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PubMed2000

Peroxidases.

The family of human peroxidases described includes myeloperoxidase, eosinophil peroxidase, uterine peroxidase, lactoperoxidase, salivary peroxidase, thyroid peroxidase and prostaglandin H1/2 synthases. The chemical identity of the peroxidase compound I and II oxidation states for the different peroxidases are compared. The identities of the distal and proximal amino acids of the catalytic site of each peroxidase are also compared. The gene characteristics and chromosomal location of the human peroxidase family have been tabulated and their molecular evolution discussed. Myeloperoxidase polymorphism and the mutations identified so far that affect myeloperoxidase activity and modulate their susceptibility to disease is described. The mechanisms for hypohalous and hypothiocyanate formation by the various peroxidases have been compared. The cellular function of the peroxidases and their hypohalites have been described as well as their inflammatory effects. The peroxidase catalysed cooxidation of drugs and xenobiotics that results in oxygen activation by redox cycling has been included. Low-density lipoprotein oxidation (initiation of atherosclerosis), chemical carcinogenesis, idiosyncratic drug reactions (e.g. agranulocytosis), liver necrosis or teratogenicity initiated by the cooxidation of endogenous substrates, plasma amino acids, drugs and xenobiotics catalysed by peroxidases or peroxidase containing cells have also been compared. Finally, peroxidase inhibitors currently in use for treating various diseases are described.

Animals↗
PubMed1992

Catalytic sites of hemoprotein peroxidases.

The structures of the active sites of horseradish and cytochrome c peroxidase, prototypical peroxidases with an imidazole heme ligand, suggest that small substrates are generally oxidized by peroxidases at the delta-meso edge of the heme group. This inference is supported by experimental results on the Coprinus macrorhizus peroxidase (52), manganese peroxidase (51), lignin peroxidase (50) and, less definitively, lactoperoxidase (90). Macromolecular substrates, exemplified by the cytochrome c peroxidase-cytochrome c interaction, are likely to be oxidized at peroxidase surface sites bearing no specific relationship to the delta-meso heme edge. The second oxidation equivalent in the two-electron Compound I states of the peroxidases is stored either as a porphyrin radical or as a protein radical, although some peroxidases have both types of compound I. The factors that control the location of the second oxidation equivalent remain unclear. Classical peroxidases do not generally catalyze olefin epoxidation and other monooxygenations but do catalyze sulfoxidation reactions. This is best rationalized by physical separation of the substrate from the ferryl oxygen, possibly by a protein barrier, because results with cytochrome c peroxidase show that there is no inherent mechanistic reason for the inability of peroxidases to epoxidize olefins. It is not yet clear why the barrier to oxygen transfer reactions is circumvented during sulfur oxidation reactions, although one possibility is that the relatively stable sulfur cation radical that is initially formed disrupts the barrier. Chloroperoxidase, the principal nonclassical hemoprotein peroxidase so far examined, has an open active site that readily catalyzes P450-like monooxygenation reactions. The active site of chloroperoxidase is a potentially useful model for that of myeloperoxidase, but caution must be used in extrapolating from one to the other because myeloperoxidase has a histidine rather than thiolate fifth heme ligand and therefore is a classical rather than nonclassical peroxidase.

Animals↗
PubMed1984

The distribution and function of peroxidases in the respiratory tract.

Heme-containing peroxidases have been demonstrated both biochemically and cytochemically in a variety of cells that either reside in the respiratory tract or circulate through it via the vasculature. The peroxidases in neutrophils and eosinophils have long been known to function in lung defense through their participation in an antimicrobial system involving hydrogen peroxide and chloride ions. Recent studies indicate that this system is also toxic to tumor cells and, as such, it may have a protective or mitigative effect on tumor formation in the lung. Eosinophil peroxidase may be involved in immediate hypersensitivity reactions in the lung because of its secretory effect on mast cells. Platelets contain peroxidases, but how they function is unknown. Whether peroxidase occurs in lymphocytes is controversial, but until more compelling evidence is presented they should be considered peroxidase-negative. A number of cells indigenous to the respiratory tract contain peroxidase activity, but there is considerable variability among species as to its presence and amount. When careful consideration is given to fixation and incubation conditions, peroxidase can be demonstrated cytochemically in the nuclear envelope and endoplasmic reticulum of some endothelial cells and type II cells of certain rodents, but its physiological role is speculative. The alveolar macrophages of most species possess little or no peroxidase activity apart from catalase which can function as a peroxidase under certain conditions. Mast cells in the respiratory tract contain peroxidase, but it is more easily demonstrated biochemically than cytochemically. The function of mast cell peroxidase is unknown, but two hypotheses worthy of investigation are its possible role in modulation of atopic allergic reactions and involvement in an antitumor defense mechanism similar to that of myeloperoxidase. Peroxidase is most abundant in the secretory cells of the tracheobronchial epithelium and glands where, in a number of species, it is synthesized and secreted as a component of mucus. Its possible contribution to lung defense is discussed in view of its morphologic similarity to the antibacterial peroxidase of milk and saliva. Because of the ease with which peroxidases can be demonstrated cytochemically, it is not surprising that morphologic information regarding their distribution in the respiratory tract has greatly exceeded insights into their functional significance. It is hoped that advancements in cell dissociation and culture, along with biochemical isolation and purification techniques, will lead to definitive conclusions concerning their physiologic roles in lung metabolism and defense.

Basophils↗
PubMed1988

Specificity of an HPETE peroxidase from rat PMN.

The 15,000xg supernatant of sonicated rat PMN contains 5-lipoxygenase that converts arachidonic acid to 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and leukotriene A4 and an HPETE peroxidase that catalyzes reduction of the 5-HPETE. The specificity of this HPETE peroxidase for peroxides, reducing agents, and inhibitors has been characterized to distinguish this enzyme from other peroxidase activities. In addition to 5-HPETE, the HPETE peroxidase will catalyze reduction of 15-hydroperoxyeicosatetraenoic acid, 13-hydroperoxyoctadecadienoic acid, and 15-hydroperoxy-8,11,13-eicosatrienoic acid, but not cumene or t-butylhydroperoxides. The HPETE peroxidase accepted 5 of 11 thiols tested as reducing agents. However, glutathione is greater than 15 times more effective than any other thiol tested. Other reducing agents, ascorbate, NADH, NADPH, phenol, p-cresol, and homovanillic acid, were not accepted by HPETE peroxidase. This enzyme is not inhibited by 10 mM KCN, 2 mM aspirin, 2 mM salicylic acid, or 0.5 mM indomethacin. When 5-[14C]HPETE is generated from [14C]arachidonic acid in the presence of unlabeled 5-HPETE and the HPETE peroxidase, the 5-[14C]HETE produced is of much lower specific activity than the [14C]arachidonic acid. This indicates that the 5-[14C]HPETE leaves the active site of 5-lipoxygenase and mixes with the unlabeled 5-HPETE in solution prior to reduction and is a kinetic demonstration that 5-lipoxygenase has no peroxidase activity. Specificity for peroxides, reducing agents, and inhibitors differentiates HPETE peroxidase from glutathione peroxidase, phospholipid-hydroperoxide glutathione peroxidase, a 12-HPETE peroxidase, and heme peroxidases. The HPETE peroxidase could be a glutathione S-transferase selective for fatty acid hydroperoxides.

Animals↗
PubMed1983

Selenium-dependent and non-selenium-dependent glutathione peroxidases in human tissue extracts.

A method for the assessment of both selenium-dependent and non-selenium-dependent glutathione peroxidases on crude tissue extracts in human is described. The enzyme activity is measured by the coupled assay system in which oxidation of reduced glutathione (GSH) is coupled to NADPH oxidation catalyzed by glutathione reductase. Total glutathione peroxidase activity is measured with cumene hydroperoxide as substrate. Selenium-dependent glutathione peroxidase is measured with tert-butyl hydroperoxide. This substrate is preferable to H2O2 which gives too high blank values compared to the assay values. The difference between total glutathione peroxidase and selenium-dependent glutathione peroxidase activities represents the non-selenium-dependent glutathione peroxidase activity. Studies of substrate specificity of the two glutathione peroxidases separated by gel filtration as well as linearity and recovery studies are presented. For a given tissue, the relative amounts of the two glutathione peroxidases given by our assay are identical to those estimated by quantifying the elution peaks after gel filtration. Based on the percentages of the two glutathione peroxidases, human tissues can be classified in four groups: (1) the non-selenium-dependent glutathione peroxidase is predominant in liver, in renal cortex and skeletal muscle; (2) non-selenium-dependent and selenium-dependent glutathione peroxidases are in equal amounts in renal medulla; (3) the selenium-dependent glutathione peroxidase is predominant in adrenal glands and platelets; (4) the selenium-dependent glutathione peroxidase represents 100% of the glutathione peroxidase activity in the other organs. The heart and the brain are of special interest in this group because of the physiological role and the regulation of the selenoenzyme.

Chromatography, Gel↗
PubMed1999

Molecular evolution of thyroid peroxidase.

Thyroid peroxidase is a member of a family of mammalian peroxidases that includes myeloperoxidase, lactoperoxidase, eosinophil peroxidase, and salivary peroxidase. Protein sequences showing a high degree of sequence similarity with mammalian peroxidases have recently been observed in several invertebrate species. A multiple sequence alignment prepared with five mammalian and six invertebrate peroxidases shows complete conservation of amino acid residues considered to be important in the formation of peroxidase compound 1. These include the distal and proximal histidines, a catalytic arginine residue, and an asparagine residue hydrogen bonded to the proximal histidine. TPO-2, an alternatively spliced form of TPO, lacks the essential asparagine (Asn 579). It is now possible to speak more broadly of the family of animal peroxidases, rather than mammalian peroxidases. The animal peroxidases comprise a group of homologous proteins that differ markedly from the plant/fungal/bacterial peroxidases in primary, secondary and tertiary structure, but which share with them a common function. Animal peroxidases probably arose independently of the plant/fungal/bacterial peroxidase superfamily and most likely belong to a different gene family. The relationship between animal and non-animal peroxidases probably represents an example of convergent evolution to a common enzymatic mechanism.

Amino Acid Sequence↗
PubMed2000

Biosensors based on novel peroxidases with improved properties in direct and mediated electron transfer.

Native horseradish peroxidase (HRP) on graphite has revealed approximately 50% of the active enzyme molecules to be in direct electron transfer (ET) contact with the electrode surface. Some novel plant peroxidases from tobacco, peanut and sweet potato were kinetically characterised on graphite in order to find promising candidates for biosensor applications and to understand the nature of the direct ET in the case of plant peroxidases. From measurements of the mediated and mediatorless currents of hydrogen peroxide reduction at the peroxidase-modified rotating disk electrodes (RDE), it was concluded that the fraction of enzyme molecules in direct ET varies substantially for the different plant peroxidases. It was observed that the anionic peroxidases (from sweet potato and tobacco) demonstrated a higher percentage of molecules in direct ET than the cationic ones (HRP and peanut peroxidase). The peroxidases with a high degree of glycosylation demonstrated a lower percentage of molecules in direct ET. It could, thus, be concluded that glycosylation of the peroxidases hinders direct ET and that a net negative charge on the peroxidase (low pI value) is beneficial for direct ET. Especially noticeable are the values obtained for sweet potato peroxidase (SPP), revealing both a high percentage in direct ET and a high rate constant of direct ET. The peroxidase electrodes were used for determination of hydrogen peroxide in RDE mode (mediatorless). SPP gave the lowest detection limit (40 nM) followed by HRP and peanut peroxidase.

Arachis↗
PubMed1996

FTIR study of the thermal denaturation of horseradish and cytochrome c peroxidases in D2O.

Fourier transform infrared (FTIR) spectroscopy was employed to examine the thermal denaturation of the Fe(III), Fe(II), and Fe(II)-CO forms of cytochrome c peroxidase and horseradish peroxidase in phosphate buffer at pD 7.0. The amide I' regions of the deconvolved spectra are consistent with predominantly alpha-helical secondary structure around room temperature, but the alpha-helical absorption of the two peroxidases differs significantly; bands assigned to alpha-helical components occur at 1659 and 1649 cm-1 in horseradish peroxidase and at 1652 and 1637 cm-1 in cytochrome c peroxidase. The thermal denaturation mechanisms of the peroxidases also vary. All three forms of cytochrome c peroxidase retain their secondary structure up to 50 degrees C, when bands characteristic of aggregation (1616 and 1684 cm-1) appear in the amide I' region, and above 55 degrees C rapid loss of secondary structure is accompanied by enhanced aggregation. In horseradish peroxidase, on the other hand, the Fe(III) and Fe(II) states exhibit dissimilar denaturation mechanisms. Slow, gradual alteration of secondary structure is observed for Fe(III) horseradish peroxidase on heating, and polypeptide unfolding appears to be complete around 90 degrees C, without aggregation. In Fe(II) and Fe(II)-CO horseradish peroxidase, aggregation bands appear at approximately 55 degrees C, signaling the onset of denaturation. Frequency shifts in the v(CO) bands above room temperature reveal the conformational changes in the heme cavity precede global conformational changes in cytochrome c peroxidase but not in horseradish peroxidase. The reduction in amide II intensities, due to peptide H-D exchange on heating the peroxidases in D2O, indicates the formation above room temperature of partially unfolded states with increased solvent accessibility but intact secondary structures.

Cytochrome-c Peroxidase↗
PubMed1962

Colorimetric investigation of the uptake of an intravenously injected protein (horseradish peroxidase) by rat kidney and effects of competition by egg white.

After intravenous injection of horseradish peroxidase into rats, the foreign protein appeared in the kidney first in the small phagosomes and its concentration there decreased quickly; it then was concentrated and "stored" for several days in the large phagosomes. After injection of 10 mg of peroxidase per 100 gm of body weight, the concentration of peroxidase in blood and urine decreased exponentially during the first 6 hours; small amounts of peroxidase were excreted in the urine for several days. When 0.05 to 1.0 mg of peroxidase per 100 gm were administered, most of the peroxidase was taken up by the liver and little by the kidney, and a portion was excreted in the urine even at the lowest dose. At doses above 1.5 mg per 100 gm, the liver cells were saturated, and large reabsorption droplets appeared in the tubule cells of the kidney. With further dosage increase, the concentration of peroxidase in the phagosomes of the kidney increased rapidly until saturation was reached at doses of 13 mg per 100 gm. After intraperitoneal injection of egg white 18 hours prior to the administration of peroxidase, the concentration of peroxidase in all kidney fractions was only 10 to 25 per cent of the values for the untreated animals, the disappearance of peroxidase from the blood was delayed, and 81 percent more peroxidase was excreted in the urine. The treatment with egg white had no effect on the uptake of peroxidase by the liver. The ability of kidney tissue to degrade and adsorb peroxidase in vitro was tested.

Animals↗
PubMed2006

An inserted loop region of stromal ascorbate peroxidase is involved in its hydrogen peroxide-mediated inactivation.

Ascorbate peroxidase isoforms localized in the stroma and thylakoid of higher plant chloroplasts are rapidly inactivated by hydrogen peroxide if the second substrate, ascorbate, is depleted. However, cytosolic and microbody-localized isoforms from higher plants as well as ascorbate peroxidase B, an ascorbate peroxidase of a red alga Galdieria partita, are relatively tolerant. We constructed various chimeric ascorbate peroxidases in which regions of ascorbate peroxidase B, from sites internal to the C-terminal end, were exchanged with corresponding regions of the stromal ascorbate peroxidase of spinach. Analysis of these showed that a region between residues 245 and 287 was involved in the inactivation by hydrogen peroxide. A 16-residue amino acid sequence (249-264) found in this region of the stromal ascorbate peroxidase was not found in other ascorbate peroxidase isoforms. A chimeric ascorbate peroxidase B with this sequence inserted was inactivated by hydrogen peroxide within a few minutes. The sequence forms a loop that binds noncovalently to heme in cytosolic ascorbate peroxidase of pea but does not bind to it in stromal ascorbate peroxidase of tobacco, and binds to cations in both ascorbate peroxidases. The higher susceptibility of the stromal ascorbate peroxidase may be due to a distorted interaction of the loop with the cation and/or the heme.

Amino Acid Sequence↗
PubMed1996

The crystal structure of peanut peroxidase.

BACKGROUND: Peroxidases catalyze a wide variety of peroxide-dependent oxidations. Based on sequence alignments, heme peroxidases have been divided into three classes. Crystal structures are available for peroxidases of classes I and II, but until now no structure has been determined for class III, the classical extracellular plant peroxidases. RESULTS: The crystal structure of peanut peroxidase has been solved to 2.7 A resolution. The helical fold is similar to that of known peroxidase structures. The 294-residue polypeptide chain is accompanied by a heme and two calcium ions, and there is some evidence of glycosylation. CONCLUSIONS: This is the first complete structure of a class III peroxidase and as such should serve as a model for other class III enzymes including the much-studied horseradish peroxidase. It may also aid in the interpretation of functional differences between the peroxidase classes. Ten helices conserved in class I and II peroxidases are also found in peanut peroxidase. Key residues of the heme environment and the location of two calcium ions are shared with class II peroxidases. Peanut peroxidase contains three unique helices, two of which contribute to the substrate access channel leading to the heme edge.

Amino Acid Sequence↗
PubMed1991

Lignin peroxidase of Phanerochaete chrysosporium. Evidence for an acidic ionization controlling activity.

The active site amino acid residues of lignin peroxidase are homologous to those of other peroxidases; however, in contrast to other peroxidases, no pH dependence is observed for the reaction of ferric lignin peroxidase with H2O2 to form compound I (Andrawis, A., Johnson, K.A., and Tien, M. (1988) J. Biol. Chem. 263, 1195-1198). Chloride binding is used in the present study to investigate this reaction further. Chloride binds to lignin peroxidase at the same site as cyanide and hydrogen peroxide. This is indicated by the following. 1) Chloride competes with cyanide in binding to lignin peroxidase. 2) Chloride is a competitive inhibitor of lignin peroxidase with respect to H2O2. The inhibition constant (Ki) is equal to the dissociation constant (Kd) of chloride at all pH values studied. Chloride binding is pH dependent: chloride binds only to the protonated form of lignin peroxidase. Transient-state kinetic studies demonstrate that chloride inhibits lignin peroxidase compound I formation in a pH-dependent manner with maximum inhibition at low pH. An apparent pKa was calculated at each chloride concentration; the pKa increased as the chloride concentration increased. Extrapolation to zero chloride concentration allowed us to estimate the intrinsic pKa for the ionization in the lignin peroxidase active site. The results reported here provide evidence that an acidic ionizable group (pKa approximately 1) at the active site controls both lignin peroxidase compound I formation and chloride binding. We propose that the mechanism for lignin peroxidase compound I formation is similar to that of other peroxidases in that it requires the deprotonated form of an ionizable group near the active site.

Benzyl Alcohols↗
PubMed1979

A spin label study of horseradish peroxidase.

The topography of the active sites of native horseradish peroxidase and manganic horseradish peroxidase has been studied with the aid of a spin-labeled analog of benzhydroxamic acid (N-(1-oxyl-2,2,5,5-tetramethylpyrroline-3-carboxy)-p-aminobenzhydroxamic acid). The optical spectra of complexes between the spin-labeled analog of benzhydroxamic acid and Fe3+ or Mn3+ horseradish peroxidase resembled the spectra of the corresponding enzyme complexes with benzhydroxamic acid. Electron spin resonance (ESR) measurement indicated that at pH 7 the nitroxide moiety of the spin-labeled analog of benzhydroxamic acid became strongly immobilized when this label bound to either ferric or manganic horseradish peroxidase. The titration of horseradish peroxidase with the spin-labeled analog of benzhydroxamic acid revealed a single binding site with association constant Ka approximately 4.7 . 10(5) M-1. Since the interaction of ligands (e.g. F-, CN-) and H2O2 with horseradish peroxidase was found to displace the spin label, it was concluded that the spin label did not indeed bind to the active site of horseradish peroxidase. At alkaline pH values, the high spin iron of native horseradish peroxidase is converted to the low spin form and the binding of the spin-labeled analog of benzhydroxamic acid to horseradish peroxidase is completely inhibited. From the changes in the concentration of both bound and free spin label with pH, the pK value of the acid-alkali transition of horseradish peroxidase was found to be 10.5. The 2Tm value of the bound spin label varied inversely with temperature, reaching a value of 68.25 G at 0 degree C and 46.5 G at 52 degrees C. The dipolar interaction between the iron atom and the free radical accounted for a 12% decrease in the ESR signal intensity of the spin label bound to horseradish peroxidase. From this finding, the minimum distance between the iron atom and nitroxide group and hence a lower limit to the depth of the heme pocket of horseradish peroxidase was estimated to be 22 A.

Binding Sites↗
PubMed2000

Expression and characterization of recombinant human eosinophil peroxidase. Impact of the R286H substitution on the biosynthesis and activity of the enzyme.

Hereditary eosinophil peroxidase deficiency is a genetic abnormality characterized by a decrease or absence of peroxidase activity and a reduction of the granule matrix volume. Recently, we identified two mutations associated with eosinophil peroxidase deficiency in a subject and his siblings, i.e. a base insertion causing the appearance of a premature stop codon and a base transition causing the replacement of an Arg at codon 286 with a His (R286H). In this article we report the stable expression of both the recombinant wild-type and the R286H eosinophil peroxidase precursor in the K-562 cell line, and the effects of the R286H substitution on the structure and function of the eosinophil peroxidase precursor. Heme group incorporation into both the recombinant wild-type and the recombinant R286H eosinophil peroxidase precursor was comparable, as was the stability of both proteins. Instead, the recombinant R286H eosinophil peroxidase precursor exhibited marked alterations of the catalytic properties and an increased sensitivity to four peroxidase inhibitors with respect to both the recombinant wild-type eosinophil peroxidase precursor and the native enzyme. In addition, the recombinant wild-type, but not the R286H, eosinophil peroxidase precursor was immunoprecipitated by two anti-(eosinophil peroxidase) mAbs. Altogether, our results suggest a protein misfolding of the R286H eosinophil peroxidase precursor which might account for its altered catalytic properties and the absence of expression of some epitopes.

Amino Acid Substitution↗
PubMed1982

Affinity chromatography of thyroid peroxidase using tyrosine coupled to Agarose.

A selective adsorbent for thyroid peroxidase was prepared by attaching tyrosine, a possible substrate of peroxidase, to agarose beads. When partially purified calf thyroid peroxidase was passed through a column containing this adsorbent, the peroxidase activity present was bound to the agarose. The binding of thyroid peroxidase on the adsorbent was inhibited by tyrosine and iodotyrosines. Quantitative elution was readily achieved by modifying the pH of eluting buffers. The peroxidase eluted at pH 8.5 or pH 9.8 was slowly inactivated. During this inactivation, enzyme activity assayed by triiodide formation was not affected, while peroxidase activity assayed by guaiacol oxidation and tyrosine iodination were slowly reduced. Enzyme activity was protected by elution under a partially anaerobic state. Iodide and guaiacol did not interfere with the adsorption of thyroid peroxidase by tyrosine residues on agarose. These data indicate the following characteristics of thyroid peroxidase. 1. Tyrosine and iodotyrosines are the substrates of thyroid peroxidase. 2. Thyroid peroxidase has specific active site(s) for tyrosine and iodide which are independent of each other. 3. The active site(s) for tyrosine and iodotyrosines are common. 4. The active site(s) for tyrosine and guaiacol are similar but are not identical. 5. Thyroid peroxidase is able to bind tyrosine before it is activated to "Complex I" by hydrogen peroxide.

Animals↗
PubMed1977

Interaction of thyroid peroxidase with concanavalin A covalently coupled to agarose.

We have investigated the interaction between concanavalin A-agarose (Con A-agarose) and thyroid peroxidase, an integral membrane protein found in the 105,000 X g, 1-h particulate fraction of thyroid tissue. An intact form of porcine thyroid peroxidase was obtained by solubilization with the nonionic detergent Triton X-100 and two fragmented, hydrophilic forms of the enzyme were prepared by trypsin treatment of the membrane. The three types of thyroid peroxidase bind to Con A-agarose and can be eluted with alpha-methyl-D-mannoside. The alpha-methyl-D-mannoside eluate of the most purified thyroid peroxidase preparation has been analyzed by polyacrylamide gel electrophoresis. Peroxidase activity corresponds with a glycoprotein band. The binding of thyroid peroxidase to Con A-agarose can be inhibited by sugars in the following order: alpha-methyl-D-mannoside greater than D-mannose greater than alpha-methyl-D-glucoside greater than D-glucose greater than D-galactose. This order of specificity is typical of Con A-sugar interactions. Furthermore, inactivation of the carbohydrate binding site of Con A by demetallization greatly reduces the extent of thyroid peroxidase binding. Reactivation of the carbohydrate binding site by the addition of Ca2+ and Mn2+ to demetallized Con A-agarose restores thyroid peroxidase binding. These and other experiments suggest that htyroid peroxidase is, like several other peroxidases, a glycoprotein. In addition, the interaction between thyroid peroxidase and Con A-agarose may provide a new purification tool for thyroid peroxidase.

Animals↗
PubMed1999

Controlled layer-by-layer immobilization of horseradish peroxidase.

Horseradish peroxidase (HRP) was biotinylated with biotinamidocaproate N-hydroxysuccinimide ester (BcapNHS) in a controlled manner to obtain biotinylated horseradish peroxidase (Bcap-HRP) with two biotin moieties per enzyme molecule. Avidin-mediated immobilization of HRP was achieved by first coupling avidin on carboxy-derivatized polystyrene beads using a carbodiimide, followed by the attachment of the disubstituted biotinylated horseradish peroxidase from one of the two biotin moieties through the avidin-biotin interaction (controlled immobilization). Another layer of avidin can be attached to the second biotin on Bcap-HRP, which can serve as a protein linker with additional Bcap-HRP, leading to a layer-by-layer protein assembly of the enzyme. Horseradish peroxidase was also immobilized directly on carboxy-derivatized polystyrene beads by carbodiimide chemistry (conventional method). The reaction kinetics of the native horseradish peroxidase, immobilized horseradish peroxidase (conventional method), controlled immobilized biotinylated horseradish peroxidase on avidin-coated beads, and biotinylated horseradish peroxidase crosslinked to avidin-coated polystyrene beads were all compared. It was observed that in solution the biotinylated horseradish peroxidase retained 81% of the unconjugated enzyme's activity. Also, in solution, horseradish peroxidase and Bcap-HRP were inhibited by high concentrations of the substrate hydrogen peroxide. The controlled immobilized horseradish peroxidase could tolerate much higher concentrations of hydrogen peroxide and, thus, it demonstrates reduced substrate inhibition. Because of this, the activity of controlled immobilized horseradish peroxidase was higher than the activity of Bcap-HRP in solution. It is shown that a layer-by-layer assembly of the immobilized enzyme yields HRP of higher activity per unit surface area of the immobilization support compared to conventionally immobilized enzyme.

Avidin↗
PubMed1993

Selenium regulation of glutathione peroxidase in human hepatoma cell line Hep3B.

Glutathione peroxidase is an important enzyme in cellular antioxidant defense systems, detoxifying peroxides and hydroperoxides. As a component of the glutathione cycle, it protects the liver from reactive oxygen metabolites. Selenocysteine is present at the catalytic site of glutathione peroxidase, and selenium availability regulates glutathione peroxidase enzyme activity. Hep3B cells, a well-differentiated human hepatoma-derived cell line, exhibited time-dependent decrease in glutathione peroxidase activity (nmol NADPH oxidized/min/mg protein, mean +/- SE) when incubated in selenium-free medium for 10 days (Day 0, 21.8 +/- 7.3; Day 2, 10.9 +/- 1.2; Day 4, 7.9 +/- 0.8; Day 6, 4.0 +/- 0.7; Day 8, 4.5 +/- 0.6; Day 10, 1.6 +/- 0.4). With the reintroduction of selenium, glutathione peroxidase activity returned. A second human hepatoma cell line, HepG2, demonstrated a similar pattern when depleted of and then repleted with selenium. To assess protein synthesis, glutathione peroxidase activity was measured in deficient and replete Hep3B cells incubated with and without selenium and with and without cycloheximide. Deficient cells (mean +/- SE) (4.9 +/- 0.2) showed an increase in glutathione peroxidase activity after 24 h in selenium-containing medium (11.6 +/- 0.2), but not when cycloheximide was included in the medium (6.9 +/- 0.5) or when cycloheximide and no selenium was included (5.3 +/- 0.8). Replete Hep3B cells (40.1 +/- 1.1) demonstrated decreased glutathione peroxidase after 24 h in medium without selenium (34.0 +/- 1.4), medium with both cycloheximide and selenium (34.0 +/- 2.6), and medium without selenium and containing cycloheximide (37.6 +/- 1.3). These data suggest that protein synthesis is needed for selenium repletion to exert control on glutathione peroxidase activity. Using a cDNA for human glutathione peroxidase (GPx1), selenium-deficient and replete Hep3B cell RNA was analyzed by Northern blot. mRNA for GPx was quantified by densitometry. The steady-state mRNA level for glutathione peroxidase in deficient cells was 40% of that in replete cells. Nuclear run-on studies to determine the rate of GPx-specific mRNA synthesis showed no difference between nuclei from selenium-replete and selenium-deficient cells. This finding eliminated the possibility of differential transcription rates as an explanation for the observed reduction in mRNA brought about by selenium deficiency and suggested instead a stabilization of mRNA in selenium-replete cells. While selenium deficiency decreased mRNA levels by 60%, glutathione peroxidase enzyme activity decreased by 93%, suggesting a co- and/or post-translational control mechanism in addition to the effect on mRNA stability.

Carcinoma, Hepatocellular↗