Human eosinophil peroxidase: a novel isolation procedure, spectral properties and chlorinating activity

Wever, Ron; Plat, H.; Hamers, Mic N.

doi:10.1016/0014-5793(81)80320-1

Cited by 80 publications

(43 citation statements)

References 16 publications

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“…of at least 0n7, which indicates that it was greater than 85 % pure. 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].…”

Section: Purity Of Myeloperoxidasesupporting

confidence: 75%

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 Myeloperoxidasesupporting

confidence: 75%

Thiocyanate and chloride as competing substrates for myeloperoxidase

Dalen

Whitehouse

Winterbourn

et al. 1997

Biochemical Journal

384

293

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“…Except for the M243T mutant, none of the Met 243 mutants shows chlorination activity. In EPO a threonine is present at this position instead of a methionine, and it has been reported that EPO is also able to carry out the peroxidative chlorination of monochlorodimedon, although the kinetic properties differ (45,46). In this respect it is interesting that the M243T mutant also still has some chlorinating activity.…”

Section: Discussionmentioning

confidence: 99%

The Sulfonium Ion Linkage in Myeloperoxidase

Kooter¹,

Moguilevsky²,

Bollen³

et al. 1999

Journal of Biological Chemistry

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The heme group of myeloperoxidase is covalently linked via two ester bonds to the protein and a unique sulfonium ion linkage involving Met 243 . Mutation of Met 243 into Thr, Gln, and Val, which are the corresponding residues of eosinophil peroxidase, lactoperoxidase, and thyroid peroxidase, respectively, and into Cys was performed. The Soret band in the optical absorbance spectrum in the oxidized mutants is now found at approximately 411 nm. Both the pyridine hemochrome spectra and resonance Raman spectra of the mutants are affected by the mutation. In the Met 243 mutants the affinity for chloride has decreased 100-fold. All mutants have lost their chlorination activity, except for the M243T mutant, which still has 15% activity left. By Fourier transform infared difference spectroscopy it was possible to specifically detect the 13 CD 3 -labeled methionyl sulfonium ion linkage. We conclude that the sulfonium ion linkage serves two roles. First, it serves as an electron-withdrawing substituent via its positive charge, and, second, together with its neighboring residue Glu 242 , it appears to be responsible for the lower symmetry of the heme group and distortion from the planar conformation normally seen in heme-containing proteins.In the family of mammalian peroxidases, myeloperoxidase (MPO) 1 is an extraordinary peroxidase. First of all, the enzyme is the only mammalian peroxidase known to peroxidize chloride to hypochlorous acid at a substantial rate. Secondly, MPO differs in its spectroscopic characteristics by its unusual redshifted Soret band in the optical absorbance as well in its pyridine hemochrome spectrum, its complicated resonance Raman spectrum, and its inverted sign pattern of the Soret band in the MCD spectrum (1-6). Those differences have been attributed to the special nature or structure of the heme group in MPO. Because this heme is covalently bound to the protein, characterization of this chromophore has been difficult. Based on different spectroscopic techniques, a formyl-containing heme a, a chlorin, and a heme b prosthetic group have been proposed in the past (3,5,(7)(8)(9).Although the enzyme differs in spectroscopic and catalytic properties, the homology between MPO and the other mammalian peroxidases is high. MPO shares respectively 70, 61, and 47% identical residues with eosinophil peroxidase (EPO), lactoperoxidase (LPO), and thyroid peroxidase (TPO) (10 -12), and an even higher homology can be found among the residues in the active site. MPO is the only mammalian peroxidase for which a crystal structure is known, at 2.3 Å resolution (13). The structural data for human MPO suggested that three heme substituents form covalent bonds with amino acid side chains in the protein. Two ester bonds were claimed to be present, between modified methyl groups on pyrrole rings A and C and the amino acids Glu 242 and Asp 94 . In a recent study, we have provided the first direct evidence by FTIR difference spectroscopy that ester bonds link the heme groups in all mammalian peroxidases via the conser...

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“…Trace levels of contaminating eosinophil peroxidase were then removed by passage over a sulfopropylSephadex column (50). The purity of isolated MPO was established by demonstrating a Reinheitszahl (RZ) value of Ͼ0.85 (A 430 /A 280 ) by means of SDS-PAGE analysis with Coomassie Blue staining and in-gel tetramethylbenzidine peroxidase staining to confirm no observable contaminating eosinophil peroxidase activity (51).…”

Section: Methodsmentioning

confidence: 99%

Thiocyanate Modulates the Catalytic Activity of Mammalian Peroxidases

Tahboub¹,

Galijašević²,

Diamond³

et al. 2005

Journal of Biological Chemistry

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We investigated the potential role of the co-substrate, thiocyanate (SCN ؊ ), in modulating the catalytic activity of myeloperoxidase (MPO) and other members of the mammalian peroxidase superfamily (lactoperoxidase (LPO) and eosinophil peroxidase (EPO) ؊ concentration, depending on the experimental conditions. Collectively, these results illustrate for the first time the potential mechanistic differences of these three enzymes. A modified kinetic model, which incorporates our current findings with the mammalian peroxidases classic cycle, is presented. Myeloperoxidase (MPO)1 and other members of the mammalian peroxidase superfamily (eosinophil peroxidase (EPO) and lactoperoxidase (LPO)) display a crucial difference (within a wide range of biological processes) in their unique ability in catalyzing the H 2 O 2 -dependent peroxidation of halides and pseudohalides to produce antimicrobial agents and hypohalous acids (1-7). These heme-containing enzymes share 50 -70%

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Human eosinophil peroxidase: a novel isolation procedure, spectral properties and chlorinating activity

Cited by 80 publications

References 16 publications

Thiocyanate and chloride as competing substrates for myeloperoxidase

Thiocyanate and chloride as competing substrates for myeloperoxidase

The Sulfonium Ion Linkage in Myeloperoxidase

Thiocyanate Modulates the Catalytic Activity of Mammalian Peroxidases

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