Molecular mimicry of the receptor-binding domain of the SARS-CoV-2 spike protein: from the interaction of spike-specific antibodies with transferrin and lactoferrin to the antiviral effects of human recombinant lactoferrin.

The pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection involves dysregulations of iron metabolism, and although the mechanism of this pathology is not yet fully understood, correction of iron metabolism pathways seems a promising pharmacological target. The previously observed effect of inhibiting SARS-CoV-2 infection by ferristatin II, an inducer of transferrin receptor 1 (TfR1) degradation, prompted the study of competition between Spike protein and TfR1 ligands, especially lactoferrin (Lf) and transferrin (Tf). We hypothesized molecular mimicry of Spike protein as cross-reactivity of Spike-specific antibodies with Tf and Lf. Thus, strong positive correlations (R2 > 0.95) were found between the level of Spike-specific IgG antibodies present in serum samples of COVID-19-recovered and Sputnik V-vaccinated individuals and their Tf-binding activity assayed with peroxidase-labeled anti-Tf. In addition, we observed cross-reactivity of Lf-specific murine monoclonal antibody (mAb) towards the SARS-CoV-2 Spike protein. On the other hand, the interaction of mAbs produced to the receptor-binding domain (RBD) of the Spike protein with recombinant RBD protein was disrupted by Tf, Lf, soluble TfR1, anti-TfR1 aptamer, as well as by peptides RGD and GHAIYPRH. Furthermore, direct interaction of RBD protein with Lf, but not Tf, was observed, with affinity of binding estimated by KD to be 23 nM and 16 nM for apo-Lf and holo-Lf, respectively. Treatment of Vero E6 cells with apo-Lf and holo-Lf (1-4 mg/mL) significantly inhibited SARS-CoV-2 replication of both Wuhan and Delta lineages. Protective effects of Lf on different arms of SARS-CoV-2-induced pathogenesis and possible consequences of cross-reactivity of Spike-specific antibodies are discussed.

Analysis of Clinical and Biochemical Characteristics of Patients With Genetically Confirmed Familial Hypercholesterolemia in Russian North Western District Residents.

Aim      To compare results of clinical, laboratory, and genetic examination of patients with familial hypercholesterolemia (FHC).Material and methods  112 patients aged 40.2±17.9 years (49 men) were examined. The gene of low-density lipoprotein receptor (LDLR) was analyzed and evaluated using the Dutch Lipid Clinic Network (DLCN) criterion of lipid score ≥6. The LDLR gene mutation was searched for using the conformational polymorphism analysis followed by sequencing of the DNA of isolated LDLR gene exons.Results Mean variables of the blood lipid profile were total cholesterol (C), 10.12±2.32 mmol/l, LDL-C, 7.72±2.3 mmol/l. Corneal arcus was observed in 15 % of patients, tendon xanthomas in 31.8 %, and xanthelasma palpebrarum in 5.3 %. The types of LDLR gene mutations included missense mutations (42.8 %), mutations causing a premature termination of protein synthesis (41.1 %), and frameshift mutations (16.1 %). In the presence of a mutation in exon 4, patients with IHD compared to patients with no IHD had significantly higher levels of total C (10.88±2.08 mmol/l vs. 8.74±1.57 mmol/l, respectively, р=0.001) and LDL-C (8.60±2.14 mmol/l vs. 6.62±1.79 mmol/l, respectively, р=0.005). Patients with IHD compared to patients with no IHD and a mutation in LDLR gene exon 9 had only a higher LDL-C level (8.96±1.53 mmol/l vs. 6.92±1.59 mmol/l, respectively, р=0.022). A differentiated comparison of IHD patients using a logistic regression depending on the identified type of LDLR gene mutation produced formulas for calculating the odds ratio of IHD and myocardial infarction (MI) with adjustments for the patient's age and baseline LDL.Conclusion      The detection rate of the LDLR gene mutations was 42.8 % for missense mutations, 41.1 % for mutations causing a premature termination of protein synthesis, and 16.1 % for frameshift mutations. Blood lipid profiles did not differ between patients from different cities and with different types of LDLR gene mutations. Blood lipid profiles were different in IHD patients depending on the mutation type.

Structure-biological activity relationships of myeloperoxidase to effect on platelet activation.

Myeloperoxidase (MPO), an oxidant-producing enzyme of neutrophils, has been shown to prime platelet activity promoting immunothrombosis. Native MPO is a homodimer, consisting of two identical protomers (monomer) connected by a single disulfide bond. But in inflammatory foci, MPO can be found both in the form of a monomer and in the form of a dimer. Beside MPO can also be in complexes with other molecules and be modified by oxidants, which ultimately affect its physicochemical properties and functions. Here we compared the effects of various forms of MPO as well as MPO in complex with ceruloplasmin (CP), a physiological inhibitor of MPO, on the platelet activity. Monomeric MPO (hemi-MPO) was obtained by treating the dimeric MPO by reductive alkylation. MPO was modified with HOCl in a molar ratio of 1:100 (MPO-HOCl). Using surface-enhanced Raman scattering (SERS) spectroscopy we showed that peaks at about 510 and 526 cm-1 corresponded to disulfide bond was recognizable in the SERS-spectra of dimeric MPO, absent in the spectrum of hemi-MPO and less intense in the spectra of MPO-HOCl, which indicates the partial decomposition of dimeric MPO with a disulfide bond cleavage under the HOCl modification. It was shown hemi-MPO to a lesser extent than dimeric MPO bound to platelets and enhanced their agonist-induced aggregation and platelet-neutrophil aggregate formation. MPO modified by HOCl and MPO in complex with CP did not bind to platelets and have no effect on platelet activity. Thus, the modification of MPO by HOCl, its presence in monomeric form as well as in complex with CP reduces MPO effect on platelet function and consequently decreases the risk of thrombosis in inflammatory foci.

Analysis of the low density lipoprotein receptor gene (LDLR) mutation spectrum in Russian familial hypercholesterolemia.

Familial hypercholesterolemia (FH) is a very common human hereditary disease in Russia and in the whole world with most of mutations localized in the gene coding for the low density lipoprotein receptor (LDLR). The object of this review is to systematize the knowledge about LDLR mutations in Russia. With this aim we analyzed all available literature on the subject and tabulated the data. More than 1/3 (80 out of 203, i. e. 39.4 %) of all mutations reported from Russia were not described in other populations. To date, most LDLR gene mutations have been characterized in large cities: Moscow (130 entries), Saint Petersburg (50 entries), Novosibirsk (34 mutations) and Petrozavodsk (19 mutations). Other regions are poorly studied. The majority of pathogenic mutations (142 out of 203 reported here or 70 %) were revealed in single pedigrees; 61 variants of mutations were described in two or more genealogies; only 5 mutations were found in 10 or more families. As everywhere, missense mutations prevail among all types of nucleotide substitutions in LDLR, but the highest national specificity is imparted by frameshift mutations: out of 27 variants reported, 19 (or 70 %) are specific for Russia. The most abundant in mutations are exons 4 and 9 of the gene due to their largest size and higher occurrence of mutations in them. Poland,the Czech Republic, Italy and the Netherlands share the highest number of mutations with the Russian population. Target sequencing significantly accelerates the characterization of mutation spectra in FH, but due to the absence of systematic investigations in the regions, one may suggest that most of LDLR mutations in the Russian population have not been described yet.

Binding of lactoferrin to the surface of low-density lipoproteins modified by myeloperoxidase prevents intracellular cholesterol accumulation by human blood monocytes.

Myeloperoxidase (MPO) is a unique heme-containing peroxidase that can catalyze the formation of hypochlorous acid (HOCl). The strong interaction of MPO with low-density lipoproteins (LDL) promotes proatherogenic modification of LDL by HOCl. The MPO-modified LDL (Mox-LDL) accumulate in macrophages, resulting in the formation of foam cells, which is the pathognomonic symptom of atherosclerosis. A promising approach to prophylaxis and atherosclerosis therapy is searching for remedies that prevent the modification or accumulation of LDL in macrophages. Lactoferrin (LF) has several application points in obesity pathogenesis. We aimed to study LF binding to Mox-LDL and their accumulation in monocytes transformed into macrophages. Using surface plasmon resonance and ELISA techniques, we observed no LF interaction with intact LDL, whereas Mox-LDL strongly interacted with LF. The affinity of Mox-LDL to LF increased with the degree of oxidative modification of LDL. Moreover, an excess of MPO did not prevent interaction of Mox-LDL with LF. LF inhibits accumulation of cholesterol in macrophages exposed to Mox-LDL. The results obtained reinforce the notion of LF potency as a remedy against atherosclerosis.

Myeloperoxidase-Induced Oxidation of Albumin and Ceruloplasmin: Role of Tyrosines.

Neutrophil myeloperoxidase (MPO) plays an important role in protecting the body against infections. MPO products - hypohalous acids and phenoxyl radicals - are strong oxidants that can damage not only foreign intruders but also host tissues, including blood plasma proteins. Here, we compared the MPO-induced oxidation of two plasma proteins with antioxidant properties - human serum albumin (HSA) and ceruloplasmin (CP). Incubation of both proteins with hypochlorite (NaOCl) or catalytically active MPO (MPO + H2O2), which synthesizes hypochlorous acid (HOCl) in the presence of chloride ions, resulted in the quenching of protein tryptophan fluorescence. Oxidation-induced changes in the structures of HSA and CP were different. HSA efficiently neutralized MPO-generated oxidants without protein aggregation, while CP oxidation resulted in the formation of large aggregates stabilized by strong covalent bonds between the aromatic amino acid residues. Tyrosine is present in the plasma as free amino acid and also as a component of the polypeptide chains of the proteins. The number of tyrosine residues in a protein does not determine its propensity for aggregate formation. In the case of CP, protein aggregation was primarily due to the high content of tryptophan residues in its polypeptide chain. MPO-dependent oxidation of free tyrosine results in the formation of tyrosyl radicals, that do not oxidize aromatic amino acid residues in proteins because of the high rate of recombination with dityrosine formation. At the same time, free tyrosine can influence MPO-induced protein oxidation due to its ability to modulate HOCl synthesis in the MPO active site.

Structural Study of the Complex Formed by Ceruloplasmin and Macrophage Migration Inhibitory Factor.

Macrophage migration inhibitory factor (MIF) is a key proinflammatory cytokine. Inhibitors of tautomerase activity of MIF are perspective antiinflammatory compounds. Ceruloplasmin, the copper-containing ferroxidase of blood plasma, is a noncompetitive inhibitor of tautomerase activity of MIF in the reaction with p-hydroxyphenylpyruvate. Small-angle X-ray scattering established a model of the complex formed by MIF and ceruloplasmin. Crystallographic analysis of MIF with a modified active site supports the model. The stoichiometry of 3 CP/MIF trimer complex was established using gel filtration. Conformity of novel data concerning the interaction regions in the studied proteins with previous biochemical data is discussed.

[Enzymatic and bactericidal activity of monomeric and dimeric forms of myeloperoxidase]. / Fermentativnaia i bakteritsidnaia aktivnost' monomernoi i dimernoi form mieloperoksidazy.

This study was carried out to compare the enzymatic and bactericidal activity of mature, dimeric myeloperoxidase (MPO) and its monomeric form. Dimeric MPO was isolated from HL-60 cells. Hemi-MPO obtained from dimeric MPO by reductive cleavage of a disulfide bond between protomeric subunits was used as the monomeric form. Both peroxidase and halogenating (chlorinating) activities of MPO were assayed, each of them by two methods. Bactericidal activity of the MPO/Н2О2/Cl- system was tested using the Escherichia coli laboratory strain DH5a. No difference in the enzymatic and bactericidal activity between dimeric MPO and hemi-MPO was found. Both forms of the enzyme also did not differ in the resistance to HOCl, the main product of MPO. HOCl caused a dose-dependent decrease in peroxidase and chlorinating activity, and the pattern of this decrease was identical for dimeric MPO and hemi-MPO. At equal heme concentration, a somewhat higher bactericidal effect was observed for the hemi-MPO/Н2О2/Cl- system compared with the dimeric MPO/Н2О2/Cl- system. However, this is most likely not related to some specific property of hemi-MPO and can be accounted for by the higher probability of contacting between bacterial surface and hemi-MPO molecules due to their two-fold greater number relative to that of dimeric MPO molecules at the same heme concentration. By using Western-blotting with antibodies to MPO, we showed, for the first time, that the dimeric molecule of MPO could be cleaved into two monomeric subunits by HOCl, most probably due to oxidation of the disulfide bond between these subunits. This finding suggests that appearance in blood of MPO corresponding in mass to its monomer may result from the damage of dimeric MPO by reactive halogen species, especially upon their overproduction underlying oxidative/halogenative stress in inflammatory diseases.

Erythropoietin and Nrf2: key factors in the neuroprotection provided by apo-lactoferrin.

Among the properties of lactoferrin (LF) are bactericidal, antianemic, immunomodulatory, antitumour, antiphlogistic effects. Previously we demonstrated its capacity to stabilize in vivo HIF-1-alpha and HIF-2-alpha, which are redox-sensitive multiaimed transcription factors. Various tissues of animals receiving recombinant human LF (rhLF) responded by expressing the HIF-1-alpha target genes, hence such proteins as erythropoietin (EPO), ceruloplasmin, etc. were synthesized in noticeable amounts. Among organs in which EPO synthesis occurred were brain, heart, spleen, liver, kidneys and lungs. Other researchers showed that EPO can act as a protectant against severe brain injury and status epilepticus in rats. Therefore, we tried rhLF as a protector against the severe neurologic disorders developed in rats, such as the rotenone-induced model of Parkinson's disease and experimental autoimmune encephalomyelitis as a model of multiple sclerosis, and observed its capacity to mitigate the grave symptoms. Moreover, an intraperitoneal injection of rhLF into mice 1 h after occlusion of the medial cerebral artery significantly diminished the necrosis area measured on the third day in the ischaemic brain. During this period EPO was synthesized in various murine tissues. It was known that EPO induces nuclear translocation of Nrf2, which, like HIF-1-alpha, is a transcription factor. In view that under conditions of hypoxia both factors demonstrate a synergistic protective effect, we suggested that LF activates the Keap1/Nrf2 signaling pathway, an important link in proliferation and differentiation of normal and malignant cells. J774 macrophages were cultured for 3 days without or in the presence of ferric and ferrous ions (RPMI-1640 and DMEM/F12, respectively). Then cells were incubated with rhLF or Deferiprone. Confocal microscopy revealed nuclear translocation of Nrf2 (the key event in Keap1/Nrf2 signaling) induced by apo-rhLF (iron-free, RPMI-1640). The reference compound Deferiprone (iron chelator) had the similar effect. Upon iron binding (in DMEM/F12) rhLF did not activate the Keap1/Nrf2 pathway. Added to J774, apo-rhLF enhanced transcription of Nrf2-dependent genes coding for glutathione S-transferase P and heme oxygenase-1. Western blotting revealed presence of Nrf2 in mice brain after 6 days of oral administration of apo-rhLF, but not Fe-rhLF or equivalent amount of PBS. Hence, apo-LF, but not holo-LF, induces the translocation of Nrf2 from cytoplasm to the nucleus, probably due to its capacity to induce EPO synthesis.

[Exocytosis of myeloperoxidase from activated neutrophils in the presence of heparin]. / Ékzotsitoz mieloperoksidazy pri aktivatsii neitrofilov v prisutstvii geparina.

Exocytosis of myeloperoxidase (MPO) from activated neutrophils in the presence of the anionic polysaccharide heparin was studied. It was determined that the optimal concentration of heparin (0.1 u/ml), at which there is no additional activation of cells (absence of amplification of exocytosis of lysozyme contained in specific and azurophilic granules). It was found that after preincubation of cells with heparin (0.1 u/ml) the exocytosis of MPO from neutrophils activated by various stimulants (fMLP, PMA, plant lectins CABA and PHA-L) increased compared to that under the action of activators alone. In addition, it was shown that heparin in the range of concentrations 0.1-50 u/ml did not affect on the peroxidase activity of the MPO isolated from leukocytes. Thus, the use of heparin at a concentration of 0.1 u/ml avoids the artifact caused by the "loss" of MPO in a result of its binding to neutrophils, and increases the accuracy of the method of registration the degranulation of azurophilic granules of neutrophils based on determination of the concentration or peroxidase activity of MPO in cell supernatants.

Capacity of ceruloplasmin to scavenge products of the respiratory burst of neutrophils is not altered by the products of reactions catalyzed by myeloperoxidase.

CP is a copper-containing ferroxidase of blood plasma, which acts as an acute phase reactant during inflammation. The effect of oxidative modification of CP induced by oxidants produced by MPO, such as HOCl, HOBr, and HOSCN, on its spectral, enzymatic, and anti-inflammatory properties was studied. We monitored the chemiluminescence of lucigenin and luminol along with fluorescence of hydroethidine and scopoletin to assay the inhibition by CP of the neutrophilic respiratory burst induced by PMA or fMLP. Superoxide dismutase activity of CP and its capacity to reduce the production of oxidants in respiratory burst of neutrophils remained virtually unchanged upon modifications caused by HOCl, HOBr, and HOSCN. Meanwhile, the absorption of type I copper ions at 610 nm became reduced, along with a drop in the ferroxidase and amino oxidase activities of CP. Likewise, its inhibitory effect on the halogenating activity of MPO was diminished. Sera of either healthy donors or patients with Wilson disease were co-incubated with neutrophils from healthy volunteers. In these experiments, we observed an inverse relationship between the content of CP in sera and the rate of H2O2 production by activated neutrophils. In conclusion, CP is likely to play a role of an anti-inflammatory factor tempering the neutrophil respiratory burst in the bloodstream despite the MPO-mediated oxidative modifications.

Rat ceruloplasmin: a new labile copper binding site and zinc/copper mosaic.

Ceruloplasmin (Cp) is a copper-containing multifunctional oxidase of plasma, an antioxidant, an acute-phase protein and a free radical scavenger. The structural organization of Cp causes its sensitivity to proteolysis and ROS (reactive oxygen species), which can alter some of the important Cp functions. Elucidation of the orthorhombic crystal structure of rat Cp at 2.3 Å resolution revealed the basis for stronger resistance of rat Cp to proteolysis and a new labile copper binding site. The presence of this site appears as a very rare and distinctive feature of rat Cp as was shown by sequence alignment of ceruloplasmin, hephaestin and zyklopen in the Deuterostomia taxonomic group. The trigonal crystal form of rat Cp at 3.2 Å demonstrates unexpected partial substitution of copper by zinc.

Comparison of Interaction between Ceruloplasmin and Lactoferrin/Transferrin: to Bind or Not to Bind.

The year 2016 marked the 50th anniversary of the discovery by S. Osaki who first showed that ceruloplasmin (CP, ferro:O2-oxidoreductase or ferroxidase) is capable of oxidizing Fe(II) to Fe(III) and favors the incorporation of the latter into transferrin (TF). However, much debate remains in the literature concerning the existence of a complex between the enzyme oxidizing iron and the protein facilitating its transport in plasma. We studied CP in exocrine fluids and demonstrated its high-affinity interaction with transferrin found in breast milk and in lacrimal fluid, i.e. with lactoferrin (LF). Here we present data obtained by comparing the interaction of CP with LF and TF using surface plasmon resonance and Hummel-Dreyer chromatography. Binding of apo-LF within the range of concentrations 1.6-51.3 µM with CP immobilized on a CM5-chip is characterized by KD = 1.07 µM. Under similar conditions, the KD for apo-TF was measured and appeared to be higher than 51.3 µM. Hummel-Dreyer chromatography of CP with 51 µM apo-LF/apo-TF in the effluent demonstrated the absence of interaction between apo-TF and CP in solution, contrary to efficient interaction between apo-LF and CP. In contrast to LF, the interaction of apo-TF with CP is probably not stable within the physiological range of concentrations of TF.

Cholesterol Levels in Genetically Determined Familial Hypercholesterolaemia in Russian Karelia.

Familial hypercholesterolaemia (FH) is a rare disease that tends to be diagnosed lately. In Russia, the genetic and phenotypic characteristics of the disease are not well defined. We investigated 102 patients with definite FH. In 52 of these patients (50.9%) genetic analysis was performed, revealing pathogenic mutations of the low density lipoprotein (LDL) receptor gene in 22 patients. We report here five mutations of the LDL receptor gene found in the Karelian FH sample for the first time. The detection rate of mutations in definite FH patients was 42.3%. Two groups of patients with a definite diagnosis of FH according to the Dutch Lipid Clinic Network criteria were compared: the first group had putatively functionally important LDL receptor gene mutations, while in the second group LDL receptor gene mutations were excluded by single-strand conformation polymorphism analysis. Total and LDL cholesterol levels were higher in the group with LDL receptor mutations compared to the mutation-free population. The frequency of mutations in patients with LDL cholesterol > 6.5 mmol/L was more than 3 times higher than that in patients with LDL < 6.5 mmol/L. Total and LDL cholesterol levels and the frequency of coronary heart disease and myocardial infarction were higher in the group with definite FH compared to groups with probable and possible FH. Cholesterol figures in FH patients of different age and sex from the Karelian population were comparable.

[The Familial Hypercholesterolemia Caused by a Novel Human Low Density Lipoprotein Receptor Gene Mutation c.1327 T>C (p.W433R)].

During investigation of molecular nature of familial hypercholesterolemia (FH) in Petrozavodsk (Russia) cohort of patients a novel low density lipoprotein (LDL) receptor gene mutation was found. This mutation designated c.1327 T>C (W443R [W422R]) was predicted to cause substitution of arginine for tryptophan residue in the very conservative -propeller domain of the LDL receptor. Inheritance of the new mutation was traced in four generations and its cosegregation with hypercholesterolemia phenotype was observed. Despite the predicted pathogenic effect of the mutation, ischemic heart disease in the pedigree was mild or absent. We consider identification of this mutation in the pedigree extremely helpful to start preventive medical treatment in affected patients.

Myeloperoxidase Stimulates Neutrophil Degranulation.

Myeloperoxidase, heme enzyme of azurophilic granules in neutrophils, is released into the extracellular space in the inflammation foci. In neutrophils, it stimulates a dose-dependent release of lactoferrin (a protein of specific granules), lysozyme (a protein of specific and azurophilic granules), and elastase (a protein of azurophilic granules). 4-Aminobenzoic acid hydrazide, a potent inhibitor of peroxidase activity of myeloperoxidase, produced no effect on neutrophil degranulation. Using signal transduction inhibitors (genistein, methoxyverapamil, wortmannin, and NiCl2), we demonstrated that myeloperoxidase-induced degranulation of neutrophils resulted from enzyme interaction with the plasma membrane and depends on activation of tyrosine kinases, phosphatidylinositol 3-kinases (PI3K), and calcium signaling. Myeloperoxidase modified by oxidative/halogenation stress (chlorinated and monomeric forms of the enzyme) lost the potency to activate neutrophil degranulation.

[Myeloperoxidase activity in blood plasma as a criterion of therapy for patients with cardiovascular disease]. / Aktivnost' mieloperoksidazy v plazme krovi kak kriterii éffektivnosti lecheniia patsientov s serdechno-sosudistymi zabolevaniiami.

A significant increase in the myeloperoxidase (MPO) activity has been found in plasma of patients with stable angina and with acute coronary syndrome (ACS) in comparison with the control group. MPO concentration was significantly increased in plasma of ACS patients. Reduced MPO activity in the treated ACS patients correlated with a favorable outcome of the disease. Generally, changes in plasma MPO concentration coincided with changes in lactoferrin concentration thus confirming the role of neutrophil degranulation in the increase of plasma concentrations of these proteins. The increase in MPO activity was obviously determined by modification of the MPO protein caused by reactive oxygen species and halogen in the molar ratio of 1 : 25 and 1 : 50. The decrease in plasma MPO activity may be associated with increased plasma concentrations of the physiological inhibitor of its activity, ceruloplasmin, and also with modification of the MPO protein with reactive oxygen species and halogen at their molar ratio of 1 : 100 and higher. Thus, MPO activity may be used for evaluation of effectiveness of the treatment of cardiovascular diseases.

The biodegradation of fullerene C60 by myeloperoxidase.

It is shown for the first time that the mammalian enzymes can cause the degradation of the C60 fullerene molecules. This biodegradation is caused by the action of а hypochlorite generated neutrophil enzyme myeloperoxidase of fullerene molecule and leads to the loss of the topology of the fullerene core.

Kinetic method for assaying the halogenating activity of myeloperoxidase based on reaction of celestine blue B with taurine halogenamines.

Myeloperoxidase (MPO) is a challenging molecular target which, if put under control, may allow regulating the development of inflammatory reactions associated with oxidative/halogenative stress. In this paper, a new kinetic method for assaying the halogenating activity of MPO is described. The method is based on measuring the rate of iodide-catalyzed oxidation of celestine blue B (CB) by oxygen and taurine N-chloramine (bromamine). The latter is produced in a reaction of taurine with HOCl (HOBr). CB is not a substrate for the peroxidase activity of MPO and does not react with hydrogen peroxide and superoxide anion radical. Taurine N-chloramine (bromamine) reacts with CB in molar ratio of 1:2. Using the new method, we studied the dependence of MPO activity on concentration of substrates and inhibitors. The specificity of MPO inhibition by non-proteolyzed ceruloplasmin is characterized. The inhibition of taurine N-chloramine production by neutrophils and HL-60 cells in the presence of MPO-affecting substances is demonstrated. The new method allows determining the kinetic parameters of MPO halogenating activity and studying its inhibition by various substances, as well as screening for potential inhibitors of the enzyme.

Interaction of ceruloplasmin with eosinophil peroxidase as compared to its interplay with myeloperoxidase: Reciprocal effect on enzymatic properties.

Myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are involved in the development of halogenative stress during inflammation. We previously described a complex between MPO and ceruloplasmin (CP). Considering the high structural homology between MPO and EPO, we studied the latter's interaction with CP and checked whether EPO becomes inhibited in a complex with CP. Disc-electrophoresis and gel filtration showed that CP and EPO form a complex with the stoichiometry 1:1. Affinity chromatography of EPO on CP-agarose (150 mM NaCl, 10 mM Na-phosphate buffer, of pH 7.4) resulted in retention of EPO. EPO protects ceruloplasmin from limited proteolysis by plasmin. Only intact CP shifted the Soret band typical of EPO from 413 to 408 nm. The contact with CP likely causes changes in the heme pocket of EPO. Peroxidase activity of EPO with substrates such as guaiacol, orcinol, o-dianisidine, 4-chloro-1-naphtol, 3,3',5,5'-tetramethylbenzidine, and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonate) is inhibited by CP in a dose-dependent manner. Similar to the interaction with MPO, the larger a substrate molecule, the stronger the inhibitory effect of CP upon EPO. The limited proteolysis of CP abrogates its capacity to inhibit the peroxidase activity of EPO. The peptide RPYLKVFNPR (corresponding to amino acids 883-892 in CP) inhibits the peroxidase and chlorinating activity of EPO. Only the chlorinating activity of EPO is efficiently inhibited by CP, while the capacity of EPO to oxidize bromide and thiocyanate practically does not depend on the presence of CP. EPO enhances the p-phenylenediamine-oxidase activity of CP. The structural homology between the sites in the MPO and EPO molecules enabling them to contact CP is discussed.