Some properties of human eosinophil peroxidase, a comparison with other peroxidases

Eosinophil peroxidase (donor:hydrogen peroxide oxidoreductase, EC 1.11.1.7) was isolated from outdated human white blood cells. The purified enzyme has a molecular weight of 71000 +/- 1000. The enzyme is composed of two subunits, of Mr 58000 and 14000, in a 1:1 stoichiometry. Amino-acid analyses showed that eosinophil peroxidase has a high content of the amino acids arginine, leucine and aspartic acid. The millimolar absorbance coefficient of the Soret band at 412 nm of eosinophil peroxidase was determined. Three independent methods yield a value for epsilon 412nm of 110 +/- 4 mm-1 X cm-1. Purified eosinophil peroxidase showed a homogeneous high-spin EPR signal with rhombic symmetry (gx = 6.50; gy = 5.40; gz = 1.982) for the haem group. EPR spectroscopy of low-spin cyanide and azide derivatives of eosinophil peroxidase, lactoperoxidase, myeloperoxidase and catalase revealed that the haem-ligand structure of eosinophil peroxidase is closely related to lactoperoxidase, whereas that of myeloperoxidase shows great resemblance to catalase.     

Axial ligand coordination in intestinal peroxidase

EPR spectra of intestinal peroxidase are reported for the first time. The resting state of intestinal peroxidase exhibits only a high spin EPR spectrum with pH-dependent rhombicity. Addition of chloride shifts the equilibrium between an acidic and a neutral form of the enzyme. In contrast, resting lactoperoxidase shows EPR spectra of both low spin and high spin species, indicating a different heme environment between these two peroxidases. The high spin signal of lactoperoxidase consists of multiple components; the major component exhibits pH-dependent rhombicity similar to intestinal peroxidase and the equilibrium between the acidic and the neutral forms is also shifted by chloride ion. EPR features of the low spin cyanide complex of intestinal peroxidase and lactoperoxidase are compared with those of other hemeproteins, whose proximal axial ligands are known to be histidine residues. The g-values of the cyanide adducts of the mammalian peroxidases are similar. The relationship between the g-value anisotropy and imidazolate character of the proximal histidine is discussed.     

Reversible carbon monoxide binding and inhibition at the active site of the Fe-only hydrogenase

Carbon monoxide binding and inhibition have been investigated by electron paramagnetic resonance (EPR) spectroscopy in solution and in crystals of structurally described states of the Fe-only hydrogenase (CpI) from Clostridium pasteurianum. Simulation of the EPR spectrum of the as-isolated state indicates that the main component of the EPR spectrum consists of the oxidized state of the "H cluster" and components due to reduced accessory FeS clusters. Addition of carbon monoxide to CpI in the presence of dithionite results in the inhibition of hydrogen evolution activity, and a characteristic axial EPR signal [g(eff(1)), g(eff(2)), and g(eff(3)) = 2.0725, 2.0061, and 2.0061, respectively] was observed. Hydrogen evolution activity was restored by successive sparging with hydrogen and argon and resulted in samples that exhibited the native oxidized EPR signature that could be converted to the reduced form upon addition of sodium dithionite and hydrogen. To examine the relationship between the spectroscopically defined states of CpI and those observed structurally by X-ray crystallography, we have examined the CpI crystals using EPR spectroscopy. EPR spectra of the crystals in the CO-bound state exhibit the previously described axial signal associated with CO binding. The results indicate that the addition of carbon monoxide to CpI results in a single reversible carbon monoxide-bound species characterized by loss of enzyme activity and the distinctive axial EPR signal.     

Acetaminophen stimulates the peroxidative metabolism of anthracyclines

Acetaminophen, a common analgesic and antipyretic drug, is frequently administered to individuals undergoing anthracycline chemotherapy. Here, the effect of acetaminophen on the metabolism of daunorubicin and doxorubicin by isolated enzymes lactoperoxidase and myeloperoxidase, and by myeloperoxidase-containing human leukemia HL-60 cells was investigated using spectrophotometric and EPR techniques. We report that at pharmacological concentrations acetaminophen strongly stimulates oxidation of the anthracyclines by lactoperoxidase and myeloperoxidase systems, which results in irreversibly altered (colorless) products. The initial rate and efficacy of daunorubicin oxidation depends on pH. While at pH approximately 7 the oxidation is rapid and extensive, almost no oxidation occurs at pH approximately 5. In the absence of daunorubicin, oxidation of acetaminophen by lactoperoxidase/hydrogen peroxide is only weakly dependent on pH, however, at pH 7.4 it strongly depends on [daunorubicin]. Ascorbate and reduced glutathione strongly inhibited oxidation of anthracyclines by lactoperoxidase and HL-60 systems. Using EPR, a daunorubicin-derived radical was detected in a daunorubicin/acetaminophen/peroxidase/hydrogen peroxide system as a narrow single line (0.175 mT) with g = 2.0047. When daunorubicin was omitted, only an acetaminophen-melanin EPR signal was detected (g = 2.0043, line width approximately 0.5 mT). Similar results were obtained with doxorubicin. We suggest that the stimulation by acetaminophen is primarily due to its preferential oxidation by peroxidases to the corresponding phenoxyl radical, which subsequently reacts with daunorubicin (doxorubicin). Because biological properties of oxidatively transformed anthracyclines will certainly be different from those of their parent compounds, the possible acetaminophen-enhanced degradation of the anthracyclines in vivo is likely to interfere with anticancer and/or cardiotoxic activities of these agents.     

Characterization of hog thyroid peroxidase

Several fundamental properties of purified hog thyroid peroxidase (A413 nm/A280 nm = 0.55) were investigated in comparison with bovine lactoperoxidase. The Mr of thyroid peroxidase was 71,000. The prosthetic group of thyroid peroxidase was identified spectrophotometrically as protoheme IX after the enzyme was hydrolyzed with Pronase. Optical spectra of oxidized and reduced thyroid peroxidases and their complexes with azide and cyanide were very similar to lactoperoxidase, except that lactoperoxidase had two reduced forms with the Soret band either at 446 or 435 nm, and thyroid peroxidase lacked a reduced form having the 446-nm band. From comparison of their pyridine hemochrome spectra, epsilon mM at 413 nm of thyroid peroxidase was estimated to be 114, being the same as that of lactoperoxidase. The cyanide inhibition for the reaction of thyroid peroxidase was competitive with hydrogen peroxide and the inhibition constant was in rough accord with the dissociation constant of its cyanide complex measured from spectrophotometric titration. Azide inhibited the reaction with an inhibition constant which was about one one-thousandth of the dissociation constant for its spectrally discernible complex. The azide inhibition was not competitive with hydrogen peroxide and decreased as the reaction proceeded. Aminotriazole inhibited the reaction strongly, and the inhibition was augmented during the reaction. These inhibition patterns of azide and aminotriazole were more or less observed in the reaction of lactoperoxidase, but not in the case of horseradish peroxidase. Characteristics of animal peroxidases are discussed.     

Characterization of one- and two-electron oxidations of glutathione coupled with lactoperoxidase and thyroid peroxidase reactions

Glutathione (GSH) was oxidized to GSSG in the presence of H2O2, tyrosine, and peroxidase. During the GSH oxidation catalyzed by lactoperoxidase, O2 was consumed and the formation of glutathione free radical was confirmed by ESR of its 5,5'-dimethyl-1-pyrroline-N-oxide adduct. When lactoperoxidase was replaced by thyroid peroxidase in the reaction system, the consumption of O2 and the formation of the free radical became negligibly small. These results led us to conclude that, in the presence of H2O2 and tyrosine, lactoperoxidase and thyroid peroxidase caused the one-electron and two-electron oxidations of GSH, respectively. It was assumed that GSH is oxidized by primary oxidation products of tyrosine, which are phenoxyl free radicals in lactoperoxidase reactions and phenoxyl cations in thyroid peroxidase reactions. When tyrosine was replaced by diiodotyrosine or 2,6-dichlorophenol, the difference in the mechanism between lactoperoxidase and thyroid peroxidase disappeared and both caused the one-electron oxidation of GSH. Iodides also served as an effective mediator of GSH oxidation coupled with the peroxidase reactions. In this case the two peroxidases both caused the two-electron oxidation of GSH.     

Spectral studies with lactoperoxidase and thyroid peroxidase: interconversions between native enzyme, compound II, and compound III

Spectral scans in both the visible (650-450 nm) and the Soret (450-380 nm) regions were recorded for the native enzyme, Compound II, and Compound III of lactoperoxidase and thyroid peroxidase. Compound II for each enzyme (1.7 microM) was prepared by adding a slight excess of H2O2 (6 microM), whereas Compound III was prepared by adding a large excess of H2O2 (200 microM). After these compounds had been formed it was observed that they were slowly reconverted to the native enzyme in the absence of exogenous donors. The pathway of Compound III back to the native enzyme involved Compound II as an intermediate. Reconversion of Compound III to native enzyme was accompanied by the disappearance of H2O2 and generation of O2, with approximately 1 mol of O2 formed for each 2 mol of H2O2 that disappeared. A scheme is proposed to explain these observations, involving intermediate formation of the ferrous enzyme. According to the scheme, Compound III participates in a reaction cycle that effectively converts H2O2 to O2. Iodide markedly affected the interconversions between native enzyme, Compound II, and Compound III for lactoperoxidase and thyroid peroxidase. A low concentration of iodide (4 microM) completely blocked the formation of Compound II when lactoperoxidase or thyroid peroxidase was treated with 6 microM H2O2. When the enzymes were treated with 200 microM H2O2, the same low concentration of iodide completely blocked the formation of Compound III and largely prevented the enzyme degradation that otherwise occurred in the absence of iodide. These effects of iodide are readily explained by (i) the two-electron oxidation of iodide to hypoiodite by Compound I, which bypasses Compound II as an intermediate, and (ii) the rapid oxidation of H2O2 to O2 by the hypoiodite formed in the reaction between Compound I and iodide.     

Thyroid peroxidase selects the mechanism of either 1- or 2-electron oxidation of phenols, depending on their substituents

Unlike lactoperoxidase and horseradish peroxidase, thyroid peroxidase catalyzed the oxidation of hydroquinone mostly by way of 2-electron transfer. This conclusion could be derived from three independent experiments: ESR measurements of p-benzosemiquinone, trapping the unpaired electron by cytochrome c, and spectrophotometric analysis of catalytic intermediates of the enzymes. The 1-electron flux for hydroquinone oxidation was found to be 15-19% in the reaction of thyroid peroxidase, while it was nearly 100% in the reactions of lactoperoxidase and horseradish peroxidase. From the spectrophotometric analysis of the catalytic intermediates of enzyme, it was suggested that the mechanism of oxidation catalyzed by thyroid peroxidase changes from a 2-electron to a 1-electron type as the substituents at 2- and 6-positions of phenol become bulky or heavy. On the other hand, the mechanism was invariably a 1-electron type when the oxidation of phenols was catalyzed by lactoperoxidase or horseradish peroxidase. These three peroxidases all catalyzed 1-electron oxidation of ascorbate.     

Spectral properties of the oestrogen-induced rat uterus peroxidase II and some of its derivatives.

1. The visible absorption spectrum of peroxidase II, isolated from the uterine tissue of oestradiol-treated rats, and some of its derivatives were recorded. The spectral properties of this enzyme are very similar to eosinophile peroxidase and lactoperoxidase, suggesting that these enzymes may have a similar form of haem as prosthetic group. 2. The uterine peroxidase is modified upon interaction with H2O2 and the difference spectrum of this modified enzyme is similar to that of complex II of lactoperoxidase. The modified enzyme was found to revert spontaneously to the native enzyme at rates which depended on the concentration of free enzyme and H2O2.     

Reactions of purified hog thyroid peroxidase with H2O2, tyrosine, and methylmercaptoimidazole (goitrogen) in comparison with bovine lactoperoxidase

Stopped flow experiments were carried out with purified hog thyroid peroxidase (A413 nm/A280 nm = 0.42). It reacted with H2O2 to form Compound I with a rate constant of 7.8 X 10(6) M-1 s-1. Compound I was reduced to Compound II by endogeneous donor with a half-life of 0.36 s. Compound I was reduced by tyrosine directly to the ferric enzyme with a rate constant of 7.5 X 10(4) M-1 s-1. Tyrosine could also reduce Compound II to the ferric enzyme with a rate constant of 4.3 X 10(2) M-1 s-1. Methylmercaptoimidazole accelerated the conversion of Compound I to Compound II and reacted with Compound II to form an inactivated form, which was discernible spectrophotometrically. The reactions of thyroid peroxidase with methylmercaptoimidazole quite resembled those of lactoperoxidase, but occurred at higher speeds. The absorption spectra of thyroid peroxidase were similar to those of lactoperoxidase and intestinal peroxidase, but obviously different from those of metmyoglobin, horseradish peroxidase, and chloroperoxidase. Similarity and dissimilarity between thyroid peroxidase and lactoperoxidase are discussed.     

Spectroscopic Properties of Siroheme Extracted from Sulfite Reductases

Siroheme has been extracted from sulfite reductases and its properties in aqueous solution have been investigated by optical absorption, electron paramagnetic resonance (EPR), and magnetic circular dichroism (MDC) spectroscopy. The absorption spectrum of siroheme exhibits a marked pH dependence, and two pK values, 4.2 and 9.0, were determined by pH titration in the range 2–12. The first pK (4.2) is thought to correspond to the ionization of the carboxylic acid side-chains on the tetrapyrrole rings, and the second pK (9.0) is attributed to displacement of the axial ligand chloride by hydroxide. The binding of the strong field ligands, CO, NO, and cyanide, were investigated by UV-visible absorption and, in the case of the cyanide complex, by low-temperature EPR and MCD spectroscopies. CO and NO were able to reduce and bind to siroheme without additional reducing agent. The EPR spectrum of the isolated siroheme (chloride-ferrisiroheme) exhibits an axial signal with g_XXX = 6.0 and ^g= 2.0, typical of high-spin ferric hemes (S = 5/2), whereas the cyanide-complexed siroheme exhibits an approximately axial signal with g_XXX = 2.38 and _g = 1.76 that is indicative of a low-spin ferric heme (S = 1/2). The low-temperature MCD spectra and magnetization data for the as-isolated and cyanide-complexed ferrisiroheme are entirely consistent with the interpretation of the EPR spectra. The results for ferrosiroheme indicate that the siroheme remains high spin (S = 2) and low spin (S = 0) on reduction of the as-isolated and cyanide-complexed siroheme, respectively. The isolated siroheme expressed sulfite reductase activity but the assessable catalytic cycle was much less than that of the native enzyme, showing the importance of the protein environment.     

Tissue distribution of constitutive and induced soluble peroxidase in rat. Purification and characterization from lacrimal gland.

A thorough search for a soluble peroxidase in 31 different tissues of rat indicated the presence of a constitutive activity only in lacrimal, preputial and submaxillary gland. An induced soluble peroxidase activity was also detected in the lactating mammary gland and in the estrogen-induced uterine secretory fluid. The lacrimal gland was the richest source of the enzyme. No peroxidase activity was detected in the lactating mammary gland of mouse and hamster nor in the preputial gland of mouse and uterine fluid of hamster. The three constitutive and two induced soluble peroxidases of rat had a native molecular mass of 73 kDa by gel filtration and they showed a similar mobility in native PAGE. Lactoperoxidase of cow's milk and solubilized rat membrane-bound peroxidases of uterus, intestine and bone marrow showed in native PAGE a mobility which was distinctly different from that of rat soluble peroxidases. As the lacrimal gland of rat was the richest source of soluble peroxidase, the enzyme was purified from this gland to apparent homogeneity; SDS/PAGE then showed a single band of molecular mass 75 kDa which was similar to that obtained by gel filtration. Peroxidase also purified from preputial and submaxillary gland, as well as commercial lactoperoxidase, had a similar molecular mass on SDS/PAGE to purified lacrimal peroxidase. The visible spectrum of lacrimal peroxidase was similar to that of lactoperoxidase but different from membrane-bound peroxidase of rat neutrophils. On isoelectric focussing, purified lacrimal peroxidase resolved into about 14 multiple forms spanning a pI range of 6.5-3.5 while lactoperoxidase focussed at the cathode. Evidence presented suggests that the multiple forms are possibly due to differences in glycosylation. Immunodiffusion, immunoprecipitation and Western blot using antilacrimal peroxidase serum showed a similar interacting species for all five soluble peroxidases of rat while membrane-bound peroxidases showed no interaction. Although in immunodiffusion, the antiserum failed to cross-react with lactoperoxidase it did interact with lactoperoxidase on Western blot. The results indicate that the various constitutive and induced soluble peroxidases of rat tissues are similar to lacrimal peroxidase but are distinctly different from the known membrane-bound peroxidases of rat. However the lacrimal peroxidase shows both similarities as well as dissimilarities with bovine lactoperoxidase. This soluble peroxidase system of rat could be useful to study tissue-specific regulation of gene expression at the molecular level.     

In vitro stimulatory effect of anti-apoptotic seminal vesicle protein 4 on purified peroxidase enzymes

The enzymatic activities of purified horseradish peroxidase, selenium-dependent glutathione peroxidase, thyroid peroxidase and myeloperoxidase, but not that of lactoperoxidase, were markedly enhanced when added into a reaction mixture containing 5 mum native seminal vesicle protein 4, a major protein secreted from rat seminal vesicle epithelium. A further increase of horseradish peroxidase activity was obtained using Ser58-phosphorylated or acetylated seminal vesicle protein 4. The activating effect of native seminal vesicle protein 4 was highest (about 60-fold) on horseradish peroxidase when 4-chloro-1-naphtol was used as the electron donor substrate. The main kinetics parameters of the stimulatory effect on horseradish peroxidase were evaluated and the enzyme-electron donor substrate interaction was investigated by HPLC and electrospray-MS. A native seminal vesicle protein 4/4-chloro-1-naphtol noncovalent adduct was detected when the protein and 4-chloro-1-naphtol were present in the appropriate molar ratio in the horseradish peroxidase-catalyzed reaction. By contrast, no adducts were formed between native seminal vesicle protein 4 and horseradish peroxidase. This native seminal vesicle protein 4/4-chloro-1-naphtol interaction might underlie the native seminal vesicle protein 4-induced horseradish peroxidase stimulation. Furthermore, native seminal vesicle protein 4 was shown by spectrophotometric and electrospray-MS analysis to interact with NADPH, an electron donor substrate of the selenium-dependent glutathione peroxidase/glutathione reductase redox system, with formation of an adduct between them. Although further investigation is required to elucidate the mechanism of adduct formation, this interaction, probably by promoting the release of the NADPH electrons required for glutathione disulphide reduction, could explain the stimulatory effect of seminal vesicle protein 4 on mammalian peroxidases possibly involved in its physiological function on the selenium-dependent glutathione peroxidase/glutathione reductase system. The biological significance of these properties of native seminal vesicle protein 4 might be related to its ability to downregulate reactive oxygen species and oxidative stress-induced apoptosis.     

Antigenic relation between thyroid peroxidase and the microsomal antigen implicated in auto-immune diseases of the thyroid

Pools of sera from patients with Graves' disease or Hashimoto's thyroiditis highly inhibit the binding to human thyroid membranes of one of 19 monoclonal antibodies raised against preparations of human thyroid membranes. This monoclonal antibody reacts with human and bovine thyroid peroxidase and bovine lactoperoxidase but not with human hemoglobin, cytochrome c and other related molecules. These results indicate that the thyroid peroxidase and the microsomal antigen are antigenically related. These data taken together with those from other groups, highly suggest that thyroid peroxidase is the microsomal antigen involved in autoimmune thyroid diseases.     

Effects of cyanogen bromide modification of the distal histidine on the spectroscopic and ligand binding properties of myoglobin: magnetic circular dichroism spectroscopy as a probe of distal water ligation in ferric high-spin histidine-bound heme proteins

Magnetic circular dichroism (MCD) spectroscopy has been utilized to characterize the change in coordination structure in native ferric sperm whale myoglobin upon cyanogen bromide-modification. Comparison of the MCD properties of the ferric high-spin state of cyanogen bromide-modified myoglobin (BrCN-Mb) with those of native ferric horseradish peroxidase and Aplysia myoglobin suggests that ferric BrCN-Mb is a potential MCD model for the pentacoordinate state of ferric high-spin histidine-ligated heme proteins. These five-coordinate heme proteins afford a relatively weak and unsymmetric signal in the Soret region of the MCD spectrum. In contrast, native ferric myoglobin and the benzohydroxamic acid adduct of ferric horseradish peroxidase show a strong and symmetric derivative-shaped Soret MCD signal which is indicative of hexacoordination with water and histidine axial ligands. Therefore it seems that MCD spectroscopy could be used to probe the presence of water ligated to the distal side of ferric high-spin heme proteins. The MCD spectra of the ferric-azide, ferrous-deoxy and ferrous-CO forms of BrCN-Mb have also been measured and compared to those of analogous native myoglobin complexes. The present MCD study has been extended to include new ligands, NO, thiocyanate and cyanate, which bind to ferric BrCN-Mb. With exogenous ligands such as CO, NO and thiocyanate, the coordination structures of the BrCN-Mb complexes are similar to those of the respective native myoglobin adducts. In the case of ferrous-deoxy and ferric-cyanate BrCN-Mb, however, the altered MCD spectra (and EPR for the latter) reveal changes in electronic structure which likely correlate with alterations of the coordination environment of these BrCN-Mb derivatives. Data are also presented which support the proposed tetrazole-bound structure for azide-treated BrCN-Mb (Hori, H., Fujii, M., Shiro, Y., Iizuka, T., Adachi, S. and Morishima, I. (1989) J. Biol. Chem. 264, 5715-5719) and the inability of the distal histidine of BrCN-Mb to stabilize the ferric ligand-bound state.     

The detection of two electron paramagnetic resonance radical signals associated with chloroperoxidase compound I

The electron paramagnetic resonance spectra of chloroperoxidase Compound I and native enzyme are compared. Upon the formation of Compound I, the g = 2.62, 2.26, and 1.82 signals associated with native enzyme disappear and are replaced by two new EPR signals, a sharp signal at g = 2.008 and a broad signal at g = 1.73. The g = 2.008 signal accounts for only 2% of the theoretical spins while the broad signal at g = 1.73 accounts for 60 to 70% of the theoretical spins in Compound I. The g = 1.73 broad signal is reminiscent of the broad EPR signal associated with horseradish peroxidase Compound I. however, the chloroperoxidase Compound I signal has a significantly different g value. The results suggest that the g = 1.73 signal represents a porphyrin pi cation radical which has a stronger coupling to the heme ferryl iron than is the case with horseradish peroxidase Compound I.     

Magnetic resonance spectral characterization of the heme active site of Coprinus cinereus peroxidase

Examination of the peroxidase isolated from the inkcap Basidiomycete Coprinus cinereus shows that the 42,000-dalton enzyme contains a protoheme IX prosthetic group. Reactivity assays and the electronic absorption spectra of native Coprinus peroxidase and several of its ligand complexes indicate that this enzyme has characteristics similar to those reported for horseradish peroxidase. In this paper, we characterize the H2O2-oxidized forms of Coprinus peroxidase compounds I, II, and III by electronic absorption and magnetic resonance spectroscopies. Electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) studies of this Coprinus peroxidase indicate the presence of high-spin Fe(III) in the native protein and a number of differences between the heme site of Coprinus peroxidase and horseradish peroxidase. Carbon-13 (of the ferrous CO adduct) and nitrogen-15 (of the cyanide complex) NMR studies together with proton NMR studies of the native and cyanide-complexed Coprinus peroxidase are consistent with coordination of a proximal histidine ligand. The EPR spectrum of the ferrous NO complex is also reported. Protein reconstitution with deuterated hemin has facilitated the assignment of the heme methyl resonances in the proton NMR spectrum.     

Resonance Raman microspectroscopic characterization of eosinophil peroxidase in human eosinophilic granulocytes.

A resonance Raman microspectroscopic study is presented of eosinophil peroxidase (EPO) in human eosinophilic granulocytes. Experiments were carried out at the single cell level with laser excitation in Soret-, Qv-, and charge transfer absorption bands of the active site heme of the enzyme. The Raman signal obtained from the cells was almost exclusively due to EPO. Methods were developed to determine depolarization ratios and excitation profiles of Raman bands of EPO in situ. A number of Raman band assignments based on earlier experiments with isolated EPO have been revised. The results show that in agreement with literature on isolated eosinophil peroxidase, the prosthetic group of the enzyme in the (unactivated) cells is a high spin, 6-coordinated, ferric protoporphyrin IX. The core size of the heme is about 2.04 A. The proximal and distal axial ligands are most likely a histidine with the strong imidazolate character typical for peroxidases, and a weakly bound water molecule, respectively. The data furthermore indicate that the central iron is displaced from the plane of the heme ring. The unusual low wavenumber Raman spectrum of EPO, strongly resembling that of lactoperoxidase, intestinal peroxidase and myeloperoxidase, suggests that these mammalian peroxidases are closely related, and characterized by, as yet unspecified, interactions between the peripheral substituents and the protein, different from those found in other protoheme proteins.     

Iodination and oxidation of thyroglobulin catalyzed by thyroid peroxidase

The kinetics of iodination and oxidation of hog thyroglobulin were studied with purified hog thyroid peroxidase and the results were compared with the reactions of free tyrosine. From Lineweaver-Burk plots and on the basis of a value of 0.83 for delta epsilon mM at 289 nm/iodine atom incorporated, the rate constant for transfer of an assumed enzyme-bound iodinium cation to thyroglobulin was estimated to be 6.7 X 10(7) and 2.3 X 10(7) M-1 s-1 in native (iodine content = 1.0%) and more iodinated (iodine content = 1.2%) thyroglobulins, respectively. This iodine-transferring reaction was stimulated by iodothyronines, similarly as observed in the reaction with free tyrosine. The iodination of thyroglobulin was inhibited by GSH, the inhibition being competitive with thyroglobulin. Thyroglobulin was oxidized in the presence of a thyroid peroxidase system without giving any appreciable change in absorbance around 300 nm. From stopped flow data, the oxidation was concluded to occur by way of two-electron transfer and the rate constant for the reaction of thyroid peroxidase Compound I with thyroglobulin was estimated to be 1.0 X 10(7) M-1 s-1. The stopped flow kinetic pattern was similar to that observed on the reaction with free tyrosine and monoiodotyrosine. About 6 mol of hydrogen peroxide were consumed per mol of thyroglobulin. Thyroid peroxidase catalyzed thyroglobulin-mediated oxidation of GSH, but lactoperoxidase did not.