A sensitive and versatile chromogenic assay for peroxidase and peroxidase-coupled reactions

Ngo, T. T.; Lenhoff, Howard M.

doi:10.1016/0003-2697(80)90475-3

Cited by 355 publications

(164 citation statements)

References 11 publications

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“…A similar oxidative product involving ferric chloride or peroxidase in the presence of H2O2 for PPDD and MQ suggests that the enzymatic mechanism is analogous to that suggested by Ngo and Lenhoff 21 for the HRP-catalyzed oxidative coupling of 3-methyl-2-benzothiazolinone hydrazone hydrochloride and aromatic amines with the formation of an indamine dye. The mechanism for the peroxidase-catalyzed reaction of PPDD and MQ is proposed in Scheme 1.…”

Section: Mechanistic Approach For the Enzyme Activity Responsesupporting

confidence: 60%

Development and evaluation of kinetic spectrophotometric assays for horseradish Peroxidase by Catalytic Coupling of Paraphenylenediamine and mequinol

2009

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This paper presents a novel spectrophotometric method to measure peroxidase activity using paraphenylenediamine dihydrochloride (PPDD) and Mequinol (MQ). The PPDD traps the free radical, and gets oxidized to electrophilic 1,4-diimine; this couples with MQ to an give intense violet-colored chromogenic species with the maximum absorbance at 560 nm. This assay was adopted for the quantification of hydrogen peroxide between 10 × 10 -6 to 80 × 10 -6 M. From the kinetic data, a two-substrate ping-pong mechanism of peroxidase was established. Catalytic efficiency and catalytic power of commercial peroxidase were 0.204 × 10 6 M -1 min -1 and 2.86 × 10 -4 min -1 , respectively. The catalytic constant (kcat) of the proposed method was 0.2080 × 10 3 min -1. As a simple, rapid, precise and sensitive technique, PPDD-MQ stands as a potential replacement for the traditional guaiacol method. Applications to the plant extracts increase its relevance in the field of biochemical analysis.

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Section: Mechanistic Approach For the Enzyme Activity Responsesupporting

confidence: 60%

Development and evaluation of kinetic spectrophotometric assays for horseradish Peroxidase by Catalytic Coupling of Paraphenylenediamine and mequinol

2009

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“…The color reaction chosen for the assay of H202 in the present study was originally developed as part of a sensitive assay for peroxidase activity (5). When reducing agents, such as ascorbate or GSH, that are present in a given extract are removed, the method can be used as a very sensitive and specific assay for peroxides (Table I).…”

Section: Discussionmentioning

confidence: 99%

Abrupt Increase in the Level of Hydrogen Peroxide in Leaves of Winter Wheat Is Caused by Cold Treatment

Okuda

Matsuda²,

Yamanaka³

et al. 1991

Plant Physiol.

408

193

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MATERIALS AND METHODSAfter cold treatment of seedlings of winter wheat (Triticum aestivum L.), levels of hydrogen peroxide in the leaves were measured. The concentration of hydrogen peroxide increased to about three times the control level within a few minutes, and retumed to the normal level in 15 to 20 minutes. The elevated level of hydrogen peroxide was found to be equivalent to 1.5 micromoles per gram fresh weight tissues of leaves.Chemicals and Enzyme DMAB and MBTH were purchased from Nakarai Tesque,

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“…Activities of peroxidases, including Mnperoxidase were assayed using 3,3-dimethylaminobenzoic acid and 3-methyl-2-benzothiazolinone hydrazone in succinate/lactate buffer (100 mM, pH 4?5), as described previously (Ngo & Lenhoff, 1980;Bourbonnais & Paice, 1990;Baldrian et al, 2000). All spectrophotometric measurements were done in a microplate reader (Sunrise, Tecan) or a UV-VIS spectrophotometer (Lambda 11, Perkin-Elmer).…”

Section: Methodsmentioning

confidence: 99%

Degradation of cellulose and hemicelluloses by the brown rot fungus Piptoporus betulinus – production of extracellular enzymes and characterization of the major cellulases

2006

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Piptoporus betulinus is a common wood-rotting fungus parasitic for birch (Betula species). It is able to cause fast mass loss of birch wood or other lignocellulose substrates. When grown on wheat straw, P. betulinus caused 65 % loss of dry mass within 98 days, and it produced endo-1,4-b-glucanase (EG), endo-1,4-b-xylanase, endo-1,4-b-mannanase, 1,4-b-glucosidase (BG), 1,4-b-xylosidase, 1,4-b-mannosidase and cellobiohydrolase activities. The fungus was not able to efficiently degrade crystalline cellulose. The major glycosyl hydrolases, endoglucanase EG1 and b-glucosidase BG1, were purified. EG1 was a protein of 62 kDa with a pI of 2?6-2?8. It cleaved cellulose internally, produced cellobiose and glucose from cellulose and cellooligosaccharides, and also showed b-xylosidase and endoxylanase activities. The K m for carboxymethylcellulose was 3?5 g l "1 , with the highest activity at pH 3?5 and 70 6C. BG1 was a protein of 36 kDa with a pI around 2?6. It was able to produce glucose from cellobiose and cellooligosaccharides, but also produced galactose, mannose and xylose from the respective oligosaccharides and showed some cellobiohydrolase activity. The K m for p-nitrophenyl-1,4-b-glucoside was 1?8 mM, with the highest activity at pH 4 and 60 6C, and the enzyme was competitively inhibited by glucose (K i =5?8 mM). The fungus produced mainly b-glucosidase and b-mannosidase activity in its fruit bodies, while higher activities of endoglucanase, endoxylanase and b-xylosidase were found in fungus-colonized wood.

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A sensitive and versatile chromogenic assay for peroxidase and peroxidase-coupled reactions

Cited by 355 publications

References 11 publications

Development and evaluation of kinetic spectrophotometric assays for horseradish Peroxidase by Catalytic Coupling of Paraphenylenediamine and mequinol

Development and evaluation of kinetic spectrophotometric assays for horseradish Peroxidase by Catalytic Coupling of Paraphenylenediamine and mequinol

Abrupt Increase in the Level of Hydrogen Peroxide in Leaves of Winter Wheat Is Caused by Cold Treatment

Degradation of cellulose and hemicelluloses by the brown rot fungus Piptoporus betulinus – production of extracellular enzymes and characterization of the major cellulases

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