Fluorometric determination of hydrogen peroxide using resorufin and peroxidase

Brotea, G.P.; Thibert, R.J.

doi:10.1016/0026-265x(88)90150-6

Cited by 21 publications

(13 citation statements)

References 15 publications

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“…The competing reaction might or might not involve the enzyme molecule. For example, it was shown that resorufin can be further oxidized to a much less fluorescent compound resazurin (46), albeit with a much slower rate than its rate of formation (47). A second possibility is that for some reason the substrates become limiting precluding further reaction cycles.…”

Section: Product Molecules Allosterically Inhibit Individual Enzyme Mmentioning

confidence: 99%

Allosteric inhibition of individual enzyme molecules trapped in lipid vesicles

Piwoński

Goomanovsky²,

Bensimon³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Enzymatic inhibition by product molecules is an important and widespread phenomenon. We describe an approach to study product inhibition at the single-molecule level. Individual HRP molecules are trapped within surface-tethered lipid vesicles, and their reaction with a fluorogenic substrate is probed. While the substrate readily penetrates into the vesicles, the charged product (resorufin) gets trapped and accumulates inside the vesicles. Surprisingly, individual enzyme molecules are found to stall when a few tens of product molecules accumulate. Bulk enzymology experiments verify that the enzyme is noncompetitively inhibited by resorufin. The initial reaction velocity of individual enzyme molecules and the number of product molecules required for their complete inhibition are broadly distributed and dynamically disordered. The two seemingly unrelated parameters, however, are found to be substantially correlated with each other in each enzyme molecule and over long times. These results suggest that, as a way to counter disorder, enzymes have evolved the means to correlate fluctuations at structurally distinct functional sites.allostery | protein dynamics | single-molecule enzymology

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Section: Product Molecules Allosterically Inhibit Individual Enzyme Mmentioning

confidence: 99%

Allosteric inhibition of individual enzyme molecules trapped in lipid vesicles

Piwoński

Goomanovsky²,

Bensimon³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…rescence assay to detect H 2 O 2 by measuring the dis-to its use either to quantitate H 2 O 2 in solution or to measure horseradish peroxidase activity, we have sucappearance of its fluorescence upon horseradish peroxidase-mediated oxidation to a nonfluorescent derivative cessfully applied this substrate in measuring the activity of NADPH oxidase in phagocytes and a variety of (4). However, the determination of H 2 O 2 using this inverse fluorescence measurement has intrinsic limita-other oxidases in solution.…”

mentioning

confidence: 99%

A Stable Nonfluorescent Derivative of Resorufin for the Fluorometric Determination of Trace Hydrogen Peroxide: Applications in Detecting the Activity of Phagocyte NADPH Oxidase and Other Oxidases

Zhou¹,

Diwu²,

Panchuk-Voloshina³

et al. 1997

Analytical Biochemistry

1,258

932

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“…Enzymatic consumption and release of 2 have been demonstrated to function as useful indicator reactions for spectrophotometric and fluorometric analyses, since the dye exhibits strong absorption (l max 571 nm, e 4-7ϫ10 4 ) and emission (excitation maximum at 563 nm and emission maximum at 587 nm at pH 7.4) at wavelengthsϾ550 nm, where potential interference in analysis of colored or turbid serum components can be avoided. [25][26][27][28][29][30][31][32][33][34][35] These observations imply that the transformation of 1 to 2 as an indicator reaction for glucose analysis using only GOD takes full advantage of 2 as an analytically useful dye.…”

Section: Resultsmentioning

confidence: 99%

“…As mentioned above, several analytical methods with spectrophotometry or fluorometry have been designed based on enzymatic release of 2 from its derivatives such as 1. [25][26][27][28][29][30][31][32][33][34][35] However, to our knowledge, generation of 2 from 1 has been used as an indicator reaction only for studies of esterase activity of cytosolic aldehyde dehydrogenase and chymotrypsin. [20][21][22][23] Thus, this is the first report to show that transformation of 1 to 2 is induced by H 2 O 2 .…”

Section: Resultsmentioning

confidence: 99%

Hydrogen Peroxide-Induced Deacetylation of Acetyl Resorufin as a Novel Indicator Reaction for Fluorometric Detection of Glucose Using Only Glucose Oxidase.

Maeda

Matsu-Ura

Nishida

et al. 2001

CHEMICAL & PHARMACEUTICAL BULLETIN

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Spectrophotometric or fluorometric methods with glucose oxidase (GOD)-peroxidase (POD)-chromogen(s) systems are well known as useful tools for determination of glucose. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15] Indicator reactions in these methods are based on oxidative formation of a colored or fluorescent substance from a chromogen(s) with H 2 O 2 , generated through GODcatalyzed oxidation of glucose, in the presence of POD. These color reactions are well-characterized chemical processes. However, the POD-dependent indicator reactions are known to be inevitably disturbed by electron donors present in biological samples such as ascorbic acid, uric acid, and bilirubin.3) Major processes of interference by these compounds are as follows: 1) reduction of oxidatively formed colored or fluorescent substance to its original chromogen(s); and 2) competition with a chromogen in reduction of H 2 O 2 . Several methodologies have been developed for elimination of such interference in glucose determination with GOD-POD-chromogen(s) systems.3) However, the most straightforward approach to glucose determination free from such interference is design of a novel indicator reaction without recourse to redox reactions coupled with H 2 O 2 and POD. To our knowledge, only a few methods with non-redox color reactions for glucose determination using only GOD have been reported. These methods utilize transformation of a vanadium(V), 16) titanium(IV) 17) or dinuclear iron(III) 18) complex to its adduct with H 2 O 2 accompanied by a bathochromic shift as a spectrophotometric indicator reaction. As expected, it was demonstrated that glucose determination with vanadium(V) and titanium(IV) complexes was not affected by various substances usually present in serum or added to test solution. 16,17) However, spectrophotometric or fluorometric determination of glucose with high accuracy should use formation of a colored or fluorescent substance from a chromogen rather than a color change of a dye as an indicator reaction.Recently, resorufin (2) was shown to be able to reoxidize a reduced form of GOD at 37°C, being transformed to a colorless dihydro derivative, although the reductive bleaching of the dye is of no use for GOD-based determination of glucose as an indicator reaction. 19) This finding prompted us to examine how resorufin derivatives such as acetyl resorufin (1) would behave in GOD-catalyzed oxidation of glucose, and it was found that 1 behaves in a manner different from 2 in the enzymatic reaction. Here, we report that deacetylation of non-fluorescent 1 to fluorescent 2 is induced by H 2 O 2 generated in GOD-catalyzed oxidation of glucose, and the transformation is promising as an indicator reaction for fluorometric determination of glucose without significant effects of ascorbic acid, uric acid, or bilirubin. ExperimentalMaterials GOD from Aspergillus niger (EC 1.1.3.4) and glucose were used as supplied from Wako Pure Chemical Industries, Ltd. Acetyl resorufin (1) [20][21][22][23] was prepared by reaction of resorufin sod...

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Fluorometric determination of hydrogen peroxide using resorufin and peroxidase

Cited by 21 publications

References 15 publications

Allosteric inhibition of individual enzyme molecules trapped in lipid vesicles

Allosteric inhibition of individual enzyme molecules trapped in lipid vesicles

A Stable Nonfluorescent Derivative of Resorufin for the Fluorometric Determination of Trace Hydrogen Peroxide: Applications in Detecting the Activity of Phagocyte NADPH Oxidase and Other Oxidases

Hydrogen Peroxide-Induced Deacetylation of Acetyl Resorufin as a Novel Indicator Reaction for Fluorometric Detection of Glucose Using Only Glucose Oxidase.

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