the Formation of Myeloperoxidase Compounds I and II: Roles of Hydrogen Peroxide and Superoxide

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