Ferric Myeloperoxidase Research Articles

Compound I of myeloperoxidase

Summary The optical spectrum of the primary peroxide compound of myeloperoxidase (compound I) is reported. The spectrum, obtained in 1 msec after mixing native ferric myeloperoxidase with excess hydrogen peroxide, exhibits a Soret maximum at 425 nm (c = 52 mM−1 cm−1) and an increase in extinction of the ferric peroxidase at wavelengths higher than 607 nm. The spectrum suggests a structure for compound I of myeloperoxidase similar to those of horseradish peroxidase and catalase. Compound I spontaneously decays to the secondary compound (compound II) in a half time of ⋍ 100 msec. The role of compound I in chloride peroxidation is discussed.

Isolation procedure and some properties of myeloperoxidase from human leucocytes

1. 1. A rapid isolation procedure with a high yield for pure myeloperoxidase (donor:H 2O 2 oxidoreductase, EC 1.11.1.7) from normal human leucocytes is described. The enzyme was solubilized from leucocytes with the detergent, cetyltrimethylammonium bromide, and purified to apparent homogeneity. The yield of the enzyme was 17% with an absorbance ratio A 430nm/ A 280nm = 0.85. 2. 2. The purified enzyme showed three isoenzyme bands after polyacrylamide gel electrophoresis; ultracentrifuge studies indicated one homogeneous band with a molecular weight of 144 000. After reduction of myeloperoxidase, sodium dodecyl sulfate gel electrophoresis resolved an intense band (63 000 daltons) and a weak band (81 000 daltons). 3. 3. The carbohydrate content of the enzyme was at least 2.5%. Mannose, glucose and N-acetylglucosamine were present. The amino acid composition is reported. 4. 4. The EPR spectrum exhibited a high-spin heme signal with rhombic symmetry ( g x = 6.92, g y = 5.07 and g z = 1.95). Upon acidification this signal was converted into a signal with more axial symmetry ( g ⊥ = 5.89). At high pH (9.5) the EPR spectrum of the enzyme only shows low-spin ferric heme resonances. The circular dichroism spectra of ferric and ferrous myeloperoxidase in the visible and ultraviolet region show maxima and minima in ellipticity.

An EPR study of myeloperoxidase in human granulocytes

1. 1. EPR spectra of human granulocytes (4 · 10 8 cells per ml) show an intense high-spin ferric heme signal with rhombic symmetry ( g x = 6.90 and g y = 5.07) for the heme group. These g-values are identical to those of partially purified myeloperoxidase and thus the signal is derived from ferric myeloperoxidase. In chicken granulocytes, which contain little or no myeloperoxidase, only an axial type of heme iron signal, weak in intensity, can be detected at g = 6.0. 2. 2. Upon phagocytosis of latex particles by human granulocytes the high-spin heme signal with rhombic symmetry is slowly converted into a signal with axial symmetry ( g x = g y = 6.0), showing that the EPR signals of myeloperoxidase in the intact cell can be used to study the involvement of the enzyme in metabolic changes during phagocytosis.

Myeloperoxidase of the leukocyte of normal blood III. The reaction of ferric myeloperoxidase with superoxide anion

Myeloperoxidase (donor:H 2O 2 oxidoreductase, EC 1.11.1.7) Compound III was formed by the addition of superoxide anions (O 2 −), which had been produced from the electrolytic reduction of molecular oxygen in an aprotic solvent, to a solution of ferric myeloperoxidase. The formation of Compound III was inhibited by superoxide dismutase from pig erythrocytes. It is concluded that myeloperoxidase Compound III can be formed from the direct reaction between ferric myeloperoxidase and (O 2 −. By the use of electron spin resonance the concentration of O 2 − in a cathodic chamber was found to reach approx. 10 mM.

Myeloperoxidase of the leukocyte of normal blood. II. The oxidation-reduction reaction mechanism of the myeloperoxidase system

The initial reaction of the aerobic oxidation of NADH in the myeloperoxidase (donor: H 2O 2 oxidoreductase, EC 1.11.1.7) system was inhibited by superoxide dismutase when the latter had been added to the reaction mixture previously, but the oxidation of NADH was not inhibited when it was added halfway during the reaction. The formation of myeloperoxidase compound III in the aerobic solution containing NADH and 2,4-dichlorophenol was also inhibited by superoxide dismutase. From these results it was concluded that a superoxide anion radical and myeloperoxidase compound III are very important active intermediates for the aerobic myeloperoxidase-catalyzed oxidation of NADH. Myeloperoxidase compound III can be formed by direct reaction between ferric myeloperoxidase and O 2 −, and the electronic structure of myeloperoxidase compound III can be considered to be Fe 3+O 2 −. Cytochrome b 5 prepared from thrombocytes was effectively reduced by the NADH-myeloperoxidase system. Myeloperoxidase catalyzed a reaction between epinephrine and H 2O 2. The light absorption maxima of a product of the reaction between epinphrine and H 2O 2 corresponds to those of adrenochrome. Myeloperoxidase also catalyzed the reaction between epinephrine analogues (norepinephrine, dopamine, DOPA, tyrosine and phenylalanine) and H 2O 2.

Myeloperoxidase of the leukocyte of normal blood. I. Reaction of myeloperoxidase with hydrogen peroxide

Myeloperoxidase (donor:H 2O 2 oxidoreductase, EC 1.11.1.7; also known as verdoperoxidase, neutrophil peroxidase) prepared from normal peripheral blood leukocyte of pig reacted with hydrogen peroxide to form three compounds which correspond to Compounds I, II and III, well characterized in the reaction between hydrogen peroxide and peroxidases of horseradish and milk. The light absorption spectra of these compounds were found to be quite peculiar. Myeloperoxidase Compound III was formed during the aerobic oxidation of NADH and also from a direct reaction between ferrous myeloperoxidase and molecular oxygen. Ferrous myeloperoxidase formed a complex with CO under restricted conditions. From the results obtained it is concluded that, in spite of the remarkable anomaly in the absorption spectra, the reaction mechanism of myeloperoxidase is similar to that of peroxidases of horseradish and milk.