Identification, purification, and characterization of a non-heme lactoperoxidase in bovine milk

A purification procedure for a protein related to lactoperoxidase devoid of the heme prosthetic group under conditions also yielding enzymatically active lactoperoxidase is described. These two forms were separated from bovine milk according to their respective behaviors on cation exchange. Lactoperoxidase and non-heme lactoperoxidase had the same apparent molecular weight in the denatured (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and native form (velocity sedimentation on sucrose gradient) about 85,000; but unlike lactoperoxidase, non-heme lactoperoxidase was devoid of light absorption properties in the Soret region and of enzyme activity. Lactoperoxidase and non-heme lactoperoxidase contained a similar amount of carbohydrate and gave very similar peptide maps after limited proteolysis by subtilisin or trypsin. The two forms appeared to be immunologically related since they gave a single line in immunodiffusion using anti-lactoperoxidase antibodies and since 125I-labeled non-heme lactoperoxidase and 125I-labeled lactoperoxidase reacted with anti-lactoperoxidase antibodies in radioimmunoassay. Lactoperoxidase and nonheme lactoperoxidase were compared in their ability to interact with diiodotyrosine and tubulin (Rousset, B., and Wolff, J. (1980) J. Biol. Chem. 255, 2514-2523). 125I-labeled diiodotyrosine bound specifically to lactoperoxidase. No detectable binding has been observed with nonheme lactoperoxidase. In contrast, lactoperoxidase and non-heme lactoperoxidase coupled to an insoluble matrix were able to bind rat brain tubulin, indicating that both forms of lactoperoxidase can be used for an affinity chromatography purification procedure of brain tubulin. Non-heme lactoperoxidase was found in milk from several origins, cow, goat, sheep, and human. In bovine milk, lactoperoxidase and non-heme lactoperoxidase were found in comparable amounts.     

Ionic strength and pH effect on the Fe(III)-imidazolate bond in the heme pocket of horseradish peroxidase: an EPR and UV-visible combined approach

The effects of chloride, dihydrogenphosphate and ionic strength on the spectroscopic properties of horseradish peroxidase in aqueous solution at pH=3.0 were investigated. A red-shift (lambda=408 nm) of the Soret band was observed in the presence of 40 mM chloride; 500 mM dihydrogenphosphate or chloride brought about a blue shift of the same band (lambda=370 nm). The EPR spectrum of the native enzyme at pH 3.0 was characterized by the presence of two additional absorption bands in the region around g=6, with respect to pH 6.5. Chloride addition resulted in the loss of these features and in a lower rhombicity of the signal. A unique EPR band at g=6.0 was obtained as a result of the interaction between HRP and dihydrogenphosphate, both in the absence and presence of 40 mM Cl-. We suggest that a synergistic effect of low pH, Cl- and ionic strength is responsible for dramatic modifications of the enzyme conformation consistent with the Fe(II)-His170 bond cleavage. Dihydrogenphosphate as well as high chloride concentrations are shown to display an unspecific effect, related to ionic strength. A mechanistic explanation for the acid transition of HRP, previously observed by Smulevich et al. [Biochemistry 36 (1997) 640] and interpreted as a pure pH effect, is proposed.     

Evidence for two H2O2-binding sites in ferric cytochrome c oxidase. Indication to the O-cycle?

H2O2 addition to the oxidized cytochrome c oxidase reconstituted in liposomes brings about a red shift of the Soret band of the enzyme and an increased absorption in the visible region with two distinct peaks at approximately 570 and 605 nm. Throughout pH range 6-8.5, the spectral changes at 570 nm and in the Soret band titrate with very similar pH-independent Kd values of 2-3 microM. At the same time, Kd of the peroxide complex measured at 605 nm increases markedly with increased H+ activity reaching the value of 18 +/- 2 microM at pH 6.0. This finding may indicate the presence of two different H2O2-binding sites in the enzyme with different affinity for the ligand at acid pH. The Soret and 570 nm band effects are suggested to report H2O2 coordination to heme iron of alpha 3, whereas the maximum at 605 nm could arise from H2O2 binding to Cu alpha 3 followed by the enzyme transition into the 'pulsed' (or '420/605') conformation. Possible implication of the two H2O2-binding sites for the cytochrome oxidase redox and proton-pumping mechanisms are discussed.     

Kinetics and equilibria of cyanide binding to prostaglandin H synthase

Cyanide binding to prostaglandin H (PGH) synthase results in a spectral shift in the Soret region. This shift was exploited to determine equilibrium and kinetic parameters of the cyanide binding process. At pH 8.0, ionic strength 0.22 M, 4 degrees C, the cyanide dissociation constant, determined from equilibrium experiments, is (65 +/- 10) microM. The binding rate constant is (2.8 +/- 0.2) x 10(3) M-1 s-1, and the dissociation rate constant is zero within experimental error. Through a kinetic study of the binding process as a function of pH, from pH 3.96 to 8.00, it was possible to determine the pKa of a heme-linked acid group on the enzyme of 4.15 +/- 0.10 with citrate buffer. An apparent pKa of 4.75 +/- 0.03 was determined with acetate buffer; this different value is attributed to complexation of the enzyme with one of the components of the acetate buffer.     

Evidence for the glycoprotein nature of radish beta-fructosidase.

Concanavalin A (Con A) was utilized free, bound to Sepharose 4 B or cross-linked to glutaraldehyde to investigate the possibility of binding this lectin to radish beta-fructosidase (E.C.3.2.1.26). The choice of cross-linked Con A as affinoadsorbent is discussed and standard conditions for binding are defined. Specificity of precipitation of this enzyme by the lectin was especially investigated. Thus, the possibility of binding was tested in the presence of high ionic strength, ethylene glycol, alpha-methyl mannoside, alpha-methyl glucoside and during periodate oxidation of the enzyme. Based on the interactions observed between beta-fructosidase and Con A under these conditions it is concluded that the saccharide binding site of the lectin is primarily involved with a secondary contribution from the hydrophobic site. The specificity of binding and the complete precipitation of beta-fructosidase activity by the insolubilized lectin imply that all beta-fructosidase activity measured in Raphanus sativus seedling extracts is linked to (a) glycoprotein form(s) of this enzyme.     

Proton interactions in the resting form of cytochrome oxidase.

The effect of pH on the near-UV absorption spectrum of cytochrome oxidase has been examined. Several lines of evidence implicate a proton binding site that can modulate the optical properties of cytochrome alpha 3 in the resting enzyme. Changing the pH within the range 6.5-10.5 was found to reversibly shift the position of the Soret band over an 11-nm range. The lower pH values caused a progressive blue shift in the Soret band, whereas the high-pH range promoted a gradual red shift. Limiting band positions were approximately 416 and 427 nm. The incubation time required to reach a stable band position varied somewhat as did the actual extent of the shift. In most cases, the shift was associated with an isosbestic point. A pH titration profile for the apparent equilibrium position of the Soret band was obtained. Nonlinear least-squares fitting to a scatter plot, assuming a single acid/base group, showed an apparent pKa of 7.8. Magnetic circular dichroism (MCD) spectra of the low-pH form at 416 nm, the high-pH form at 427 nm, and the cyanide derivative at 428 nm were compared. No evidence of a high-pH-dependent low-spin transition or a change in the redox state of cytochrome a3 was found, confirming earlier work [Baker, G. M., Noguchi, M., & Palmer, G. (1987) J. Biol. Chem. 262, 595-604]. Subtraction of ferricytochrome a [spectrum taken from Vanneste, W. H. (1966) Biochemistry 5, 838-848] from a series of blue-shifting spectra showed a band at 414 nm that progressively gained amplitude and a band at 430 nm that correspondingly lost amplitude. A series of red-shifting spectra showed the opposite behavior with a clear isosbestic point being evident in both cases. The difference extinction change at 414 and 430 nm depended linearly on the position of the Soret band, both showing a reversible dependence on pH. The 430-nm band is noted to be unusually red-shifted for high-spin ferric heme a. An additional, pH-insensitive band was observed at 408-410 nm which was eliminated by treatment with cyanide. The kinetics of the pH-induced blue shift and red shift were obtained at 416 nm by using dual-wavelength method and found to be biphasic, despite the occurrence of an isosbestic point.(ABSTRACT TRUNCATED AT 400 WORDS)     

Iodide binding and regulation of lactoperoxidase activity toward thyroid goitrogens.

The effects of the antithyroid goitrogens, methylthiouracil and methylmercaptoimidazole, on the oxidation of N-acetyltyrosylamide at pH 8.8 by lactoperoxidase have been evaluated in the presence and the absence of iodide for the purpose of elucidating the effects of iodide. At pH 8.8, iodine is not oxidized. In the absence of iodide, the two antithyroid drugs inactivate lactoperoxidase by a second order process. When iodide is added before methylthiouracil or methylmercaptoimidazole, enzyme inactivation does not occur as rapidly and both goitrogens are readily oxidized. The kinetics of the oxidation reactions have been analyzed in order to obtain the equilibrium constant of the iodide . lactoperoxidase complex. Essentially the same iodide dissociation constant, i.e. 2 x 10(-5) M, was found by studying its effects on the kinetics of oxidation of the two antithyroid drugs. A large difference absorption spectrum is observed in the Soret region between native lactoperoxidase and lactoperoxidase inactivated by methylthiouracil.     

Lactoperoxidase from human colostrum.

The present study has confirmed that human colostrum contains a lactoperoxidase (EC 1.11.1.7) [Langbakk & Flatmark (1984) FEBS Lett. 174, 300-303], which represents about 0.004% of the total protein in crude colostrum. An apparent 32-fold purification of the enzyme was obtained by a multistep procedure, as modified from that of the bovine enzyme, with a recovery of about 7%. By use of chromatography on an immunoaffinity column (directed against bovine lactoperoxidase B), an apparent 1450-fold purification was obtained in a single step, with a recovery of 21%. The enzyme behaved as a glycoprotein (binding to concanavalin A-Sepharose), and revealed spectral properties (Soret peak at 412 nm) and an Mr (80,000) similar to those of the bovine enzyme.     

A slow spectral shift of cytochrome c oxidase induced by EDTA and o-phenanthroline

Low concentrations of EDTA (in the presence of Ca2+ excess) or o-phenanthroline cause a blue shift of the oxidized cytochrome oxidase Soret absorption band. The effect develops within approximately 2 hours and does not depend on EDTA concentration provided the complexon is in a molar excess over the enzyme. It is suggested that the enzyme spectral characteristics depend on the presence of some tightly bound heavy metal ions which can stabilize one of the spectrally distinct conformations of cytochrome c oxidase.     

Spectral properties of myeloperoxidase and its ligand complexes

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

Site-directed mutagenesis of Met243, a residue of myeloperoxidase involved in binding of the prosthetic group

 The optical absorbance spectrum of reduced myeloperoxidase is red-shifted with respect to that of other haemoproteins, and has the Soret band at 472 nm and the α band at 636 nm. The origin of the red shift is poorly understood, but the interaction of the protein matrix with the chromophore is thought to play an important role. Met243 is one of the three residues in close proximity to the prosthetic group of the enzyme, and we have examined the effect of a Met243Gln mutation on the spectroscopic properties and catalytic activity of the enzyme. The mutation has a large effect on the position of the Soret band in the optical absorbance spectrum of the reduced mutated enzyme, which shifts from 472 nm to 445 nm. The alkaline pyridine haemochrome spectrum is greatly affected and similar to that of protohaem. The mutation also drastically affects the resonance Raman (RR) spectrum, which is indicative of an iron porphyrin-like chromophore. The mutant enzyme is unable to peroxidise chloride to hypochlorous acid. We conclude that there are two factors involved which account for the red-shifted Soret band. One of them is the interaction of Met243 with the prosthetic group via a special sulfonium linkage. The other factor which contributes is the presence of ester linkages between hydroxylated methyl groups on the haem and glutamate and aspartate residues, respectively. The results, combined with those of previous studies, now give us a comprehensive picture of the various factors which contribute to the unusual optical properties of myeloperoxidase. Received: 17 July 1996 / Accepted: 28 November 1996     

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.     

The reactions of horseradish peroxidase, lactoperoxidase, and myeloperoxidase with enzymatically generated superoxide

The formation and decay of intermediate compounds of horseradish peroxidase, lactoperoxidase, and myeloperoxidase formed in the presence of the superoxide/hydrogen peroxide-generating xanthine/xanthine oxidase system has been studied by observation of spectral changes in both the Soret and visible spectral regions and both on millisecond and second time scales. It is tentatively concluded that in all cases compound III is formed in a two-step reaction of native enzyme with superoxide. The presence of superoxide dismutase completely inhibited compound III formation; the presence of catalase had no effect on the process. Spectral data which indicate differences in the decay of horseradish peroxidase compound III back to the native state in comparison with compounds III of lactoperoxidase and myeloperoxidase are also presented.     

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.     

Covalent binding of peroxidase to CH- and AH-Sepharose 4 B]

The properties of peroxidase insolubilized by covalent binding to CH- and AH-Sepharose 4 B in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) are described. CH-Sepharose 4 B bound peroxidase yields an enzyme preparation with a residual specific activity of 60.6%. When bound to AH-Sepharose 4 B, the residual specific activity is to 78%. The reasons of these differences in the catalytic activity of the two insolubilized enzyme preparations are discussed. By covalent binding on CH- and AH-Sepharose 4 B, peroxidase exibits no changes in its pH optimum; it virtually keeps the same activity after being used ten times. Insolubilized peroxidase preparations, dried and reimbibed after being stored for 6 weeks at room temperature still display 50% of the initial specific activity of the insolubilized enzyme. Stored in acetate buffer, the enzyme preparations maintain their activity during all this interval.     

Intermediate filaments: Detection of lectin-like activity in purified alcoholic hyaline

Alcoholic hyaline, an intracellular, filamentous (10 nm) aggregate isolated from human alcoholic livers, bound the glycoprotein enzyme, horseradish peroxidase, in a specific and reproducible manner. Using a solid-phase assay system consisting of adsorbed alcoholic hyaline, we have shown that this binding is thermolabile, relatively insensitive to both pH extremes and high ionic strength, and highly sensitive to the presence of neutral and amino sugars. The results suggest that the binding of horseradish peroxidase is not a passive adsorption but rather an “active” phenomenon involving carbohydrate groups on the enzyme. The presence of an intracellular, filament-associated lectin is strongly indicated.     

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.     

Oxidation of cytochrome c by cytochrome c oxidase: spectroscopic binding studies and steady-state kinetics support a conformational transition mechanism

The long-known biphasic response of cytochrome c oxidase to the concentration of cytochrome c has been explained, alternatively, by the presence of a catalytic and a regulatory site on the oxidase, by negative cooperativity between adjacent active sites in dimeric oxidase, or by a transition of the enzyme molecule between different conformational states. The three mechanistic hypotheses allow testable predictions about the relationship between substrate binding and steady-state kinetics catalyzed by the monomeric and dimeric (or oligomeric) enzyme. We have tested these predictions on monomeric, dimeric, and oligomeric beef heart oxidase and on monomeric oxidase from Paracoccus denitrificans. The aggregation state of the oxidase was evaluated from the sedimentation equilibrium in the ultracentrifuge and by gel chromatography. The binding of cytochrome c to cytochrome c oxidase was measured by spectrophotometric titration of cytochrome c oxidase with cytochrome c. The procedure makes use of a small perturbation in the Soret band of the absorption spectrum of the cytochrome c-cytochrome c oxidase complex. The steady-state oxidation of cytochrome c was followed spectroscopically by an automated assay procedure, and the kinetic parameters were deduced by numerical analysis of several hundred initial rate assays in the substrate concentration range 0.15-30 microM. The following results were obtained: (1) The kinetics of cytochrome c oxidation are always biphasic at low ionic strength, independent of the aggregation state of the enzyme. (2) The kinetics become apparently monophasic at ionic strengths above 100 mM or at slightly acidic pH values.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reconstitution of pterin-free inducible nitric-oxide synthase

Inducible nitric-oxide synthase (NOS) was expressed and purified in the absence of 6(R)-tetrahydro-l-biopterin (H(4)B). Pterin-free NOS exhibits a Soret band (416-420 nm) characteristic of predominantly low spin heme and does not catalyze the formation of nitric oxide (. NO) (Rusche, K. M., Spiering, M. M., and Marletta, M. A. (1998) Biochemistry 37, 15503-15512). Reconstitution of pterin-free NOS with H(4)B was monitored by a shift in the Soret band to 396-400 nm, the recovery of.NO-forming activity, and the measurement of H(4)B bound to the enzyme. As assessed by these properties, H(4)B binding was not rapid and required the presence of a reduced thiol. Spectral changes and recovery of activity were incomplete in the absence of reduced thiol. Full reconstitution of holoenzyme activity and stoichiometric H(4)B binding was achieved in the presence of 5 mm glutathione (GSH). Preincubation with GSH before the addition of H(4)B decreased, whereas lower concentrations of GSH extended, the time required for reconstitution. Six protected cysteine residues in pterin-free NOS were identified by labeling of NOS with cysteine-directed reagents before and after reduction with GSH. Heme and metal content of pterin-free and H(4)B-reconstituted NOS were also measured and were found to be independent of H(4)B content. Additionally, pterin-free NOS was reconstituted with 6-methylpterin analogs, including redox-stable deazapterins. Reconstitution with the redox-stable pterin analogs was neither time- nor thiol-dependent. Apparent binding constants were determined for the 6-methyl- (50 microm) and 6-ethoxymethyl (200 microm) deazapterins. The redox-stable pterin analogs appear to bind to NOS in a different manner than H(4)B.