Mechanism of horseradish peroxidase-catalyzed conversion of iodine to iodide in the presence of EDTA and H2O2

EDTA not only blocks the horseradish peroxidase (HRP)-catalyzed iodide oxidation to I-3 but also causes an enzymatic conversion of oxidized iodine species to iodide (Banerjee, R. K., De, S. K., Bose, A. K., and Datta, A. G. (1986) J. Biol. Chem. 261, 10592-10597). The EDTA effect on both of these reactions can be withdrawn with a higher concentration of iodide and not with H2O2. Spectral studies indicate a possible interaction of EDTA with HRP as evidenced by the formation of modified compound 1 with H2O2 at 416 nm instead of 412 nm in the absence of EDTA. EDTA causes a hypochromic effect on HRP at 402 nm which undergoes the bathochromic red shift to 416 nm by H2O2. The addition of iodide to the 416 nm complex causes the reappearance of the Soret band of HRP at 402 nm. Among various EDTA analogues tested, N-N-N'-N'-tetramethylethylenediamine (TEMED) is 80% as effective as EDTA in the conversion of I-3 to iodide and produces a spectral shift of HRP similar to EDTA. Interaction of EDTA with HRP is further indicated by the hyperchromic effect of HRP and H2O2 on the absorption of EDTA at 212 nm. The addition of oxidized iodine species produces a new peak at 230 nm due to formation of iodide. EDTA at a higher concentration can effectively displace radioiodide specifically bound to HRP indicating its interaction at the iodide-binding site. The enzyme, after radioiodide displacement with EDTA, shows a characteristic absorption maximum at 416 nm on the addition of H2O2, indicating that EDTA is bound with the enzyme. Both positive and negative circular dichroism spectra of HRP and the HRP.H2O2 complex, characteristic of heme absorption, are altered by EDTA, suggesting an EDTA-induced conformational change at or near the heme region. This is associated with a change of affinity of heme toward H2O2 and azide. It is postulated that EDTA interacts at the iodide-binding site of the HRP inducing a new conformation that blocks iodide oxidation but is suitable to convert iodine to iodide by a redox reaction with H2O2.     

Iodide oxidation and iodine reduction mediated by horseradish peroxidase in the presence of ethylenediaminetetraacetic acid (EDTA): the superoxide effect

Ethylenediaminetetraacetic acid (EDTA) is an inhibitor of iodide (I-) oxidation that is catalyzed by horseradish peroxidase (HRP). HRP-mediated iodine (I2) reduction and triiodide (I3+) disappearance occur in the presence of this inhibitor. It is interesting that in the presence of EDTA, HRP produces superoxide radical, a reactive oxygen species that is required for iodine reduction. Substitution of potassium superoxide (KO2) or a biochemical superoxide generating system (xanthine/xanthine oxidase) for HRP and H2O2 in the reaction mixture also can reduce iodine to iodide. Thus, iodine reduction mediated by HRP occurs because HRP is able to mediate the formation of superoxide in the presence of EDTA and H2O2. Although superoxide is able to mediate iodine reduction directly, other competing reactions appear to be more important. For example, high concentrations (mM range) of EDTA are required for efficient iodine reduction in this system. Under such conditions, the concentration (microM range) of contaminating EDTA-Fe(III) becomes catalytically important. In the presence of superoxide, EDTA-Fe(III) is reduced to EDTA-Fe(II), which is able to reduce iodine and form triiodide rapidly. Also of importance is the fact that EDTA-Fe(II) reacts with hydrogen peroxide to form hydroxyl radical. Hydroxyl radical involvement is supported by the fact that a wide variety of hydroxyl radical (OH) scavengers can inhibit HRP dependent iodine reduction in the presence of EDTA and hydrogen peroxide.     

EDTA inhibits peroxidase-catalyzed iodide oxidation through interaction at the iodide binding site

EDTA inhibits the formation of I3- from iodide catalysed by various pure peroxidases. The inhibition is concentration-dependent and chloroperoxidase (CPO) is more sensitive than horseradish peroxidase (HRP) and lactoperoxidase (LPO). EDTA is more active than EGTA or other biological chelators tested. Zn2+, Mn2+ and Co2+ are equally active in reversing the effect of EDTA on both CPO and HRP almost completely, but ineffective in the case of LPO. The effect of EDTA on HRP can be reversed by a higher concentration of iodide but not by H2O2. EDTA causes a hypsochromic change in the absorption of the Soret band of HRP at 402 nm, and iodide can reverse this effect. EDTA can effectively displace radioiodide specifically bound to HRP. It is suggested that EDTA inhibits iodide oxidation by interacting at the iodide binding site of the HRP.     

Inhibition of lignin peroxidase-mediated oxidation activity by ethylenediamine tetraacetic acid and N-N-N'-N'-tetramethylenediamine

The mineralization rate of LC-[1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane] (DDT) was reduced by 90% on the 18th day in fungal cultures of Phanerochaete chrysosporium in the presence of 8 mM ethylenediamine tetraacetic acid (EDTA). In the presence of 8 mM N-N-N'-N'-tetramethylenediamine (TEMED), the mineralization rate of 14C-DDT was reduced by 80%. In the presence of 2 mM or 10 mM EDTA, 95% inhibition of lignin peroxidase (LiP) mediated veratryl alcohol oxidase activity and 97% inhibition of LiP mediated iodide oxidase activity occurred. TEMED caused 79% inhibition of veratryl alcohol oxidase activity and 92% inhibition of iodide oxidase activity when the amount used was 2 mM and 10 mM, respectively. In the presence of Zn(II) with slight molar excess of the EDTA concentration, reversed the EDTA mediated non-competitive inhibition of LiP catalyzed veratryl alcohol or iodide oxidation, Zn(II) also reversed the inhibition of LiP catalyzed veratryl alcohol oxidase activity caused by chelators other than EDTA and TEMED. In addition to Zn(II), several other metal ions also relieved EDTA mediated inhibition of veratryl alcohol and iodide oxidase activity catalyzed by LiP. The ability of veratryl alcohol to inhibit iodide oxidation catalyzed by LiP showed that veratryl alcohol could inhibit LiP mediated iodide oxidase activity. Increasing the concentration of iodide was also shown to inhibit veratryl alcohol oxidation. Kinetic analysis showed that the reaction was competitive inhibition.     

Pseudo-catalytic degradation of hydrogen peroxide in the lactoperoxidase/H2O2/iodide system

Non-stoichiometric (excessive) consumption of hydrogen peroxide (H2O2), which was observed in various lactoperoxidase-catalysed reactions, was tested in the lactoperoxidase/H2O2/iodide system. In preliminary experiments the suitability of the system was tested with special regard to the triiodide (I-3) absorption and the I2/I-3 equilibrium. Triiodide equilibrium concentrations evaluated theoretically and experimentally were compared after adding a known amount of iodine (I2) to solutions containing variable I- concentrations. A close fit of the two methods was only obtained if experiments were carried out in pure aqueous or 0.001 M H2SO4 medium. The presence of various anions, e.g. OH- and Cl-, led to a measurable decrease in I-3 and I2 equilibrium concentrations. These ions are able to displace competitively I- in forming association products with I+ and I2. When I+ and I2 were generated enzymatically by lactoperoxidase and hydrogen peroxide, additional interactions with H2O2 were observed. Depending on the enzyme and iodide concentrations, variable amounts of H2O2 disappeared nonproductively. Due to its ambivalent redox reactivity, part of the H2O2 is not reduced to H2O in the enzyme-catalysed generation of iodine, but undergoes oxidation to O2 by an oxidized iodine compound. This suggests a pseudo-catalytic side reaction which can competitively interfere with the I2/I-3 generation or (and) the iodination reaction.     

Mechanism of enzymatic and non-enzymatic tyrosine iodination. Inhibition by excess hydrogen peroxide and/or iodide

Non-enzymatic (I2-mediated) and lactoperoxidase-catalyzed iodination of tyrosine are inhibited by excess iodide (I-) and/or hydrogen peroxide (H2O2). This phenomenon is a consequence of the concentration-dependent dual role of I- and H2O2 in the iodinating system. I- and H2O2, in addition to their function as primary substrates of peroxidase, may act as alternative 'iodine acceptors' and therefore compete with tyrosine for the active iodinating agent, irrespective of whether this compound is an enzyme-associated iodinium cation (E X I delta +) or an equivalent oxidized iodine species (IOH, IC1, I2). The competitive reaction pathways resulting from excess I- and/or H2O2 in the iodination system are I2/I-3 generation and/or pseudo-catalatic degradation of H2O2, respectively. Our results also demonstrate that I2 (and alternative medium-dependent oxidized iodine species such as IOH and IC1) generated in the iodination system may play an important role as iodinating agent(s). They serve as a substitute for the enzyme-bound iodinium species (E X I delta +), if the prevailing I- concentration favours this pathway. The proposed mechanism of the various antagonistic and interactive reaction pathways is summarized in a scheme.     

The role of compound III in reversible and irreversible inactivation of lactoperoxidase

In the presence of iodide (I-, 10 mM) and hydrogen peroxide in a large excess (H2O2, 0.1-10 mM) catalytic amounts of lactoperoxidase (2 nM) are very rapidly irreversibly inactivated without forming compound III (cpd III). In contrast, in the absence of I- cpd III is formed and inactivation proceeds very slowly. Increasing the enzyme concentration up to the micromolar range significantly accelerates the rate of inactivation. The present data reveal that irreversible inactivation of the enzyme involves cleavage of the prosthetic group and liberation of heme iron. The rate of enzyme destruction is well correlated with the production of molecular oxygen (O2), which originates from the oxidation of excess H2O2. Since H2O2 and O2 per se do not affect the heme moiety of the peroxidase, we suggest that the damaging species may be a primary intermediate of the H2O2 oxidation, such as oxygen in its excited singlet state (1 delta gO2), superoxide radicals (O-.2), or consequently formed hydroxyl radicals (OH.).     

Elimination of shrimp endogenous peroxidase background in immunodot blot assays to detect white spot syndrome virus (WSSV)

False positive results were obtained in immunodot blot assays to detect white spot syndrome virus when horseradish peroxidase-conjugated sheep anti-mouse immunoglobin (Ig) serum was used as a secondary antibody with 3-3'-diaminobenzine tetrahydrochloride dihydrate as the detection substrate. The cause was considered to be a reaction of shrimp endogenous peroxidase (POD) with the substrate. In experiments designed to inhibit POD activity, 9 different reagents were used at different concentrations and for different treatment times. EDTA, sodium azide, HEPES-Na, NaHSO3, H2O2 and phenylthiourea (PTU) were able to inhibit POD activity by 44, 60, 64, 67, 79, and 90%, respectively. Phenylmethylsulfonyl fluoride did not inhibit POD, and neither periodic acid nor H2O2 in methanol were appropriate due to the formation of flocculant precipitates when added to shrimp extracts. It was concluded that of the treatments tested, 10 mM PTU for 2 h yielded optimal inhibition and that such pretreatment of samples eliminates false positive results in immunodot blot assays.     

Oxidation of hydroquinones by the versatile ligninolytic peroxidase from Pleurotus eryngii. H2O2 generation and the influence of Mn2+.

Formation of H2O2 during the oxidation of three lignin-derived hydroquinones by the ligninolytic versatile peroxidase (VP), produced by the white-rot fungus Pleurotus eryngii, was investigated. VP can oxidize a wide variety of phenols, including hydroquinones, either directly in a manner similar to horseradish peroxidase (HRP), or indirectly through Mn3+ formed from Mn2+ oxidation, in a manner similar to manganese peroxidase (MnP). From several possible buffers (all pH 5), tartrate buffer was selected to study the oxidation of hydroquinones as it did not support the Mn2+-mediated activity of VP in the absence of exogenous H2O2 (unlike glyoxylate and oxalate buffers). In the absence of Mn2+, efficient hydroquinone oxidation by VP was dependent on exogenous H2O2. Under these conditions, semiquinone radicals produced by VP autoxidized to a certain extent producing superoxide anion radical (O2*-) that spontaneously dismutated to H2O2 and O2. The use of this peroxide by VP produced quinone in an amount greater than equimolar to the initial H2O2 (a quinone/H2O2 molar ratio of 1 was only observed under anaerobic conditions). In the presence of Mn2+, exogenous H2O2 was not required for complete oxidation of hydroquinone by VP. Reaction blanks lacking VP revealed H2O2 production due to a slow conversion of hydroquinone into semiquinone radicals (probably via autooxidation catalysed by trace amounts of free metal ions), followed by O2*- production through semiquinone autooxidation and O2*- reduction by Mn2+. This peroxide was used by VP to oxidize hydroquinone that was mainly carried out through Mn2+ oxidation. By comparing the activity of VP to that of MnP and HRP, it was found that the ability of VP and MnP to oxidize Mn2+ greatly increased hydroquinone oxidation efficiency.     

Metabolism of 2-ketoaldehydes in mold: purification and characterization of glyoxalase I from Aspergillus niger

Glyoxalase I catalyzing the conversion of methylglyoxal into S-lactoylglutathione in the presence of glutathione was purified approximately 1,400-fold with 2.9% activity yield from mold, Aspergillus niger. The enzyme consisted of a single polypeptide chain with a relative molecular weight of 36,000 on both SDS-polyacrylamide gel electrophoresis and Sephadex G-150 gel filtration. The enzyme was most active at pH 7.0, 35-37 degrees C. Among the various aldehydes tested, the enzyme was active on methylglyoxal and 4,5-dioxovalerate with Km values of 1.25 and 0.87 mM, respectively. The activity of the enzyme was completely inhibited by Zn2+ at 0.5 mM. An equimolar amount of EDTA (0.5 mM) protected the enzyme from inactivation by Zn2+. EDTA competitively (K1 = 1.3 mM) inhibited the activity of the enzyme. Fe2+ was a potent activator for the enzyme, the activation being approximately 2.4-fold at 0.5 mM.     

NADPH oxidation catalyzed by the peroxidase/H2O2 system. Iodide-mediated oxidation of NADPH to iodinated NADP

Oxidation of NADPH catalyzed by the peroxidase/H2O2 system is known to require the presence of mediating molecules. Using either lactoperoxidase or horseradish peroxidase, we demonstrated that in the peroxidase/H2O2 system, NADPH oxidation was mediated by iodide. The oxidation product was the iodinated NADP. This product was shown to possess spectral characteristics different from those of NADP+ and NADPH, since for iodinated NADP, increased absorbance was observed in the 280-nm region and was directly proportional to the rate of iodination. It is suggested that oxidation and iodination of NADPH proceed via a single reaction between the intermediary iodide oxidation species and NADPH. Experiments with different molecules of NADPH analogues indicated that iodination occurred in the nicotinamide part of the NADPH molecule.     

Scavenging of H2O2 and production of oxygen by horseradish peroxidase

Peroxidases catalyze many reactions, the most common being the utilization of H2O2 to oxidize numerous substrates (peroxidative mode). Peroxidases have also been proposed to produce H2O2 via utilization of NAD(P)H, thus providing oxidant either for the first step of lignification or for the "oxidative burst" associated with plant-pathogen interactions. The current study with horseradish peroxidase characterizes a third type of peroxidase activity that mimics the action of catalase; molecular oxygen is produced at the expense of H2O2 in the absence of other reactants. The oxygen production and H2O2-scavenging activities had temperature coefficients, Q10, of nearly 3 and 2, which is consistent with enzymatic reactions. Both activities were inhibited by autoclaving the enzyme and both activities had fairly broad pH optima in the neutral-to-alkaline region. The apparent Km values for the oxygen production and H2O2-scavenging reactions were near 1.0 mM H2O2. Irreversible inactivation of horseradish peroxidase by exposure to high concentrations of H2O2 coincided with the formation of an absorbance peak at 670 nm. Addition of superoxide dismutase (SOD) to reaction mixtures accelerated the reaction, suggesting that superoxide intermediates were involved. It appears that horseradish peroxidase is capable of using H2O2 both as an oxidant and as a reductant. A model is proposed and the relevance of the mechanism in plant-bacterial systems is discussed.     

Interactions of iodide ions with isolated photosystem 2 particles

The effects of I- ions on O2 evolution by photosystem 2 particles, which were depleted of the 18-kDa and the 23-kDa extrinsic proteins of the O2 evolution complex by NaCl washing (dPS2 particles) were examined. In the absence of Cl- (incompetent dPS2) I- stimulated O2 evolution up to 3-6 mM, depending on the associated cation, and inhibited it at higher concentrations. In the presence of Cl- (competent dPS2), I- was inhibitory at all concentrations. The inhibition was reversible, it occurred at a site preceding Tyrz (Tyr residue mediating electron transfer from H2O to photosystem 2), and it interfered noncompetitively with the reactivation of incompetent dPS2 with Cl-. Furthermore, the organic salts tetrabutyl ammonium iodide and tetraphenyl phosphonium iodide proved to be stronger inhibitors than the inorganic NaI. This is interpreted as an indication of a negatively charged surface, situated behind a hydrophobic permeability barrier. Permeant organic cations, being better compensators of the inner surface charge than Na+, are also more apt in facilitating access of the I- ions to the inhibitory site in the vicinity of Tyrz.     

Mechanism-based inactivation of horseradish peroxidase by sodium azide. Formation of meso-azidoprotoporphyrin IX

Catalytic turnover of sodium azide by horseradish peroxidase, which produces the azidyl radical, results in inactivation of the enzyme with KI = 1.47 mM and kinact = 0.69 min-1. Inactivation of 80% of the enzyme requires approximately 60 equiv each of NaN3 and H2O2. The enzyme is completely inactivated by higher concentrations of these two agents. meso-Azidoheme as well as some residual heme are obtained when the prosthetic group of the partially inactivated enzyme is isolated and characterized. Reconstitution of horseradish peroxidase with meso-azidoheme yields an enzyme without detectable catalytic activity even though reconstitution with heme itself gives fully active enzyme. The finding that catalytically generated nitrogen radicals add to the meso carbon of heme shows that biological meso additions are not restricted to carbon radicals. The analogous addition of oxygen radicals may trigger the normal and/or pathological degradation of heme.     

Hydroxyl-radical production in physiological reactions. A novel function of peroxidase.

Peroxidases catalyze the dehydrogenation by hydrogen peroxide (H2O2) of various phenolic and endiolic substrates in a peroxidatic reaction cycle. In addition, these enzymes exhibit an oxidase activity mediating the reduction of O2 to superoxide (O2.-) and H2O2 by substrates such as NADH or dihydroxyfumarate. Here we show that horseradish peroxidase can also catalyze a third type of reaction that results in the production of hydroxyl radicals (.OH) from H2O2 in the presence of O2.-. We provide evidence that to mediate this reaction, the ferric form of horseradish peroxidase must be converted by O2.- into the perferryl form (Compound III), in which the haem iron can assume the ferrous state. It is concluded that the ferric/perferryl peroxidase couple constitutes an effective biochemical catalyst for the production of .OH from O2.- and H2O2 (iron-catalyzed Haber-Weiss reaction). This reaction can be measured either by the hydroxylation of benzoate or the degradation of deoxyribose. O2.- and H2O2 can be produced by the oxidase reaction of horseradish peroxidase in the presence of NADH. The .OH-producing activity of horseradish peroxidase can be inhibited by inactivators of haem iron or by various O2.- and .OH scavengers. On an equimolar Fe basis, horseradish peroxidase is 1-2 orders of magnitude more active than Fe-EDTA, an inorganic catalyst of the Haber-Weiss reaction. Particularly high .OH-producing activity was found in the alkaline horseradish peroxidase isoforms and in a ligninase-type fungal peroxidase, whereas lactoperoxidase and soybean peroxidase were less active, and myeloperoxidase was inactive. Operating in the .OH-producing mode, peroxidases may be responsible for numerous destructive and toxic effects of activated oxygen reported previously.     

Hydroxylation of p-coumaric acid by horseradish peroxidase. The role of superoxide and hydroxyl radicals.

1. In the presence of dihydroxyfumarate, horseradish peroxidase catalyses the conversion of p-coumaric acid into caffeic acid at pH 6. This hydroxylation is completely inhibited by superoxide dismutase. 2. Dihydroxyfumarate cannot be replaced by ascorbate H2O2, NADH, cysteine or sulphite. Peroxidase can be replaced by high (10 mM) concentrations of FeSO4, but this reaction is almost unaffected by superoxide dismutase. 3. Hydroxylation by the peroxidase/dihydroxyfumarate system is completely inhibited by low concentrations of Mn2+ or Cu2+. It is proposed that this is due to the ability of these metal ions to react with the superoxide radical O2--. 4. Hydroxylation is partially inhibited by mannitol, Tris or ethanol and completely inhibited by formate. This seems to be due to the ability of these reagents to react with the hydroxyl radical -OH. 5. It is concluded that O2-- is generated during the oxidation of dihydroxyfumarate by peroxidase and reacts with H2O2 to produce hydroxyl radicals, which then convert p-coumaric acid into caffeic acid.     

Mechanism of azide binding to chloroperoxidase and horseradish peroxidase: use of an iodine laser temperature-jump apparatus

The kinetics of azide binding to chloroperoxidase have been studied at eight pH values ranging from 3.0 to 6.6 at 9.5 +/- 0.2 degrees C and ionic strength of 0.4 M in H2O. The same reaction was studied in D2O at pD 4.36. In addition, results were obtained on azide binding to horseradish peroxidase at pD 4.36 and pH 4.56. Typical relaxation times were in the range 10-40 microseconds. The value of kH/kD(on) for chloroperoxidase is 1.16, and kH/kD(off) is 1.7; corresponding values for horseradish peroxidase are 1.10 and 2.4. The H/D solvent isotope effects indicate proton transfer is partially rate controlling and is more important in the dissociation of azide from the enzyme-ligand complex. A mechanism is proposed in which hydrazoic acid binds to chloroperoxidase in a concerted process in which its proton is transferred to a distal basic group. Hydrogen bonding from the newly formed distal acid to the bound azide facilitates formation of hydrazoic acid as the leaving group in the dissociation process. The binding rate constant data, kon, can be fit to the equation kon = k3/(1 + KA/[H+]), where k3 = 7.6 X 10(7) M-1 S-1 and KA, the dissociation constant of hydrazoic acid, is 2.5 X 10(-5) M. The same mechanism probably is valid for the ligand binding to horseradish peroxidase.     

Non-deleterious inhibition of endogenous peroxidase activity (EPA) by cyclopropanone hydrate: a definitive approach

Endogenous peroxidase activity (EPA) poses a serious problem in immunoperoxidase localization of antigens unable to withstand deleterious effects of aldehyde fixatives, alcohols, and various oxidative reagents. This has forced the development of more selective inhibition methods. Of these, phenylhydrazine or azide combined with small amounts of H2O2 have proved quite effective. However, the precise mechanism of the action of these compounds on EPA generating proteins is not understood. Cyclopropanone hydrate is a compound whose inhibitory action on the heme moiety of horseradish peroxidase is well understood. The aim of this study was to investigate the effect of this compound on EPA and to compare its efficiency with that of optimal phenylhydrazine and sodium azide regimens. In addition, any gross deleteriousness of cyclopropanone hydrate towards immunoperoxidase immunolocalization of three of the most delicate lymphocyte surface antigens was investigated. Cyclopropanone hydrate was found to inhibit EPA with progressing strength between 0.15-15 mM. Over this range, H2O2 was found necessary for inhibition only for cyclopropanone hydrate concentrations up to 0.15 mM. Beyond this amount, the compound inhibited EPA equally strongly in the presence or absence of H2O2, reaching near-maximum inhibition at 15 mM. This and the H2O2-requiring regimens were found to cause no gross diminution in immunoperoxidase staining of CD4, CD6, and CD8 antigens in snap-frozen, acetone-fixed human tonsil sections. Cyclopropanone hydrate therefore provides a definitive non-deleterious mode of inhibiting EPA for immunoperoxidase staining of delicate antigens.     

反相胶束体系中辣根过氧化物酶的活力和动力学性质

本文系统研究辣根过氧化物酶在ＣＴＡＢ／Ｈ２Ｏ／ＣＨＣ．３－ｉｓｏｏｃｔａｎｅ（１∶１,Ｖ／Ｖ）反相胶束体系中的催化行为。在一定条件下酶反符合Ｍｉｃｈａｅｌｉｓ－Ｍｅｎｔｅｎ动力学。研究水含量、底物浓度、ＰＨ、温度、表面活性剂的浓度等对酶反应的影响,结果表明表面活性剂对酶表现非竞争性抑制作用,高浓度的过氧化氢抑制酶活,最适ＰＨ为７．０。在低水含量（Ｗ０＜５）的胶束体系中保温后,酶的活力发生不可逆的改     

Oxidoreductase activities of polyclonal IgGs from the sera of Wistar rats are better activated by combinations of different metal ions

It was shown that IgGs purified from the sera of healthy Wistar rats contain several different bound Me2+ ions and oxidize 3,3'-diaminobenzidine through a H2O2-dependent peroxidase and H2O2-independent oxidoreductase activity. IgGs have lost these activities after removing the internal metal ions by dialysis against EDTA. External Cu2+ or Fe2+ activated significantly both activities of non-dialysed IgGs containing different internal metals (Fe > or = Pb > or = Zn > or = Cu > or = Al > or = Ca > or = Ni > or = Mn > Co > or = Mg) showing pronounced biphasic dependencies corresponding to approximately 0.1-2 and approximately 2-5 mM of Me2+, while the curves for Mn2+ were nearly linear. Cu2+ alone significantly stimulated both the peroxidase and oxidoreductase activities of dialysed IgGs only at high concentration (> or = 2 mM), while Mn2+ weakly activated peroxidase activity at concentration >3 mM but was active in the oxidoreductase oxidation at a low concentration (<1 mM). Fe2+-dependent peroxidase activity of dialysed IgGs was observed at 0.1-5 mM, but Fe2+ was completely inactive in the oxidoreductase reaction. Mg2+, Ca2+, Zn2+, Al2+ and especially Co2+ and Ni2+ were not able to activate dialysed IgGs, but slightly activated non-dialysed IgGs. The use of the combinations of Cu2+ + Mn2+, Cu2+ + Zn2+, Fe2+ + Mn2+, Fe2+ + Zn2+ led to a conversion of the biphasic curves to hyperbolic ones and in parallel to a significant increase in the activity as compared with Cu2+, Fe2+ or Mn2+ ions taken separately; the rates of the oxidation reactions, catalysed by non-dialysed and dialysed IgGs, became comparable. Mg2+, Co2+ and Ni2+ markedly activated the Cu2+-dependent oxidation reactions catalysed by dialysed IgGs, while Ca2+ inhibited these reactions. A possible role of the second metal in the oxidation reactions is discussed.