Identification of the Colored Guaiacol Oxidation Product Produced by Peroxidases

Doerge, Daniel R.; Divi, Rao L.; Churchwell, Mona I.

doi:10.1006/abio.1997.2191

Cited by 243 publications

(173 citation statements)

References 8 publications

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“…39 PDDA/PSS/Mb colloids were resuspended in 1 mL of 10 mM pH 5.5 acetate buffer containing 4 mM guaiacol, and H 2 O 2 was added to 1 mM. Production of the colored product 3,3′-dimethoxy-4,4′-biphenoquinone [39][40][41] was monitored by UV-vis spectroscopy at 470 nm. The biocolloids were centrifuged from the reaction mixture before UV-vis spectra were obtained.…”

Section: Methodsmentioning

confidence: 99%

Enzyme−DNA Biocolloids for DNA Adduct and Reactive Metabolite Detection by Chromatography−Mass Spectrometry

et al. 2008

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Silica microbead bioreactors (0.5 µm diameter) coated with DNA and enzymes were fabricated to measure reactive metabolite and DNA-adduct formation rates relevant to genotoxicity screening. Cytochrome (cyt) P450 2E1, cyt P450 cam , and myoglobin (Mb) were incorporated into thin films with DNA using the electrostatic layer-bylayer (LbL) method. The utility of these biocolloids was demonstrated by oxidation of guaiacol, styrene, and (4-methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK). Enzyme turnover rates for formation of reactive metabolites were monitored using gas chromatography/mass spectrometry (GC/MS) and liquid chromatography-mass spectrometry (LC-MS). Capillary LC-MS/MS was employed to determine DNA nucleobase adducts after catalyzing the reactive metabolite formation with DNAenzyme biocolloids and then using neutral thermal hydrolysis on the biocolloids. Dramatic improvements in surface area to volume ratio over similar films on macroscopic surfaces opens new avenues for genotoxicity screening and enabled the first use of pure cyt P450 enzymes in enzyme-DNA films to produce DNA adducts. The method makes possible identification and formation rate measurements of major and minor DNA adducts as well as the metabolites themselves in <5 min of reaction time using relevant human liver enzymes.Toxicity is often not identified sufficiently early in developing new pharmaceutical, agricultural, personal care, and dietary products. Roughly 30% of drug development failures result from toxicity issues, driving drug costs up significantly. 1-4 Conventional in vitro biological tests such as Ames, chromosome aberration, mouse lymphoma, and Comet assays provide qualitative answers to toxicity questions based on bulk DNA damage but do not give chemical structure or site-specific DNA damage information. 5,6 Animal testing may not relate to human exposure because of metabolic and physiological differences between humans and animal models. 2,4,7 While all these toxicity tests are valuable, we believe that rapid, high-throughput toxicity screening methods for new chemicals that provide chemical structure information about reactive intermediates at early stages of commercial development are important goals to complement conventional microbiological and animal tests.Bioactivation of xenobiotic molecules by metabolic enzymes often results in reactive metabolites that damage biomolecules, including DNA, in a major toxicity pathway. [8][9][10] An example is the metabolism of lipophilic compounds by liver cytochrome (cyt) P450 (or CYP) enzymes creating reactive electrophilic metabolites. 4,11 These electrophiles attack nucleophilic DNA sites, primarily guanines, potentially resulting in genotoxicity. Depending on the adduct site, additional metabolism, and DNA repair processes, covalent DNA adducts can lead to mutations, teratogenesis, and carcinogenesis. 12-14 Thus, nucleobase adducts are key biomarkers for predicting cancer risk in humans and for exposure to toxic chemicals, and sensitive liquid chromatography-mass spectromet...

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Section: Methodsmentioning

confidence: 99%

Enzyme−DNA Biocolloids for DNA Adduct and Reactive Metabolite Detection by Chromatography−Mass Spectrometry

et al. 2008

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“…The oxidation of guaiacol by peroxidases in the presence of hydrogen peroxide (H 2 O 2 ) was used as the basis for the colorimetric assay based on the development of a reddish color (Doerge et al, 1997). The assay was carried out by adding 1.0 ml of 30% H 2 O 2 (R&M, UK) to the reaction mixture consisting of 0.5 ml of sample extracts, 7.5 ml of 0.1 mol/L sodium phosphate buffer at pH 6.1, and 1.0 ml of 1% guaiacol (0.01 g/ml; Merck, Germany).…”

Section: Determination Of Specific Activity Of Peroxidasementioning

confidence: 99%

Morphological and biochemical responses of Oryza sativa L. (cultivar MR219) to ion beam irradiation

Ling

Ung

Hussein

et al. 2013

J. Zhejiang Univ. Sci. B

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Abstract:Objective: Heavy ion beam, which has emerged as a new mutagen in the mutation breeding of crops and ornamental plants, is expected to result in the induction of novel mutations. This study investigates the morphological and biochemical responses of Oryza sativa toward different doses of carbon ion beam irradiation. Methods: In this study, the dry seeds of O. sativa were irradiated at 0, 20, 40, 60, 80, 100, and 120 Gy, followed by in-vitro germination under controlled conditions. Morphological and biochemical studies were conducted to investigate the morphological and physiological responses of O. sativa towards ion beam irradiation. Results: The study demonstrated that low doses (10 Gy) of ion beam have a stimulating effect on the height, root length, and fresh weight of the plantlets but not on the number of leaves. Meanwhile, doses higher than 10 Gy caused reductions in all the morphological parameters studied as compared to the control samples. The highest total soluble protein content [(2.11±0.47) mg/g FW] was observed in plantlets irradiated at 20 Gy. All irradiated plantlets were found to have 0.85% to 58.32% higher specific activity of peroxidase as compared to the control samples. The present study also revealed that low doses of ion beam (10 and 20 Gy) had negligible effect on the total chlorophyll content of O. sativa plantlets while 40 Gy had a stimulating effect on the chlorophyll content. Plantlets irradiated between 40 to 120 Gy were shown to be 0.38% to 9.98% higher in total soluble nitrogen content which, however, was not significantly different from the control samples. Conclusions: Carbon ion beam irradiation administered at low to moderate doses of 10 to 40 Gy may induce O. sativa mutants with superior characteristics.

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“…The collected protein was desalted with PD-10 columns and eluted in 5 mM phosphate buffer, pH 7.4, and then applied to a hydroxylapatite (HA) column equilibrated with 5 mM sodium phosphate, pH 7.4. After washing the HA column with 5 mM phosphate buffer, the elution was performed stepwise with increasing phosphate concentrations (20,50, 100, and 200 mM; pH 7.4). Fractions at 50 and 100 mM sodium phosphate, which showed the Soret maximum at 412 nm, were collected.…”

Section: Methodsmentioning

confidence: 99%

“…Rates of guaiacol or ABTS oxidation were calculated by following the absorbance increase at 470 nm (⑀ 470 ϭ 26.6 mM Ϫ1 cm Ϫ1 ) (20) and 414 nm (⑀ 414 ϭ 36 mM (21), respectively. For the enzyme, 50 nM (in guaiacol oxidation assays) or 2.5 nM (in ABTS oxidation assays) was used; for H 2 O 2 , 100 M was used.…”

Section: Methodsmentioning

confidence: 99%

A Stable Bacterial Peroxidase with Novel Halogenating Activity and an Autocatalytically Linked Heme Prosthetic Group

Auer

Gruber

Bellei³

et al. 2013

Journal of Biological Chemistry

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Background: First analysis was made of bacterial ancestor of peroxidases from mammalian innate immune system. Results: Highly stable heme enzyme possesses high bromination activity and covalently bound prosthetic group. Conclusion: Post-translational autocatalytic (peroxide-driven) heme modification is found in prokaryotic and eukaryotic halogenating peroxidases. Significance: Peroxidase-mediated production of antimicrobial hypohalous acids was developed early in evolution.

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Identification of the Colored Guaiacol Oxidation Product Produced by Peroxidases

Cited by 243 publications

References 8 publications

Enzyme−DNA Biocolloids for DNA Adduct and Reactive Metabolite Detection by Chromatography−Mass Spectrometry

Enzyme−DNA Biocolloids for DNA Adduct and Reactive Metabolite Detection by Chromatography−Mass Spectrometry

Morphological and biochemical responses of Oryza sativa L. (cultivar MR219) to ion beam irradiation

A Stable Bacterial Peroxidase with Novel Halogenating Activity and an Autocatalytically Linked Heme Prosthetic Group

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