Native Myeloperoxidase Research Articles

Molecular analysis of the human myeloperoxidase promoter region.

Myeloperoxidase (MPO) is a granule protein, transiently expressed during the promyelocyte stage of myeloid differentiation. It is transcribed in a stage and lineage specific manner. Studies of MPO gene regulation can help to elucidate the mechanism of normal and abnormal myeloid differentiation. Our preliminary data indicated the lack of basal promoter activity in the region immediately 5' to the MPO cDNA. Here, we report the results of the detailed molecular studies of the human MPO promoter region. To locate potential promoter elements active in HL60 cells, we made promoter deletion constructs ranging in size from 200 bp to 4.5 kb of the 5' region of the hMPO gene, cloned into the chloramphenicol acetyl transferase (CAT) reporter vector. Following electroporation of the promoter constructs into HL60 cells, CAT enzyme production was found only in the construct containing approximately the 1 kb region upstream of the reported MPO cDNA. A separate set of constructs was made to look for putative MPO enhancer elements. Several fragments upstream of the MPO promoter showed prominent transactivation of the TK promoter, indicating a possible enhancer. Tissue specificity of MPO promoter fragments was determined in myeloid cells arrested either before induction of MPO expression (KG1), during MPO expression (HL60), or after it had ceased (U937), as well as in non-MPO expressing non-myeloid cells. The construct containing an approximate 1000 bp fragment of the 5' region of MPO was found to direct CAT expression only in HL60 cells. The 3'-truncations of this promoter region resulted in loss of tissue-specificity, while the promoter activity remained largely unchanged. A negative regulatory element was found upstream of the MPO promoter which repressed heterologous promoters in all the tested cell lines. Enhancer elements showed no tissue- or stage-specificity that were characteristic for native MPO gene. Sequence analysis of the putative MPO promoter region showed a number of potential transcription factor binding sites. Of special interest is the region containing the purine-rich site that can bind proteins from the ets-family of transcription factors and a duplicate GATA-like site. When inserted upstream of a reporter containing the minimal Herpes simplex viral thymidine kinase (HSV-TK) promoter (into pBL2CAT plasmid) this site strongly activated the TK promoter in transfected myeloid cells. Further studies showed that oligonucleotides derived from the MPO promoter region bind multiple proteins in a band-shift assay. Taken together, our experiments located the regulatory elements important for human MPO gene expression in HL60 promyelocytes.

Antibody reactivity against thyroid peroxidase and myeloperoxidase in autoimmune thyroiditis and systemic vasculitis.

Potential cross-reactivity between thyroid peroxidase (TPO) and myeloperoxidase (MPO) molecules was evaluated by analysing the binding of 199 TPO antibody- and MPO antibody-positive sera to TPO and MPO molecules. Sera from six patients with autoimmune thyroiditis (AITD) and four patients with systemic vasculitis (SV) with different TPO-MPO antibody findings were then chosen for further analyses. All six patients with AITD had TPO antibodies in enzyme immunoassay (EIA) and four of them had simultaneously MPO antibodies in EIA. In AITD patients antibody binding to TPO could not be inhibited by adding native MPO to the serum diluent, suggesting that the possible cross-reactive epitopes were exposed in the denaturated MPO molecule. Similarly, the MPO ab reactivity of patients with systemic vasculitis could not be inhibited by native TPO. To study whether TPO and MPO antibodies recognize linear epitopes, the binding of antibodies to synthetic TPO and MPO peptides was analysed. Several TPO and MPO peptides were reactive, including peptides reacting with both TPO and MPO antibody-positive sera. One of the most cross-reactive peptides contained AA 586-601 in TPO, showing also particularly high AA homology (88%) with MPO (AA 594-609). The results suggest that TPO and MPO molecules contain cross-reactive epitopes that are exposed in denaturated molecules and may thus cause false positive antibody findings in solid phase EIA assays.

Myeloperoxidase mediates cell adhesion via the alpha M beta 2 integrin (Mac-1, CD11b/CD18).

Myeloperoxidase is a leukocyte component able to generate potent microbicidal substances. A homologous invertebrate blood cell protein, peroxinectin, is not only a peroxidase but also a cell adhesion ligand. We demonstrate in this study that human myeloperoxidase also mediates cell adhesion. Both the human myeloid cell line HL-60, when differentiated by treatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or retinoic acid, and human blood leukocytes, adhered to myeloperoxidase; however, undifferentiated HL-60 cells showed only minimal adhesion. No cells adhered to horseradish peroxidase, and cell adhesion to myeloperoxidase was not decreased by catalase, thus showing that peroxidase activity, per se, was neither sufficient nor necessary for the adhesion activity. Mannan, which has been reported to inhibit the binding of peroxidases to cells, did not affect adhesion to myeloperoxidase. However, adhesion to myeloperoxidase was inhibited by monoclonal antibodies to alpha M (CD11b) or to beta2 (CD18) integrin subunits, but not by antibodies to alpha L (CD11a), alpha M (CD11c), or to other integrins. Native myeloperoxidase mediated dose-dependent cell adhesion down to relatively low concentrations, and denaturation abolished the adhesion activity. It is evident that myeloperoxidase supports cell adhesion, a function which may be of considerable importance for leukocyte migration and infiltration in inflammatory reactions, that alpha M beta2 integrin (Mac-1 or CD11b/CD18) mediates this adhesion, and that the alphaM beta2 integrin-mediated adhesion to myeloperoxidase is distinct from the previously reported ability of this integrin to bind to certain denatured proteins at high concentrations.

Peroxidase-catalyzed oxidation of beta-carotene in HL-60 cells and in model systems: involvement of phenoxyl radicals.

Recent studies provide extensive evidence for the importance of carotenoids in protecting against oxidative stress associated with a number of diseases. In particular, reactions of carotenoids with phenoxyl radicals generated by peroxidase-catalyzed one-electron metabolism of phenolic compounds may represent an important antioxidant function of carotenoids. To further our understanding of the antioxidant mechanisms of carotenoids, we used in the present work two different phenolic compounds, phenol and a polar homologue of vitamin E (2,2,5,7,8-pentamethyl-6-hydroxychromane, PMC), as representatives of two different types of phenols to study reactions of their respective phenoxyl radicals with carotenoids in cells and in model systems. We found that phenoxyl radicals of PMC did not oxidize beta-carotene in either HL-60 cells or in model systems with horseradish peroxidase (HRP)/H2O2. In contrast, the phenoxyl radicals generated from phenol (by native myeloperoxidase in HL-60 cells or HRP/H2O2 in model systems) effectively oxidized beta-carotene and other carotenoids (canthaxanthin, lutein, lycopene). One-electron reduction of the phenoxyl radical by ascorbate (assayed by electron spin resonance-detectable formation of semidehydroascorbyl radicals) prevented HRP/H2O2-induced oxidation of beta-carotene. PMC, but not phenol, protected beta-carotene against oxidation induced by a lipid-soluble azo-initiator of peroxyl radicals. No adducts of peroxidase/phenol/H2O2-induced beta-carotene oxidation intermediates with phenol were detected by high-performance liquid chromatography-mass spectrometry analysis of the reaction mixture. Since carotenoids are essential constituents of the antioxidant defenses in cells and biological fluids, their depletion through the reaction with phenoxyl radicals formed from endogenous, nutritional and environmental phenolics, as well as phenolic drugs, may be an important factor in the development of oxidative stress.

A study on the NO-myeloperoxidase complex by absorption and electron paramagnetic (spin) resonance spectroscopies

The nitric oxide (NO) complexes of native myeloperoxidase and a modified myeloperoxidase (a cytochrome oxidase-like derivative) were investigated by absorption and electron paramagnetic (spin) resonance (ESR) spectrometries. The ferrous native myeloperoxidase formed two different complexes with NO, at low and high NO concentrations, while the ferrous modified myeloperoxidase formed only one complex regardless of the concentration of NO. The ESR spectrum of the NO complex of native myoloperoxidase (ferrous form) showed a characteristic triplet-triplet hyperfine structure, indicating a six-coordinate heme, and that of the modified myeloperoxidase (ferrous from) showed a single triplet hyperfine structure, indicating a five-coordinate heme. These results reveal that the native myeloperoxidase has two binding sites to NO, probably heme iron and the formyl group (-CHO) on the porphyrin ring of heme, and that the modified myeloperoxidase has only one such binding site, probably heme iron. Thus, the modification caused the loss of one binding site, probably that corresponding to the formyl group in the native myeloperoxidase. The ESR spectra of the native and modified myeloperoxidase complexes also indicated that the heme iron in native myeloperoxidase bonds with a fifth (proximal) ligand, probably the N nucleus of a histidine residue in the protein moiety of this enzyme, and that the heme iron in modified myeloperoxidase interacts weakly or does not bond with a fifth (proximal) ligand, probably the N nucleus of an amino residue in the protein moiety of this enzyme. The myeloperoxidase in vivo may scavenge NO, resulting in elimination of the NO molecule's paramagnetism.

Spectral Analysis of Lactoperoxidase: EVIDENCE FOR A COMMON HEME IN MAMMALIAN PEROXIDASES

The identity of the non-extractable heme of mammalian lactoperoxidase (LPO) has remained unsolved for over 40 years. Accepted possibilities include a constrained heme b or an 8-thiomethylene-modified heme b. Recent studies of myeloperoxidase (MPO) (Fenna, R., Zeng, J., and Davey, C. (1995) Arch. Biochem. Biophys. 316, 653-656; Taylor, K. L., Strobel, F., Yue, K. T., Ram, P., Pohl, J., Woods, A. S., and Kinkade, J. M., Jr. (1995) Arch. Biochem. Biophys. 316, 635-642) suggest possible prosthetic group similarities between MPO and LPO. To address heme identity for LPO, we used comparative magnetic circular dichroism (MCD) spectroscopy of LPO versus myoglobin (Mb), horseradish peroxidase (HRP), and MPO. MCD spectra of native Fe3+-LPO and Fe3+-CN--LPO are approximately 10 nm red shifted from analogous forms of Mb and HRP, including the formate-Mb adduct. MCD spectra of native LPO and MPO are opposite in sign, and MCD spectra of their cyanoadducts also differ. These data indicate the LPO heme is distinct from heme b of Mb and HRP as well as from "heme m" of MPO. From this work and literature analysis, we suggest that the non-extractable "heme l" of LPO has the two vinyl groups of heme b but lacks the 2-sulfonium-vinyl linkage of heme m. The observed red shifts in LPO spectra may derive from ester linkages to protein as for MPO. Strong spectral analogies between LPO and mammalian peroxidases (e.g. from saliva, eosinophils, thyroid, intestine) indicate similar prosthetic heme moieties.

Denaturation and renaturation of myeloperoxidase. Consequences for the nature of the prosthetic group.

The effects of the chaotropic agent, guanidine HCl, on the chlorinating activity, optical absorption, EPR, and resonance Raman spectra of myeloperoxidase have been studied. In the presence of the agent the Soret optical absorption of the reduced enzyme (lambda max = 474 nm) is blue shifted to 448 nm, a position similar to heme alpha-containing enzymes. The chlorinating activity of the enzyme disappears, and EPR spectra show a loss of intensity of the rhombic high spin heme signals (gx = 6.9; gy = 5.4) and the appearance of a more axial high spin signal (gx = gy = 6.0). Surprisingly the effects of guanidine HCl are partly reversible. Upon decreasing the concentration of the chaotropic agents by dilution, both the chlorinating activity and the original optical spectrum of native reduced enzyme (lambda max = 474 nm) are partly restored. The resonance Raman spectra of denatured cyanomyeloperoxidase are less complicated than those of native myeloperoxidase, which have been interpreted previously to suggest an iron chlorin chromophore. The multiple lines in the oxidation state marker region are not seen in the spectra of the denatured species. The changes suggest that upon denaturation the macrocycle is converted into a more symmetric structure. Since the effects on the optical absorption spectrum are reversible we speculate that, in the native enzyme, an apparent porphyrin macrocycle undergoes a reversible interaction with amino acid residues in the protein which creates an asymmetry in the electronic distribution of the macrocycle. Comparison of the Raman spectra of denatured cyanomyeloperoxidase with those of analogous heme alpha model complexes suggests the presence of a formyl group in the denatured species; our data, however, demonstrate that the chromophore structure is not identical to heme alpha and may contain a different C beta substitution on the ring macrocycle.

Autoantibodies to native myeloperoxidase in patients with pulmonary hemorrhage and acute renal failure.

Sera from 245 patients were screened by indirect immunofluorescence for perinuclear/nuclear staining (P-ANCA) of ethanol-fixed neutrophils, a staining pattern which is associated with the presence of antibodies to myeloperoxidase. Using immunoblot and immunoprecipitation techniques on 15 P-ANCA-positive sera, 13 patients demonstrated antibody to purified or native myeloperoxidase but not to denatured myeloperoxidase. In patients with P-ANCA, the most frequent reason for medical attention was hemoptysis (8/13; 62%). Of the 15 sera with P-ANCA, acute renal failure was identified in 9 patients (60%). Five patients (33%) had both. All patients (eight of eight) with hemoptysis had antibodies which bound functional MPO as compared to three of seven P-ANCA-positive patients without hemoptysis (P less than 0.001), suggesting that antibodies which recognize conformational sites on native myeloperoxidase occur in a subgroup of patients with alveolar hemorrhage as their presenting clinical sign. These findings may provide insight into the disease process associated with P-ANCA. We further identify a subgroup of patients with a severe pulmonorenal syndrome and antibodies recognizing native myeloperoxidase.

Photochemically modified myeloperoxidase, with optical spectral properties analogous to those of lactoperoxidase, retains its original catalytic activity

During the course of a reducing reaction using ketyl radicals generated from ketone photoreduction with ultraviolet light, a photoinduced chemical modification of the chromophore group in myeloperoxidase has been found. Light absorption and resonance Raman spectra for this modified enzyme indicated an iron porphyrin chromophore group. The alkaline pyridine hemochrome of the modified enzyme exhibited an optical spectrum closely related to that of iron protoporphyrin IX. The chromophore group of the modified myeloperoxidase was cleaved from the protein by methoxide. Proton magnetic resonance of the diamagnetic bis(cyanide) compound of the extracted heme group showed the presence of two vinyl and three methyl side chains associated with a porphyrin macrocycle. These data provide further insight into the structure of the active site in myeloperoxidase. The EPR spectral properties and enzymatic activities of the native myeloperoxidase are essentially conserved in the modified enzyme. Our present results indicate that the heme peripheral substituent is modified while the stereochemical structure surrounding the chromophore group is not altered by the photochemical modification.

A steady-state study on the formation of Compounds II and III of myeloperoxidase

The reaction between native myeloperoxidase and hydrogen peroxide, yielding Compound II, was investigated using the stopped-flow technique. The pH dependence of the apparent second-order rate constant showed the existence of a protonatable group on the enzyme with a p K a of 4.9. This group is ascribed to the distal histidine imidazole, which must be deprotonated to enable the reaction of Compound I with hydrogen peroxidase to take place. The rate constant for the formation of Compound II by hydrogen peroxide was 3.5 · 10 4 M −1 · s −1. During the reaction of myeloperoxidase with H 2O 2, rapid reduction of added cytochrome c was observed. This reduction was inhibitable by superoxide dismutase, and this demonstrates that superoxide anion radicals are generated. When potassium ferrocyanide was used as an electron donor to generate Compound II from Compound I, the pH dependence of the apparent second-order rate constant indicated involvement of a group with a p K a of 4.5. However, with ferrocyanide as an electron donor, protonation of the group was necessary to enable the reaction to take place. The rate constant for the generation of Compound II by ferrocyanide was 1.6 · 10 7 M −1 · s −1. We also investigated the reaction of Compound II with hydrogen peroxide, yielding Compound III. Formation of Compound III ( k = 50M −1 ·s −1) proceeded via two different pathways, one of which was inhibitable by tetranitomethane. We further investigated the stability of Compound II and Compound III as a function of pH, ionic strength and enzyme concentration. The half-life values of both Compound II and Compound III were independent of the enzyme concentration and ionic strength. The half-life value of Compound III was pH-dependent, showing a decreasing stability with increasing pH, whereas the stability of Compound II was independent of pH over the range 3–11.

Superoxide modulates the activity of myeloperoxidase and optimizes the production of hypochlorous acid.

Myeloperoxidase catalyses the conversion of H2O2 and Cl- to hypochlorous acid (HOCl). It also reacts with O2- to form the oxy adduct (compound III). To determine how O2- affects the formation of HOCl, chlorination of monochlorodimedon by myeloperoxidase was investigated using xanthine oxidase and hypoxanthine as a source of O2- and H2O2. Myeloperoxidase was mostly converted to compound III, and H2O2 was essential for chlorination. At pH 5.4, superoxide dismutase (SOD) enhanced chlorination and prevented formation of compound III. However, at pH 7.8, SOD inhibited chlorination and promoted formation of the ferrous peroxide adduct (compound II) instead of compound III. We present spectral evidence for a direct reaction between compound III and H2O2 to form compound II, and for the reduction of compound II by O2- to regenerate native myeloperoxidase. These reactions enable compound III and compound II to participate in the chlorination reaction. Myeloperoxidase catalytically inhibited O2- -dependent reduction of Nitro Blue Tetrazolium. This inhibition is explained by myeloperoxidase undergoing a cycle of reactions with O2-, H2O2 and O2-, with compounds III and II as intermediates, i.e., by myeloperoxidase acting as a combined SOD/catalase enzyme. By preventing the accumulation of inactive compound II, O2- enhances the activity of myeloperoxidase. We propose that, under physiological conditions, this optimizes the production of HOCl and may potentiate oxidant damage by stimulated neutrophils.

Biosynthesis and processing of myeloperoxidase--a marker for myeloid cell differentiation.

Myeloperoxidase (MPO), a heme protein, is a major component of azurophilic granules of neutrophils. Optimal oxygen-dependent microbicidal activity depends on MPO as the critical enzyme for the generation of hypochlorous acid and other toxic oxygen products. MPO is synthesized during the promyelocytic stage of myeloid differentiation, the stage at which azurophilic granules are formed. Like other lysosomal enzymes, MPO is synthesized as a larger precursor which is subsequently processed and transported intracellularly to the lysosomes. The primary translation product is a single 80-kDa protein which undergoes cotranslational N-linked glycosylation to produce a 92-kDa glycoprotein. Glucosidases in the endoplasmic reticulum or early cis Golgi convert the proMPO to a 90-kDa form which is sorted into a prelysosomal compartment that undergoes final proteolytic maturation to native MPO, a pair of heavy-light protomers with subunits of 60 kDa and a 12 kDa. These events contrast with similar processes seen with other lysosomal enzymes in two ways. First, alkalinization of lysosomes with NH4+ does not alter processing or transport, in contrast to the pH dependence of these processes for other lysosomal enzymes. However, some studies indicate retardation of processing in the presence of the proton ionophore monensin. Second, intracellular transport of MPO is not apparently mediated by the mannose-6-phosphate receptor system. The gene for MPO is on the long arm of chromosome 17 (17q22, 23) near the breakpoint of the 15, 17 translocation of acute promyelocytic leukemia. The gene spans approximately 14 kb and contains 11 irons and 12 exons. The cloned full-length cDNA is approximately 2.2 kb and both normal bone marrow and cultured promyelocytic leukemia cells express two species of mRNA. Inherited MPO deficiency, a relatively common disorder, is associated with the absence of mature MPO but the presence of proMPO, consistent with a post-translational defect. Studies at the molecular level aimed at identifying the underlying genetic defect are thus far consistent with that hypothesis. In addition, the basis for the observed association between acquired MPO deficiency and some myeloid leukemias can now be studied at the molecular level using these probes.

Role of high-avidity binding of human neutrophil myeloperoxidase in the killing of Actinobacillus actinomycetemcomitans.

The binding of the neutrophil enzyme myeloperoxidase (MPO) to microbial surfaces is believed to be the first step in its microbicidal activity. The MPO-H2O2-Cl- system is responsible for most oxidative killing of Actinobacillus actinomycetemcomitans by human neutrophils. There appear to be three forms of MPO (MPO I, II, and III), all of which can kill this organism in the presence of H2O2 and chloride. In this study, we characterized the binding of native human neutrophil MPO to A. actinomycetemcomitans by an elution procedure dependent on the cationic detergent cetyltrimethylammonium bromide. Binding of native MPO was rapid and reached apparent equilibrium within 1 min. A proportion of binding under equilibrium conditions was saturable and highly avid, with a capacity of 4,500 sites per cell and a dissociation constant of 7.9 X 10(-10) M. At equal protein concentrations, more MPO III bound than MPO II, and more MPO II bound than MPO I. The high-avidity interaction was inhibitable with yeast mannan and with the serotype-defining mannan of A. actinomycetemcomitans. Binding was also partially reversible with yeast mannan. MPO bound to the high-avidity sites did not oxidize guaiacol but oxidized chloride, as detected by the chlorination of taurine. MPO bound to the high-avidity sites was incapable of killing A. actinomycetemcomitans alone in the presence of H2O2 and Cl-, but potentiated killing when sufficient additional MPO was provided. The killing of A. actinomycetemcomitans by the MPO-H2O2-Cl- system was inhibited by yeast mannan and a serotype-defining mannan of A. actinomycetemcomitans. We conclude that high-avidity binding of MPO to the surface of A. actinomycetemcomitans is a mannan-specific interaction and that MPO bound to the high-avidity sites is essential but not alone sufficient to kill A. actinomycetemcomitans.

20] Human myeloperoxidase and hemi-myeloperoxidase

Publisher Summary This chapter describes human myeloperoxidase (MPO) and hemi-MPO. MPO is an enzyme found in the azurophilic granules of mammalian neutrophils and is also identified in human monocytes. Its function is to kill bacteria that have been phagocytosed by these cells. This bactericidal activity comes from its ability to oxidize halides to their hypohalous acids. In a halide-dependent reaction, MPO can lead to the oxidation of methionine to its sulfoxide derivative. In assay system for MPO, a noncarcinogcnic, nonmutagenic derivative of benzidine, 3,3′,5,5′-tetramethylbenzidine (TMB) is utilized as substrate. The assay is designed to measure MPO activity in the presence or absence of halides. As MPO oxidizes halides to hypohalous acids such as hypochlorous acid (HOCl), the assay is stoichiometric in as much as each H2O2 molecule produces either one molecule of HOCl or two oxidized dye molecules. The isolation and purification of native MPO from human white blood cell pellets consist of detergent solubilization, ammonium sulfate precipitation, and gel filtration.

Comparison of myeloperoxidase and hemi-myeloperoxidase with respect to catalysis, regulation, and bactericidal activity

It has been demonstrated previously (P. C. Andrews and N. I. Krinsky (1981) J. Biol. Chem. 256, 4211–4218) that human leukocyte myeloperoxidase, an α 2 β 2 enzyme, can be cleaved by mild reduction and alkylation to an α 1 β 1 structure that we have termed hemi-myeloperoxidase. The native enzyme and hemi-myeloperoxidase have the same specific activity in a Cl −-independent peroxidase assay and identical visible spectra under either oxidized or reduced conditions. This paper compares other properties of native and hemi-myeloperoxidase. Both enzymes are inhibited by high concentrations of H 2O 2 in an identical fashion. Both enzymes showed identical regulation by pH and Cl −. The utilization of Cl −, as assayed by chlorination of diethanolamine, was moderately decreased in hemi-myeloperoxidase. This reduction in chlorination was not reflected in a bactericidal assay, where again, hemi-myeloperoxidase was identical in activity to native myeloperoxidase.

Spectral properties of myeloperoxidase and its ligand complexes

The effects of ligands with various field strengths on the optical absorption spectrum of myeloperoxidase have been investigated. As is the case with other hemoproteins, the Soret peak in the optical absorption spectra at 77 K moves to longer wavelengths when strong-field ligands are present, whereas binding of such ligands as chloride and fluoride, which stabilize the high-spin state, shows the opposite effect. With a ligand of intermediate field strength, such as azide, the optical spectrum is not affected at room temperature, but lowering of the temperature results in the formation of the low-spin form of the enzyme. Similarly, in native myeloperoxidase a spin state equilibrium is found in which the low-spin state is favoured at high ionic strength and displays corresponding changes in the optical spectra. From the ligand- and the temperature-induced changes in the optical spectra of the ferric enzyme it is concluded that the band at 620–630 nm is an α band of the low-spin heme iron species, whereas the bands at 500 and 690 nm are probably ‘charge-transfer’ bands ∗ ∗ ‘Charge transfer’ does not refer to the pure charge-transfer transitions, which occur in the near infrared, but to mixed transitions of ligand and charge transfer. of the heme with the iron in the high-spin state.

The reductive cleavage of myeloperoxidase in half, producing enzymically active hemi-myeloperoxidase.

Reduction and alkylation of human myeloperoxidase under nondenaturing conditions results in the cleavage of this enzyme. Sedimentation equilibrium data is presented which shows that the molecular weight of the cleavage product (78,000 +/- 2,000) is half that of the native enzyme (153,000 +/- 4,000). We conclude that the cleavage product is the half-enzyme hemi-myeloperoxidase. Hemi-myeloperoxidase retains both heme groups and contains both subunit types (Mr = 57,500 and 14,000) in the same ratio as native myeloperoxidase. The two halves of native myeloperoxidase are apparently not dependent upon one another for peroxidatic activity, as the specific activity of the half-enzyme is the same as that of the native enzyme. Analytical ultracentrifugation studies show native myeloperoxidase has a sedimentation coefficient of 8.0 and an axial ratio of 5:1, while hemi-myeloperoxidase has a sedimentation coefficient of 4.3 and an axial ratio of 10:1. When [3H]iodoacetic acid was used to prepare hemi-myeloperoxidase, the label incorporated with a stoichiometry of 1.2 [3H]carboxymethyl groups per hemi-myeloperoxidase, with 90% of this label associated with the heavy subunit. From these observations we conclude that native myeloperoxidase contains two heavy-light protomers, which are joined along their long axes by a single disulfide bond between the heavy subunits. Selective reduction of this disulfide bond by the use of nondenaturing conditions results in the formation of hemi-myeloperoxidase, a catalytically active heavy-light protomer of native myeloperoxidase.

Evidence that myeloperoxidase is composed of isoenzymes

Preparative disc electrophoretic separation of the myeloperoxidase of the normal human leucocyte made possible the demonstration of six isoenzymes. Studies of isolated bands suggested that each isoenzyme is a dimeric molecule, arising from three kinds of monomeric subunits, A, B, and C. The isoenzymes are referred to as AA, AB, AC, BB, BC, and CC. Oxidized spectra of the homologous isoenzymes AA, BB, and CC, were similar to the native myeloperoxidase as were the reduced spectra of AA and CC. The reduced spectrum of isoenzyme BB showed rapid autooxidation. Amino acid compositions of the isoenzymes were similar to native myeloperoxidase, but showed distinguishing features. Two inactive anodic bands were also noted whose properties suggest that they are either degradation products or contaminants. The genetic origin of the three monomers is not apparent since the myeloperoxidase was prepared from many donors.