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PubMed1993

Magnetic resonance spectroscopy, calcium content, and anion coordination studies of bovine and goat lactoperoxidase.

Bovine lactoperoxidase from two purebred strains and a commercial source as well as lactoperoxidase isolated from Alpine goat milk were examined by proton NMR spectroscopy for structural comparison of the heme site. Hyperfine shifted proton NMR spectra for both the native enzymes and cyanide complexes were equivalent for the protein obtained from the four separate sources. Activity assays (guaiacol and iodide ion oxidations) were also employed to compare the enzyme from various sources. Bovine lactoperoxidase was shown to contain 1.5 +/- 0.1 calcium ions per heme unit. Lactoperoxidase complexes with nitrite ion and thiocyanate ion were characterized for comparison with the cyanide complex. The nitrite complex exhibits a proton NMR hyperfine shift pattern at ambient temperature consistent with a low-spin ferric formulation. Interaction of lactoperoxidase with thiocyanate ion was monitored by NMR and EPR spectroscopy. Proton NMR spectra of lactoperoxidase in the presence of excess thiocyanate ion illustrated the retention of a high-spin ferric configuration consistent with predominant binding of the physiological thiocyanate substrate at a non-heme site at room temperature. However, EPR spectroscopy at cryogenic temperatures revealed the existence of a low-spin lactoperoxidase thiocyanate complex. This result may be explained by low-affinity ambient temperature thiocyanate heme binding that is greatly enhanced at liquid helium temperature.

Animals↗
PubMed1999

Contribution of the lactoperoxidase system to the keeping quality of pasteurized milk.

The lactoperoxidase system is a naturally occurring antimicrobial system found in milk, with lactoperoxidase, thiocyanate and hydrogen peroxide as its components. The keeping quality of milk pasteurized at 72 degrees C for 15 s was found to be better than that of milk heated at 80 degrees C for 15 s. This agrees with previous findings and is usually attributed to heat shocking of spores. However, complete deactivation of lactoperoxidase occurred at 80 degrees C-15 s, whereas at 72 degrees C-15 s residual lactoperoxidase activity was approximately 70%, which may provide an alternative explanation. Higher levels of hypothiocyanite (the major antimicrobial agent produced by the lactoperoxidase system) were also detected in milk processed at 72 than at 80 degrees C, which supports the theory that the lactoperoxidase system has a role in the keeping quality of pasteurized milk. Of all the methods evaluated, titratable acidity and alcohol stability gave the most consistent estimates of keeping quality, while dissolved oxygen was a good indication of the onset of spoilage. Lactoperoxidase activity decreased with temperature more rapidly between 70 and 80 degrees C than is usual for an enzyme over a 10 deg C range.

Animals↗
PubMed1977

Enzyme activity of salivary lactoperoxidase adsorbed to human enamel.

Human saliva was incubated with human enamel powder, and the lactoperoxidase activity of the saliva was measured before and after incubation. Liquidphase lactoperoxidase activity was reduced in direct proportion to the weight of enamel powder added. Lactoperoxidase molecules were adsorbed to the enamel surface in an enzymatically active conformation, and this enamel-bound lactoperoxidase activity was also measured. The adsorption of lactoperoxidase was irreversible and produced at least a 40% increase in the concentration of lactoperoxidase in the enamel surface phase as compared with its concentration in the liquid phase. Enamel-bound lactoperoxidase, as well as the free enzyme, was capable of inactivating the key glycolytic enzyme hexokinase. The implications of the adsorption phenomenon for bacterial colonization are discussed.

Adsorption↗
PubMed1979

Lactoperoxidase binding to streptococci.

There have been conflicting reports regarding the binding of lactoperoxidase to bacterial cell surfaces. We describe here the effects of cell-bound lactoperoxidase on acid production by suspensions of Streptococcus mutans (NCTC 10449) in the presence of hydrogen peroxide and thiocyanate. Saline suspensions of log-phase bacteria were treated with 0.1 mg of lactoperoxidase per ml and were then washed thoroughly. The addition of hydrogen peroxide and thiocyanate markedly reduced the acid production of these lactoperoxidase-treated bacteria but had no effect on the acid production of untreated controls. After a 3-h incubation in saline, the lactoperoxidase-treated bacteria produced acid in the presence of hydrogen peroxide and thiocyanate at the same rate as untreated bacteria. These observations suggest that lactoperoxidase is initially bound to the cell surface in an enzymatically active form at a concentration sufficient to inhibit acid production. The lactoperoxidase is slowly degraded or desorbed as the bacteria stand in saline suspension.

Cell Membrane↗
PubMed1966

The inhibition of streptococci by lactoperoxidase, thiocyanate and hydrogen peroxide. The effect of the inhibitory system on susceptible and resistant strains of group N streptococci.

1. The growth of the lactoperoxidase-sensitive Streptococcus cremoris 972 in a synthetic medium was inhibited by lactoperoxidase and thiocyanate. The glycolysis and oxygen uptake of suspensions of Strep. cremoris 972 in glucose or lactose were also inhibited. The lactoperoxidase-resistant Strep. cremoris 803 was not inhibited under these conditions but was inhibited in the absence of a source of energy. 2. Lactoperoxidase (EC 1.11.1.7), thiocyanate and hydrogen peroxide completely inhibited the hexokinases of non-metabolizing suspensions of both strains. The inhibition was reversible, hexokinase and glycolytic activities of Strep. cremoris 972 being restored by washing the cells free from inhibitor. The aldolase and 6-phosphogluconate-dehydrogenase activities of Strep. cremoris 972 were partially inhibited but several other enzymes were unaffected. 3. The resistance of Strep. cremoris 803 to inhibition was not due to the lack of hydrogen peroxide formation, to the destruction of peroxide, to the inactivation of lactoperoxidase or to the operation of alternative pathways of carbohydrate metabolism. 4. A ;reversal factor', which was partially purified from extracts of Strep. cremoris 803, reversed the inhibition of glycolysis of Strep. cremoris 972. The ;reversal factor' also catalysed the oxidation of NADH(2) in the presence of an intermediate oxidation product of thiocyanate and was therefore termed the NADH(2)-oxidizing enzyme. 5. The NADH(2)-oxidizing enzyme was present in lactoperoxidase-resistant streptococci but was absent from lactoperoxidase-sensitive streptococci.

Carbohydrate Metabolism↗
PubMed1994

Mechanism-based inactivation of lactoperoxidase and thyroid peroxidase by resorcinol derivatives.

Humans are exposed to resorcinol derivatives in the environment through ground water, foods, food additives, drugs, and hair dyes. Epidemiological studies have linked human exposure to phenolic compounds with the thyroid disorder, goiter. The results presented here demonstrate the suicide (mechanism-based) inactivation of thyroid peroxidase (TPO) and the closely related lactoperoxidase (LPO) by resorcinol derivatives. The evidence for this mechanism includes irreversible, hydrogen peroxide-dependent loss of enzymatic activity by kinetics consistent with a suicide mechanism, concomitant with changes in the visible spectrum of the prosthetic heme group and covalent binding of resorcinol (ca. 10 mol/mol of lactoperoxidase inactivated). The inactivation was specific for thyroid peroxidase and lactoperoxidase since the activity of horseradish peroxidase, myeloperoxidase, chloroperoxidase, or the pseudoperoxidase, metmyoglobin, was unaffected by incubation with resorcinol. The enzymatic oxidation of resorcinol by lactoperoxidase was linked to inactivation since the same products were observed spectrally, albeit at a much lower level, as were observed with horseradish peroxidase. The results are consistent with thyroid peroxidase- and lactoperoxidase-catalyzed oxidation of resorcinol derivatives to reactive radical species that covalently bind to amino acid residues unique to these two enzymes. The oxidation of thyroid peroxidase and lactoperoxidase by hydrogen peroxide produces catalytic intermediates containing unpaired electron density on amino acid residues similar to that seen with cytochrome c peroxidase. These results provide an explanation for the potency of resorcinol derivatives in the inhibition of LPO and TPO and the goitrogenic responses observed in humans and animals. The widespread occurrence of resorcinol derivatives in the environment suggests that exposure to these compounds may cause thyroid dysfunction in humans.

Dose-Response Relationship, Drug↗
PubMed2005

Optical spectra of lactoperoxidase as a function of solvent.

The iron of lactoperoxidase is predominantly high-spin at ambient temperature. Optical spectra of lactoperoxidase indicate that the iron changes from high-spin to low-spin in the temperature range from room temperature to 20 K. The transformation is independent of whether the enzyme is in glycerol/water or solid sugar glass. Addition of the inhibitor benzohydroxamic acid increases the amount of the low-spin form, and again the transformation is independent of whether the protein is in an aqueous solution or a nearly anhydrous sugar. In contrast to lactoperoxidase, horseradish peroxidase remains high-spin over the temperature excursion in both solvents and with addition of benzohydroxamic acid. We conclude that details of the heme pocket of lactoperoxidase allow ligation changes with temperature that are dependent upon the apoprotein but independent of solvent fluctuations. At low pH, lactoperoxidase shows a solvent-dependent transition; the high-spin form is predominant in anhydrous sugar glass, but in the presence of water, the low-spin form is also present in abundance. The active site of lactoperoxidase is not as tightly constrained at low pH as at neutrality, though the enzyme is active over a wide pH range.

Binding Sites↗
PubMedDate not supplied

Suicide inactivation of lactoperoxidase by 3-amino-1,2,4-triazole.

Amitrole (3-amino-1,2,4-triazole) meets the criteria for a suicide (mechanism-based) inhibitor of lactoperoxidase. Amitrole causes rapid inactivation of lactoperoxidase only in the presence of hydrogen peroxide, and the kinetics are consistent with a suicide mechanism. Approximately 7 mol of radiolabeled amitrole binds covalently per equivalent of lactoperoxidase activity lost. The visible spectrum of lactoperoxidase inactivated by amitrole is unchanged, suggesting that covalent modification of the heme prosthetic group does not occur. The 13C NMR spectrum of lactoperoxidase inactivated by [13C]amitrole shows unique resonances which support the hypothesis that covalent binding occurs on the protein moiety. The similarities between lactoperoxidase and thyroid peroxidase suggest a similar mechanism for inhibition of thyroid hormone synthesis by amitrole.

Amitrole↗
PubMed1994

Lysozyme and lactoperoxidase inhibit the adherence of Streptococcus mutans NCTC 10449 (serotype c) to saliva-treated hydroxyapatite in vitro.

Lysozyme, lactoperoxidase and salivary peroxidase inhibit the metabolism and growth of mutans streptococci, but any possible effects on the adherence of these bacteria are unknown. In this study the effects of lysozyme and lactoperoxidase on the adhesion of 3H-labelled Streptococcus mutans (NCTC 10449, serotype c strain) to saliva-coated hydroxyapatite were studied at pH 5.0 and 7.0. Human whole saliva was either lysozyme-depleted and centrifuged, or sterilized and dialysed to achieve no detectable lysozyme and peroxidase activities; this modified saliva was used to form experimental pellicles. The incorporation of lysozyme (50-200 micrograms/ml) to the pellicle caused a significant (p < 0.01) reduction in the adherence of S. mutans without any loss of bacterial viability. Pretreatment of either saliva-coated apatite or S. mutans cells with lysozyme did not change the results but lysozyme bound more readily to bacteria than to the experimental pellicles. Also, lactoperoxidase (10-200 micrograms/ml) reduced significantly (p < 0.001) the adherence of S. mutans but, in contrast to lysozyme, in a dose-dependent way. The strongest inhibition of adhesion was found when both saliva-coated apatite and bacteria were pretreated with lactoperoxidase. This enzyme bound to experimental pellicles in preference to streptococci. A non-specific protein control, albumin, did not block the inhibition by lysozyme or lactoperoxidase. The inhibition of adherence of a serotype c strain of S. mutans to saliva-coated hydroxyapatite is a novel antibacterial mechanism for both lysozyme and lactoperoxidase.

Albumins↗
PubMed1990

Human salivary peroxidase and bovine lactoperoxidase are cross-reactive.

Peroxidases are abundant in nature, and the primary function of mammalian peroxidases is to catalyze the peroxidation of halides and pseudohalides. Previous studies have shown that antibodies raised against bovine lactoperoxidase moderately cross-react with human salivary peroxidase, a feature that has been used in the present study to examine epitopes common to the antigen and human salivary peroxidase. Polyclonal antibodies against a highly purified preparation of bovine lactoperoxidase were raised in rabbits, and their properties were examined. In double-immunodiffusion experiments, the two enzymes showed partial identity, and in competitive radioimmunoassay and enzyme-linked immunosorbent assay, lactoperoxidase replaced the labeled and coated antigen, while salivary peroxidase did not. However, salivary peroxidase from human and rat saliva samples and the purified enzyme in its non-reduced, reduced, and de-glycosylated forms were recognized by these antibodies, as analyzed by Western blot analysis and immunodetection. The major activity of these antibodies was directed against the protein core of the antigen. Immunodetection of the peptide fragments of bovine lactoperoxidase and human salivary peroxidase revealed structural differences in the two enzymes. These antibodies also precipitated an in vitro translation product from rat-parotid-gland cell lysate that, on SDS-PAGE, compared favorably with the expected molecular weight of a de-glycosylated peroxidase. The antibodies partly inhibited the enzyme activity of salivary peroxidase and the peroxidase in rat parotid gland lysate, but the enzyme activity of lactoperoxidase was not affected by addition of anti-lactoperoxidase IgG between 25 and 400 micrograms/mL. The enzyme activity remained unchanged in all samples when pre-immune IgG was used.

Animals↗
PubMed1990

Ethyl hydroperoxide-dependent metabolism of N,N-dimethyl-p-anisidine catalysed by lactoperoxidase.

1. Chemical oxidation of N,N-dimethyl-p-anisidine (DMA) with bromine at pH 5-8 gave p-N,N-dimethylaminophenoxy radical irrespective of the DMA/Br2 ratio (1 to 100), whereas the ethyl hydroperoxide-supported oxidation of DMA catalysed by lactoperoxidase gave the phenoxy radical or the cation radical of DMA depending upon the concentration of DMA. 2. The amount of p-benzoquinone produced by the chemical oxidation increased steeply with an increase in pH above 6.0, whereas that produced by the lactoperoxidase-ethyl hydroperoxide-bromide system exhibited a pH optimum centred around pH 6.0. When the concentration of DMA was increased 10-fold, the enzymic formation of p-benzoquinone greatly decreased, whereas that of formaldehyde increased. 3. The rate of formation of the oxidized bromine species by the lactoperoxidase-ethyl hydroperoxide-bromide system showed a similar pH-profile to the formation of p-benzoquinone. 4. The oxidized bromine species is considered to be the predominant oxidizing agent in the lactoperoxidase-ethyl hydroperoxide-bromide system at pH 6.0 and below. The decrease in the amount of p-benzoquinone formed, and the increase in the amount of formaldehyde formed, by the lactoperoxidase-ethyl hydroperoxide-bromide system with increasing concentration of DMA, were interpreted in terms of competition between bromide and DMA for the reaction with compound I of lactoperoxidase.

Aniline Compounds↗
PubMed1989

Interaction of lactoperoxidase with enzymes and immunoglobulins in bovine milk.

The interaction of lactoperoxidase with lysozyme and ribonuclease as well as immunoglobulins from cow milk has been investigated. As gel filtration and enzyme kinetics experiments have shown, the lactoperoxidase was slightly activated by complexing to lysozyme, while IgA and IgM were inhibitory for the peroxidase. Oh the other hand, IgG and ribonuclease had no effect on the enzyme activity although the latter did form a complex with the lactoperoxidase. The interaction between the lysozyme and lactoperoxidase appears to be rather specific since the alteration of the lactoperoxidase sugar moiety by periodate oxidation, prevented the formation of the lactoperoxidase-lysozyme complex.

Animals↗
PubMed1979

Interaction of lactoperoxidase with thiols and diiodotyrosine.

Glutathione and cysteine bind to the heme of lactoperoxidase, thereby causing a red shift of the Soret band which is reversed upon addition of iodide or guaiacol, two substrates for lactoperoxidase. The rate of formation of the enzyme-thiol complex is enhanced by diiodotyrosine. Binding of diiodotyrosine to lactoperoxidase does not cause a shift of the Soret band which indicates binding to the protein of the enzyme. At neutral pH and low ionic strength, lactoperoxidase is adsorbed on insolubilized diiodotyrosine (diiodotyrosine-agarose). It can be eluted at slightly increased ionic strength which shows that the binding is weak. In the presence of 5 X 10(-4) M glutathione, however, the binding of the enzyme to diiodotyrosine-agarose becomes much stronger so that a high salt concentration is required for elution. Lactoperoxidase is also adsorbed on insolubilized thiols (thiol-agarose). The presence of diiodotyrosine is not required for strong binding. A simple method for the preparation of lactoperoxidase from milk by affinity chromatography is based on the interactions of the enzyme with the two ligands, thiols and diiodotyrosine.

Cysteine↗
PubMed1966

The inhibition of streptococci by lactoperoxidase, thiocyanate and hydrogen peroxide. The oxidation of thiocyanate and the nature of the inhibitory compound.

1. The products of the lactoperoxidase-catalysed oxidation of thiocyanate by hydrogen peroxide were sulphate, carbon dioxide and ammonia. Cyanate, sulphite and a compound showing increased extinction at 235mmu (the ;235 compound') were intermediate oxidation products. 2. Two of the intermediates acted as electron acceptors in the oxidation of NADH(2). Thus NADH(2) was oxidized by sulphite in the presence of lactoperoxidase (EC 1.11.1.7) and Mn(2+) and by the ;235 compound' in the presence of an enzyme, the NADH(2)-oxidizing enzyme, present in extracts of lactoperoxidase-resistant streptococci. Sulphur dicyanide also acted as an electron acceptor in the latter reaction. The ;235 compound' was also reduced non-enzymically by sulphite. 3. The glycolysis of lactoperoxidasesensitive streptococci suspended in glucose solution was not inhibited by sulphite, cyanate, cyanide or the ;235 compound' but was inhibited by sulphur dicyanide. The inhibition by 0.1mm-sulphur dicyanide could be reversed, as could that caused by lactoperoxidase, thiocyanate and hydrogen peroxide, by washing the cells or by the addition of a cell-free extract of a lactoperoxidase-resistant streptococcus. 4. The effects of 0.1mm-sulphur dicyanide on catabolic enzymes of resting streptococci were very similar to those of the lactoperoxidase-thiocyanate-hydrogen peroxide system. Thus hexokinase was completedly inhibited, glucose 6-phosphate dehydrogenase and aldolase were partially inhibited and phosphohexokinase was little affected in both cases.

Autoradiography↗
PubMed1966

Inhibitory effect of saliva on glutamic acid accumulation by Lactobacillus acidophilus and the role of the lactoperoxidase-thiocyanate system.

Clem, W. H. (University of Washington, Seattle), and S. J. Klebanoff. Inhibitory effect of saliva on glutamic acid accumulation by Lactobacillus acidophilus and the role of the lactoperoxidase-thiocyanate system. J. Bacteriol. 91:1848-1853. 1966.-Saliva contains an antimicrobial system which inhibits the growth of Lactobacillus acidophilus, as well as a number of other organisms, in complete growth medium. This antimicrobial system consists of the salivary peroxidase (lactoperoxidase) and thiocyanate ions, and requires the presence of H(2)O(2). Saliva inhibits the accumulation of glutamic acid and certain other amino acids by resting cells. This effect of saliva is decreased by dialysis, and thiocyanate ions restore the inhibitory effect of dialyzed saliva. The inhibitory effect of saliva is decreased by heat (100 C, 10 min), and lactoperoxidase restores the inhibitory effect of heated saliva. Thus, the inhibition of glutamic acid accumulation by saliva appears to be due in part to the lactoperoxidase-thiocyanate antimicrobial system. H(2)O(2) increases the inhibitory effect of both saliva and the lactoperoxidase-thiocyanate system on glutamic acid accumulation. The inhibition of glutamic acid accumulation is not preceded by a loss in microbial viability. The glutamic acid accumulated by L. acidophilus under the conditions employed remains largely (over 90%) as free glutamic acid. This suggests that saliva and the lactoperoxidase-thiocyanate-H(2)O(2) system inhibit the net transport of glutamic acid into the cell.

Carbon Isotopes↗
PubMed1986

Oxygenation of organosulfur compounds by peroxidases: evidence of an electron transfer mechanism for lactoperoxidase.

The oxygenation of benzyl methylsulfide, thioanisole, and thiobenzamide to the respective sulfoxides was found to be catalyzed by chloroperoxidase, lactoperoxidase, and horseradish peroxidase. The activities of lactoperoxidase and horseradish peroxidase were similarly low toward benzyl methylsulfide and thioanisole but lactoperoxidase efficiently catalyzed the oxygenation of thiobenzamide while horseradish peroxidase showed low activity. Chloroperoxidase had high reactivity toward all three substrates tested in halide-independent reactions and only small differences in the rates of enzymatic sulfoxidation were observed. The logarithm of lactoperoxidase activity was found to linearly correlate with the voltammetric peak potentials for oxidation of the three substrates tested. The results of this study are consistent with a one-electron transfer mechanism for lactoperoxidase-mediated sulfoxidation.

Chloride Peroxidase↗
PubMed1981

Lactoperoxidase-catalyzed inactivation of hexokinase.

The enzymatic activity of hexokinase (ATP : D-hexose 6-phosphotransferase, EC 2.7.1.1) decreased rapidly when the enzyme was exposed to the lactoperoxidase antimicrobial system (consisting of lactoperoxidase, H2O2 and SCN-). Inactivation did not begin until the reaction of one sulfhydryl group per hexokinase monomer was completed. Loss of enzyme activity accompanied the reaction of at least one additional sulfhydryl group per monomer. Covalent incorporation of 14C-labeled SCN- into hexokinase increased as the inactivation reaction progressed. The rate of the hexokinase activity loss dependent on temperature, pH and the presence of glucose and phosphate ion. When H2O2 and SCN- were applied to a Sepharose column bearing covalently attached lactoperoxidase, the column eluate inactivated hexokinase. This demonstrated that the lactoperoxidase molecule itself need not be in contact with hexokinase in order to catalyze hexokinase inactivation. The sulfhydryl-reactive oxidation product of SCN- which is generated by the column is sufficient. The results are consistent with a two-stage reaction in which the exposed, non-essential sulfhydryl groups on the hexokinase molecule react first to produce an enzymatically active but unstable form of hexokinase. This modified form of hexokinase then undergoes a spontaneous, temperature-dependent structural change, which allows reaction of previously shielded, essential sulfhydryl groups. The phenomenon described here suggests a possible mechanism for the antimicrobial effects of the lactoperoxidase system.

Glucose↗
PubMed1980

Structure of milk lactoperoxidase. A study using circular dichroism and difference absorption spectroscopy.

Circular dichroism spectra of milk lactoperoxidase, its fluoride and cyanide derivatives, and those of ferrous lactoperoxidase and its carbonyl and cyanide compounds, were recorded in the wavelength region 200-670 nm. All derivatives have split ellipticity bands, suggesting that lactoperoxidase has a narrow heme pocket that prevents ligands forming linear iron-ligand bonds. Difference absorption spectroscopy of the enzyme in the far-ultraviolet region supports the previously held view that the fifth ligand of the heme iron is histidine. The secondary structure of lactoperoxidase, calculated from the far-ultraviolet circular dichroism spectrum, contains 65% beta-structure, 23% alpha-helix and 12% unordered structure. Reduction of lactoperoxidase with dithionite gives two forms, indicating that after reduction some compound arising from dithionite binds in the vicinity of the heme iron.

Animals↗