Biomedical subjects

D P Barr

Publications and source records attributed to D P Barr.

At least 19 recordsLinked to original sources

PubMed2000

Self-association of mucin.

Aggregation phenomena in aqueous solutions of purified human tracheobronchial mucin have been studied by rheological methods, steady-state fluorescence, quasielastic light scattering, and spin probe techniques. At temperatures below 30 degrees C and concentrations above 15 mg/mL and in the absence of chaotropic agents, mucin solutions are viscoelastic gels. A gel-sol transition is observed at temperatures above 30 degrees C that is manifested by the diminishing storage modulus and a loss tangent above unity throughout the studied frequency range of the oscillatory shear. No decline in the mucin molecular weight is observed by size-exclusion chromatography above 30 degrees C in the absence of redox agents or proteolytic enzymes. Aggregation of hydrophobic protein segments of the mucin chains at 37 degrees C is indicated by QELS experiments. The decreasing polarity of the microenvironment of pyrene solubilized into mucin solutions at temperatures above 30 degrees C, concomitant with the gel-sol transition, shows the hydrophobicity of the formed aggregates. ESR spectra of the fatty acid spin probe, 16-doxylstearic acid indicate that the aggregate-aqueous interface becomes more developed at elevated temperatures.

Bronchi↗

PubMed1998

The fate of the oxidizing tyrosyl radical in the presence of glutathione and ascorbate. Implications for the radical sink hypothesis.

Cellular systems contain as much as millimolar concentrations of both ascorbate and GSH, although the GSH concentration is often 10-fold that of ascorbate. It has been proposed that GSH and superoxide dismutase (SOD) act in a concerted effort to eliminate biologically generated radicals. The tyrosyl radical (Tyr.) generated by horseradish peroxidase in the presence of hydrogen peroxide can react with GSH to form the glutathione thiyl radical (GS.). GS. can react with the glutathione anion (GS-) to form the disulfide radical anion (GSSG-). This highly reactive disulfide radical anion will reduce molecular oxygen, forming superoxide and glutathione disulfide (GSSG). In a concerted effort, SOD will catalyze the dismutation of superoxide, resulting in the elimination of the radical. The physiological relevance of this GSH/SOD concerted effort is questionable. In a tyrosyl radical-generating system containing ascorbate (100 microM) and GSH (8 mM), the ascorbate nearly eliminated oxygen consumption and diminished GS. formation. In the presence of ascorbate, the tyrosyl radical will oxidize ascorbate to form the ascorbate radical. When measuring the ascorbate radical directly using fast-flow electron spin resonance, only minor changes in the ascorbate radical electron spin resonance signal intensity occurred in the presence of GSH. These results indicate that in the presence of physiological concentrations of ascorbate and GSH, GSH is not involved in the detoxification pathway of oxidizing free radicals formed by peroxidases.

Animals↗

PubMed1998

Characterization of cytochrome c free radical reactions with peptides by mass spectrometry.

The reactions of horse heart cytochrome c, hydrogen peroxide, and the spin trap 3,5-dibromo-4-nitrosobenzenesulfonic acid with a series of polypeptides were investigated using mass spectrometry. The mass spectra obtained from these reactions revealed that after a free radical has been generated on the heme-containing protein horse heart cytochrome c, it can be transferred to other biomolecules. In addition, the number of free radicals transferred to the target molecule could be determined. Recipient peptides/proteins that contained a tyrosine and/or tryptophan amino acid residue were most susceptible to free radical transfer. Using tandem mass spectrometry, the location of the 3,5-dibromo-4-nitrosobenzenesulfonic acid radical adduct on the nonapeptide RWIILGLNK was unequivocally determined to be at the tryptophan residue. We also demonstrated that the presence of an antioxidant in the reaction mixture not only inhibits free radical formation on horse heart cytochrome c, but also interferes with the transfer of the free radical, once it has been formed on cytochrome c.

Amino Acid Sequence↗

PubMed1998

Site-specific spin trapping of tyrosine radicals in the oxidation of metmyoglobin by hydrogen peroxide.

The reaction between metmyoglobin and hydrogen peroxide produces both a ferryl-oxo heme and a globin-centred radical(s) from the two oxidizing equivalents of the hydrogen peroxide. Evidence has been presented for localization of the globin-centred radical on one tryptophan residue and tyrosines 103 and 151. When the spin-trapping agent 5,5-dimethyl-1-pyrroline N-oxide (DMPO) is included in the reaction mixture, a radical adduct has been detected, but the residue at which that adduct is formed has not been determined. Replacement of either tryptophans 7 and 14 or tyrosines 146 and 151 with phenylalanine has no effect on the formation of DMPO adduct in the reaction with hydrogen peroxide. When tyrosine 103 is replaced with phenylalanine, however, only DMPOX, a product of the oxidation of the spin-trap, is detected. Tyrosine-103 is, therefore, the site of radical adduct formation with DMPO. The spin trap 2-methyl-2-nitrosopropane (MNP), however, forms radical adducts with any recombinant sperm whale metmyoglobin that contains either tyrosine 103 or 151. Detailed spectral analysis of the DMPO and MNP radical adducts of isotopically substituted tyrosine radical yield complete structural determinations. The multiple sites of trapping support a model in which the unpaired electron density is spread over a number of residues in the population of metmyoglobin molecules, at least some of which are in equilibrium with each other.

Amino Acid Substitution↗

PubMed1997

Detection of free radicals produced from the reaction of cytochrome P-450 with linoleic acid hydroperoxide.

The ESR spin-trapping technique was employed to investigate the reaction of rabbit cytochrome P-450 1A2 (P450) with linoleic acid hydroperoxide. This system was compared with chemical systems where FeSO4 or FeCl3 was used in place of P450. The spin trap 5, 5'-dimethyl-1-pyrroline N-oxide (DMPO) was employed to detect and identify radical species. The DMPO adducts of hydroxyl, O2-., peroxyl, methyl and acyl radicals were detected in the P450 system. The reaction did not require NADPH-cytochrome P-450 reductase or NADPH. The same DMPO-radical adducts were detected in the FeSO4 system. Only DMPO-.OH radical adduct and carbon-centred radical adducts were detected in the FeCl3 system. Peroxyl radical production was completely O2-dependent. We propose that polyunsaturated fatty acids are initially reduced to form alkoxyl radicals, which then undergo intramolecular rearrangement to form epoxyalkyl radicals. Each epoxyalkyl radical reacts with O2, forming a peroxyl radical. Subsequent unimolecular decomposition of this peroxyl radical eliminates O2-. radical.

Animals↗

PubMed1997

In vivo production of nitric oxide in rats after administration of hydroxyurea.

The metabolism of nitrovasodilators such as glyceryl trinitrate and nitroprusside provides the active moiety of these drugs (that is, nitric oxide). This process is not limited to the known nitrovasodilators, but also occurs with nitroaromatic antimicrobials. Here we report that the administration of hydroxyurea, an antitumor drug, to rats at pharmacological doses formed detectable nitrosyl hemoglobin, which increased with dose. At higher doses, nitrosyl hemoprotein complexes could also be detected in liver tissue. [15N]hydroxyurea was synthesized and compared with [14N]hydroxyurea. These observations verified that nitric oxide detected as nitrosyl hemoglobin or nitrosyl hemoprotein complexes in rats was the result of the metabolism of hydroxyurea. The time course and dose-dependence of nitric oxide generation were also investigated. Hydroxyurea's antineoplastic activity is caused by its direct action on ribonucleotide reductase, the rate-limiting enzyme in DNA synthesis. Because nitric oxide also inhibits ribonucleotide reductase, this metabolite may supplement this action of hydroxyurea. In addition, the known ability of hydroxyurea to ease the pain of sickle cell anemia patients may be the result of vasodilation by the drug-derived nitric oxide.

Animals↗

PubMed1996

ESR spin-trapping of a protein-derived tyrosyl radical from the reaction of cytochrome c with hydrogen peroxide.

The reaction of horse heart cytochrome c with hydrogen peroxide was investigated using the ESR spin-trapping technique and the nitroso spin traps 3,5-dibromo-4-nitrosobenzenesulfonic acid (DBNBS) and 2-methyl-2-nitrosopropane (MNP). The ESR spectra obtained using both spin traps were typical of an immobilized nitroxide and indicated that the adduct was a macromolecule. The intensity of the ESR spectrum corresponding to the DBNBS/*cytochrome c radical adduct was greatly enhanced by performing the reaction under anaerobic conditions, which suggested that the spin trap was competing with O2 for reaction with the radical site(s). Nonspecific proteolysis of either the DBNBS or the MNP adducts revealed isotropic three-line spectra. In addition, a high resolution ESR spectrum for the protease-treated MNP cytochrome c-derived protein radical adduct was obtained. The superhyperfine couplings detected in this spectra were identical to those detected from an authentic MNP/tyrosyl adduct. Carbon-13 labeling of the aromatic ring positions of tyrosine yielded additional hyperfine coupling, demonstrating that the radical site was definitely located on the ring of tyrosine. Mass spectrometry detected as many as four DBNBS/.cytochrome c-derived adducts from the reaction of cytochrome with H2O2. Thus, it would appear four radical sites are formed during the reaction, at least one of which is tyrosine.

Animals↗

PubMed1996

ESR spin trapping investigation of radical formation from the reaction between hematin and tert-Butyl hydroperoxide.

Various mechanisms have been proposed for the reaction between heme proteins and organic hydroperoxides, including a peroxidase-type mechanism and homolytic cleavage. We used electron spin resonance (ESR) spectroscopy to investigate the formation of radicals in a hematin/tert-butyl hydroperoxide system. Spin trapping studies, using 5,5-dimethyl-1-pyrroline N-oxide (DMPO), showed the formation of peroxyl and alkoxyl radicals in this system. At higher hematin concentrations an alkyl radical adduct could also be detected, which was identified as the methyl radical by using the spin trap 2-methyl-2-nitrosopropane. Furthermore, the relative contribution of the peroxyl and alkoxyl radical adducts was determined at various DMPO concentrations using computer simulation. It was found that at low DMPO concentrations both the alkoxyl and the peroxyl radical adducts could be detected. At higher DMPO concentrations, on the other hand, the alkoxyl radical dominated, whereas the peroxyl radical adduct decreased to a small portion of the total radical adduct population. Thus, the alkoxyl radical was the initial radical, produced by homolytic scission of the O-O bond of the hydroperoxide by ferric hematin. Although some tert-butyl peroxyl radicals could be detected by direct ESR, the majority of the peroxyl radicals detected by spin trapping were methyl peroxyl radicals, formed in the reaction between methyl radicals (formed by beta-scission of the alkoxyl radicals) and oxygen.

Computer Simulation↗

PubMed1995

Lignin peroxidases can also oxidize manganese.

The peroxidase isozymes secreted by the white rot fungus Phanerochaete chrysosporium include lignin peroxidases and manganese-dependent peroxidases. The major isozymes, called lignin peroxidases, are thought to oxidize chemicals directly. The manganese-dependent peroxidases (H3, H4, H5, and H9) are relatively minor, making up only a fraction of the total peroxidase protein. However, we have found that lignin peroxidases will also catalyze the H2O2-dependent oxidation of Mn2+ to Mn3+. We have used lignin peroxidase isozyme H2 (LiPH2) to characterize the manganese peroxidase activity of lignin peroxidases. Transient state kinetic studies were used to obtain a second-order rate constant of 4.2 x 10(4) M-1 S-1 for the reaction of LiPH2-compound I with free or chelated Mn2+ at pH 6.0. This reaction was too fast to monitor at pH 4.5. Only chelated Mn2+ could reduce LiPH2-compound II to ferric enzyme. The Mn(2+)-chelate (oxalate) first bound LiPH2-compound II with a Kd of (1.5 +/- 0.3) x 10(-5) M and then reduced LiPH2-compound II to ferric enzyme with a first order rate constant of 215 +/- 6 S-1. Steady-state kinetic studies on LiPH2 were performed by directly monitoring the formation of Mn(3+)-oxalate. These results show that oxidation of Mn2+ by a lignin peroxidase does not occur through free radical mediation as proposed previously [Popp et al. (1990) Biochemistry 29, 10475-10480). Electron spin resonance and oxygen evolution studies also indicate that Mn2+ is directly oxidized by LiPH2.(ABSTRACT TRUNCATED AT 250 WORDS)

Basidiomycota↗

PubMed1995

Mechanism of radical production from the reaction of cytochrome c with organic hydroperoxides. An ESR spin trapping investigation.

The mechanism for the reaction of cytochrome c with t-butyl hydroperoxide and cumene hydroperoxide was investigated. ESR spin trapping studies using 5,5-dimethyl-1-pyrroline N-oxide were performed to demonstrate the presence of hydroperoxide-derived peroxyl, alkoxyl, and methyl radicals. Computer simulation of the experimental data obtained at various 5,5-dimethyl-1-pyrroline N-oxide concentrations was used to determine the relative contributions of each radical adduct to each composite ESR spectrum. From these analyses, it was concluded that the alkoxyl radical of the hydroperoxide was the initial radical produced, presumably by homolytic scission of the O-O bond by ferric cytochrome c. This was in contrast to a previous ESR study that proposed a heterolytic peroxidase-type mechanism for the reaction of cytochrome c with organic hydroperoxides. Methyl radicals were produced from the beta-scission of the alkoxyl radical. The peroxyl radicals are shown to be secondary products formed from the reaction of oxygen with the methyl radical to produce the methyl peroxyl radical. In separate experiments, visible absorption spectroscopy revealed that the heme center was destroyed during the reaction. Both the heme destruction and production of radical adducts were inhibited by cyanide, presumably due to the formation of a cyanoheme complex.

Alcohols↗

PubMed1994

Conversion of lignin peroxidase compound III to active enzyme by cation radicals.

It has been previously reported that the catalytically inactive compound III form of lignin peroxidase is formed during the oxidation of certain chemicals such as phenols (P.J. Harvey and J.M. Palmer, 1990, J. Biotechnol. 13, 169-179). Here we provide evidence that the cation radicals of methoxybenzenes such as 1,2,4,5-tetramethoxybenzene (TMB) and veratryl alcohol promote the oxidative conversion of compound III back to ferric enzyme. Two kinetic phases were observed during the oxidation of TMB by lignin peroxidase. In the first phase the formation of TMB cation radical and compound III were observed simultaneously. The second phase involved a rapid disappearance of compound III and the TMB cation radical. Ferric enzyme appeared concomitantly with the disappearance of compound III. The TMB cation radical, generated electrochemically, was able to convert compound III to ferric enzyme. Comparative studies using veratryl alcohol were performed and supported the idea that the cation radical of these chemicals are capable of reactivating compound III. The significance of these reactions with respect to lignin peroxidase catalysis are discussed.

Anisoles↗

PubMed1994

Effect of superoxide and superoxide dismutase on lignin peroxidase-catalyzed veratryl alcohol oxidation.

We have shown that superoxide (O2.-) is produced during the oxidation of veratryl alcohol by lignin peroxidase (LiP) by the reaction of the veratryl alcohol cation radical with hydrogen peroxide (D. B. Barr, M. M. Shah, and S. D. Aust, 1993, J. Biol. Chem. 268, 241-244). Compound III, an inactive form of peroxidases can be formed by reaction of the ferric enzyme with O2.-. We therefore studied the effects of O2.- and superoxide dismutase (SOD) on the veratryl alcohol oxidase activity of LiP. SOD enhanced the rate of veratryl alcohol oxidation by LiP and veratryl alcohol oxidation was inhibited by the addition of KO2. Upon the addition of KO2, activity was also preceded by a lag period. Under steady-state turnover conditions (i.e., for veratryl alcohol oxidation), the addition of KO2 resulted in the formation of LiP compound III. Compound II of LiP was observed following a time period that correlated with the lag prior to veratryl aldehyde formation. The extent of the lag preceding veratryl aldehyde formation increased with increasing concentrations of KO2 and decreased with increasing concentrations of veratryl alcohol. It was postulated that during the lag period the veratryl alcohol cation radical reacted with compound III to regenerate the native enzyme. In this process the veratryl alcohol cation radical was reduced to veratryl alcohol, and thus, no veratryl aldehyde was detected during the lag period.

Agaricales↗

PubMed1994

The role of oxalate in lignin peroxidase-catalyzed reduction: protection from compound III accumulation.

Reduction may be an important step in the degradation of some highly oxidized environmental pollutants by Phanerochaete chrysosporium. Lignin peroxidases (LiP) from P. chrysosporium are able to catalyze reductive reactions using veratryl alcohol (VA) as a mediator and either oxalate or EDTA as electron donors. Reduction of oxygen to superoxide, monitored by oxygen consumption, was used as a measure of the reductive activity of LiP. In the presence of EDTA, the rate of O2 reduction catalyzed by LiP decreased with time and increasing concentrations of H2O2. When oxalate replaced EDTA, LiP-catalyzed O2 reduction did not decrease with time, and increasing concentrations of H2O2 increased the duration and extent of O2 reduction. LiP was converted to the compound III state in the presence of EDTA, H2O2, and veratryl alcohol. When oxalate replaced EDTA, compound II was observed. The importance of the veratryl alcohol cation radical (VA.+) in the conversion of LiP compound III to active enzyme has been previously examined (D.P. Barr and S.D. Aust, 1994, Arch. Biochem. Biophys. 311, 378-382). We propose that rapid reduction of VA.+ by EDTA results in accumulation of LiP compound III and the loss of activity resulting in a decrease in LiP-catalyzed reduction reactions. Oxalate is less effective in reducing the VA.+, therefore, some VA.+ remains to convert compound III to active enzyme and maintain LiP-catalyzed reduction reactions. Thus oxalate, a normal secondary metabolite of P. chrysosporium, is a suitable candidate for mediating reduction reactions by LiP in vivo.

Edetic Acid↗

PubMed1994

Pollutant degradation by white rot fungi.

The white rot fungi technology is very different from other more well-established methods of bioremediation (e.g., bacterial systems). The differences are primarily due to the mechanisms discussed previously. The unusual mechanisms used by the fungi provide them with several advantages for pollutant degradation, but the complexity of these mechanisms has also made the technology slow to emerge as a viable method of bioremediation. One distinct advantage that white rot fungi have over bacterial systems is that they do not require preconditioning to a particular pollutant. Bacteria must be preexposed to a pollutant to allow the enzymes that degrade the pollutant to be induced. The pollutant must also be present in a significant concentration, otherwise induction of enzyme synthesis will not occur. Therefore, there is a finite level to which pollutants can be degraded by bacteria. In contrast, the degradative enzymes of white rot fungi are induced by nutrient limitation. Thus, cultivate the fungus on a nutrient that is limited in something, and the degradative process will be initiated. Also, because the induction of the lignin-degrading system is not dependent on the chemical, pollutants are degraded to near-nondetectable levels by white rot fungi. Another unique feature of pollutant degradation by white rot fungi involves kinetics. The process of chemical conversion by these fungi occurs via a free-radical process, and thus the degradation of chemicals often follows pseudo-first-order kinetics. In fact, in several studies, it has been found that the rate of mineralization or disappearance of a pollutant is proportional to the concentration of the pollutant. This makes the time required to achieve decontamination more important than the rate of degradation. Because the metabolism of chemicals by bacteria involves mostly enzymatic conversions, pollutant degradation often follows Michaelis-Menton-type kinetics. Therefore, Km values of various degradative enzymes with respect to the pollutant must be considered when using bacteria for bioremediation. Considering this, the solubility of a pollutant or a mixture of pollutants might also present a problem for bacterial degradation. In contrast, using a nonspecific free-radical-based mechanism, the fungi are able to degrade insoluble complex mixtures of pollutants, such as creosote (Aust and Bumpus 1989) and Arochlor (Bumpus and Aust 1987b). Inexpensive nutrient sources, such as sawdust, wood chips, surplus grains, and agricultural wastes, can be used to effectively cultivate white rot fungi.(ABSTRACT TRUNCATED AT 400 WORDS)

Basidiomycota↗

PubMed1993

Veratryl alcohol-dependent production of molecular oxygen by lignin peroxidase.

Veratryl alcohol- and H2O2-dependent production of oxygen by lignin peroxidase isozyme H2 (LiPH2) from Phanerochaete chrysosporium was investigated. Veratryl alcohol oxidation by LiPH2 decreased with increasing concentrations of H2O2 while oxygen evolution increased. The absorption spectrum of the LiPH2 in these experiments indicated that it was in the compound II state. We propose that O2 production results from the one electron oxidation of H2O2 by the veratryl alcohol cation radical to yield superoxide, as the addition of superoxide dismutase stimulated oxygen production. It has been reported previously that oxygen is consumed in reaction mixtures containing lignin peroxidase, H2O2, veratryl alcohol, and oxalate (Popp, J. L., Kalyanaraman, B., and Kirk, T.K. (1990) Biochemistry 29, 10475-10480). In the presence of oxalate, we observed oxygen consumption that was dependent on the H2O2 concentration. The ability of other methoxybenzenes to mediate oxygen production appeared to be related to their redox potential. It was concluded that cation radicals can oxidize H2O2 by one electron which results in the production of superoxide and the evolution of molecular oxygen. Thus, the rates of LiPH2-catalyzed O2 consumption or O2 production are dependent on the relative concentrations of H2O2 and oxalate.

Agaricales↗

PubMed1993

On the mechanism of peroxidase-catalyzed oxygen production.

A mechanism for oxygen production involving the reaction of cation radicals with H2O2 was investigated. The rate of oxygen production by peroxidases was greatly enhanced by the addition of ABTS [2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid)] or chlorpromazine, both of which are oxidized to cation radicals by peroxidases. We propose that enhanced oxygen evolution is due to the reaction of the cation radicals of these compounds with H2O2 to produce O2-. which then dismutates to produce oxygen. Oxygen evolution was quantitated in reaction mixtures containing electrochemically generated ABTS cation radicals and H2O2. The reaction was found to be first order with respect to the concentrations of the ABTS cation radical and H2O2 and the rate constants indicated that O2-. was an intermediate. Ferric EDTA enhanced oxygen production in both reaction systems (i.e., using ABTS+. that was generated electrochemically or with a peroxidase). These results suggest that O2-. production catalyzed by peroxidases is dependent upon a compound which is oxidized by the peroxidase to a cation radical. Superoxide is subsequently produced from the one-electron oxidation of H2O2 by the cation radical.

Benzothiazoles↗

PubMed1992

Production of hydroxyl radical by lignin peroxidase from Phanerochaete chrysosporium.

The mechanism for the production of hydroxyl radical by lignin peroxidase from the white rot fungus Phanerochaete chrysosporium was investigated. Ferric iron reduction was demonstrated in reaction mixtures containing lignin peroxidase isozyme H2 (LiPH2), H2O2, veratryl alcohol, oxalate, ferric chloride, and 1,10-phenanthroline. The rate of iron reduction was dependent on the concentration of oxalate and was inhibited by the addition of superoxide dismutase. The addition of ferric iron inhibited oxygen consumption in reaction mixtures containing LiPH2, H2O2, veratryl alcohol, and oxalate. Thus, the reduction of ferric iron was thought to be dependent on the LiPH2-catalyzed production of superoxide in which veratryl alcohol and oxalate serve as electron mediators. Oxalate production and degradation in nutrient nitrogen-limited cultures of P. chrysosporium was also studied. The concentration of oxalate in these cultures decreased during the period in which maximum lignin peroxidase activity (veratryl alcohol oxidation) was detected. Electron spin resonance studies using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide were used to obtain evidence for the production of the hydroxyl radical in reaction mixtures containing LiPH2, H2O2, veratryl alcohol, EDTA, and ferric chloride. It was concluded that the white rot fungus might produce hydroxyl radical via a mechanism that includes the secondary metabolites veratryl alcohol and oxalate. Such a mechanism may contribute to the ability of this fungus to degrade environmental pollutants.

Agaricales↗

PubMed1992

On the mechanism of inhibition of the veratryl alcohol oxidase activity of lignin peroxidase H2 by EDTA.

The mechanism of inhibition of the veratryl alcohol oxidase activity of lignin peroxidase H2 (LiPH2) by EDTA was investigated. It was found that EDTA was decarboxylated and that cytochrome c, nitro blue tetrazolium, ferric iron, and molecular oxygen were reduced in a reaction mixture containing LiPH2, H2O2, veratryl alcohol, and EDTA. The reductive activity observed with LiPH2 followed first order kinetics with respect to the concentration of EDTA. Stoichiometry studies showed that in the presence of sufficient EDTA, 1.7 mol of ferric iron were reduced per mole of H2O2 added to the reaction mixture. Superoxide- and EDTA-derived radicals were detected by ESR spin trapping upon incubation of LiPH2 with H2O2, veratryl alcohol, and EDTA. The Km values of veratryl alcohol and H2O2 remained the same for both the oxidative and reductive activities of LiPH2. Reductive activity was also observed with LiPH2 and EDTA using other free radical mediators in the place of veratryl alcohol, such as 1,4-dimethoxybenzene, 1,2,3- and 1,2,4-trimethoxybenzenes, and 1,2,4,5-tetramethoxybenzene. EDTA reduced the cation radical of 1,2,4,5-tetramethoxybenzene formed by LiPH2 in the presence of H2O2. Hence, it is proposed that the apparent inhibition of the veratryl alcohol oxidase activity of LiPH2 by EDTA is due to the reduction of the veratryl alcohol cation radical intermediate back to veratryl alcohol by EDTA. The reduction of cytochrome c, nitro blue tetrazolium, ferric ion, and molecular oxygen appears to be mediated by the EDTA radical formed by reduction of the veratryl alcohol cation radical.

Alcohol Oxidoreductases↗