Purification and some properties of myeloperoxidase and eosinophil peroxidase from human blood

Olsen, R L; Little, C. H. A.

doi:10.1042/bj2090781

Cited by 119 publications

(75 citation statements)

References 28 publications

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“…There was no shoulder at 412 nm in its absorption spectrum, which would indicate the presence of eosinophil peroxidase [29]. SDS\PAGE under reducing conditions gave three bands with molecular masses of 57, 39 and 15n5 kDa (result not shown), as reported by others [30][31][32]. We also ran native gels of our enzyme preparations and stained for peroxidase activity ( Figure 1).…”

Section: Purity Of Myeloperoxidasementioning

confidence: 88%

Thiocyanate and chloride as competing substrates for myeloperoxidase

Dalen

Whitehouse

Winterbourn

et al. 1997

Biochemical Journal

384

293

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The neutrophil enzyme myeloperoxidase uses H2O2 to oxidize chloride, bromide, iodide and thiocyanate to their respective hypohalous acids. Chloride is considered to be the physiological substrate. However, a detailed kinetic study of its substrate preference has not been undertaken. Our aim was to establish whether myeloperoxidase oxidizes thiocyanate in the presence of chloride at physiological concentrations of these substrates. We determined this by measuring the rate of H2O2 loss in reactions catalysed by the enzyme at various concentrations of each substrate. The relative specificity constants for chloride, bromide and thiocyanate were 1:60:730 respectively, indicating that thiocyanate is by far the most favoured substrate for myeloperoxidase. In the presence of 100 mM chloride, myeloperoxidase catalysed the production of hypothiocyanite at concentrations of thiocyanate as low as 25 μM. With 100 μM thiocyanate, about 50% of the H2O2 present was converted into hypothiocyanite, and the rate of hypohalous acid production equalled the sum of the individual rates obtained when each of these anions was present alone. The rate of H2O2 loss catalysed by myeloperoxidase in the presence of 100 mM chloride doubled when 100 μM thiocyanate was added, and was maximal with 1 mM thiocyanate. This indicates that at plasma concentrations of thiocyanate and chloride, myeloperoxidase is far from saturated. We conclude that thiocyanate is a major physiological substrate of myeloperoxidase, regardless of where the enzyme acts. As a consequence, more consideration should be given to the oxidation products of thiocyanate and to the role they play in host defence and inflammation.

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Section: Purity Of Myeloperoxidasementioning

confidence: 88%

Thiocyanate and chloride as competing substrates for myeloperoxidase

Dalen

Whitehouse

Winterbourn

et al. 1997

Biochemical Journal

384

293

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“…Inhibition with higher concentrations of H z 0 2 is a property also shown by eosinophil and uterine enzymes [4, 341. However, gastric peroxidase is stimulated 50% by 2 M guanidinium-HC1, a property shown by human myeloperoxidase which is activated 30-35% by 1.8-3.0 M guanidinium-HC1 [35]. Human eosinophil enzyme is not activated under identical condition [351.…”

Section: Discussionmentioning

confidence: 99%

Purification, characterization and origin of rat gastric peroxidase

Banerjee

1986

European Journal of Biochemistry

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A membrane-bound peroxidase (EC 1.1 1.1.7) from rat stomach has been solubilized by 0.2% cetyltrimethylammonium bromide in the presence of 1.2 M NH4Cl. The enzyme was purified 3355-fold to apparent homogeneity as judged by acid polyacrylamide gel electrophoresis and appears to be a cationic protein. In sodium dodecyl sulfate gel electrophoresis, the enzyme shows single polypeptide band of M , 45000. In gel permeation, the M , has been estimated as 47000. Spectral properties indicate the presence of Soret band at 412 nm which shifts to 425 nm on complexation with CN-and to 430 nm on reduction with dithionite. The velocity constant, kl for the reaction of the proxidase with H202 is 1.38 x lo7 M-' s-' and K,,, for H,O, is 0.1 mM. The enzyme contains active sulphydryl groups and is inhibited by sulphydryl reagents of which p-hydroxymercuribenzoate is more reactive than mersalyl or N-ethylmaleimide. The enzyme is very resistant to thermal denaturation up to 65°C and also to chaotropic reagents at least up to 2 M above which it is inactivated. The enzyme shows similarity with the intestinal eosinophil peroxidase as regards the molecular mass, spectral, kinetic and some of the catalytic properties. However, they differ significantly in terms of their interaction with fluoride ion, sulphydryl reagents, chaotropic reagent and also with the antiserum against the gastric peroxidase. Histochemically, the gastric peroxidase is shown to be localised in the gastric gland proper of the fundic stomach, rich in parietal and chief cells.After the suggestion of Rytomaa and Teir tha't all animal tissue peroxidases (except thyroid and myeloperoxidase) are contributed by the eosinophils [l], the properties of the peroxidases purified from various tissues have been compared [2 -161 in order to establish whether the enzyme is distinct and intrinsic to each tissue or it is derived from the contaminating eosinophils. Recently King et al. [17] and Olsen and Little [5] have shown that estrogen-induced peroxidase in rat uterus is mainly contributed by the eosinophils. The enzyme from pig and rat intestine has also been characterized and shown to have some similarities with eosinophil peroxidase [2, 31. Very recently we have established that rat intestinal peroxidase is not an endogenous enzyme and is mainly derived from the eosinophils accumulating in the core of the intestinal villi [18].The presence of a highly active peroxidase in mouse gastric mucosa and its cellular and subcellular localization was reported earlier from our labortory [19]. Recently we have shown that this enzyme in rat gastric mucosa is modulated by adrenal glucocorticoids [20]. However, it is not established yet whether gastric peroxidase is an enzyme intrinsic to the tissue or is contributed by the eosinophls, as in the case of intestinal peroxidase [18]. We have isolated homogeneous gastric peroxidase and its molecular, spectral, kinetic, catalytic and immunological properties have been studied and compared with those of the intestinal eosinophil peroxidase. The data...

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“…To avoid permanently staining the Scotch Brite pads with crystal violet, we often electrophorese the dye front off the gel (800 -1000 V-h) when subsequently blotting native gels. One gel was washed and stained with trimethylbenzidine to assess peroxidase activity (33)(34)(35). The other gel was soaked in transfer buffer for 1 h and then subjected to electroblotting to a nitrocellulose filter for 850 V-h.…”

Section: Methodsmentioning

confidence: 99%

Impact of Two Novel Mutations on the Structure and Function of Human Myeloperoxidase

Goedken

McCormick

Leidal

et al. 2007

Journal of Biological Chemistry

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The heme protein myeloperoxidase (MPO) contributes critically to O 2 -dependent neutrophil antimicrobial activity. Two Japanese adults were identified with inherited MPO deficiency because of mutations at Arg-499 or Gly-501, conserved residues near the proximal histidine in the heme pocket. Because of the proximity of these residues to a critical histidine in the heme pocket, we examined the biosynthesis, function, and spectral properties of the peroxidase stably expressed in human embryonic kidney cells. Biosynthesis of normal MPO by human embryonic kidney cells faithfully mirrored events previously identified in cells expressing endogenous MPO. Mutant apopro-MPO was 90 kDa and interacted normally with the molecular chaperones ERp57, calreticulin, and calnexin in the endoplasmic reticulum. However, mutant precursors were not proteolytically processed into subunits of MPO, although secretion of the unprocessed precursors occurred normally. Although ␦-[14 C]aminolevulinic acid incorporation demonstrated formation of pro-MPO in both mutants, neither protein was enzymatically active. The Soret band for each mutant was shifted from the normal 430 to ϳ412 nm, confirming that heme was incorporated but suggesting that the number of covalent bonds or other structural aspects of the heme pocket were disrupted by the mutations. These studies demonstrate that despite heme incorporation, mutations in the heme environs compromised the oxidizing potential of MPO.

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Purification and some properties of myeloperoxidase and eosinophil peroxidase from human blood

Cited by 119 publications

References 28 publications

Thiocyanate and chloride as competing substrates for myeloperoxidase

Thiocyanate and chloride as competing substrates for myeloperoxidase

Purification, characterization and origin of rat gastric peroxidase

Impact of Two Novel Mutations on the Structure and Function of Human Myeloperoxidase

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