Employment of a Phenoxy-Substituted Acridinium Ester as a Long-Lived Chemiluminescent Indicator of Glucose Oxidase Activity and Its Application in an Alkaline Phosphatase Amplification Cascade Immunoassay

Rc, Brown; Weeks, Ian; Fisher, M; Harbron, Stuart; Cj, Taylorson; Js, Woodhead

doi:10.1006/abio.1998.2604

Cited by 23 publications

(13 citation statements)

References 7 publications

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“…The assay is a homogeneous chemiluminescence-based HPA (hybridization protection assay) ( [24,25] and reviewed in [26]). This type of assay has been applied successfully to measure the activities of enzymes, including human telomerase prepared from cell-free extracts [27,28] and glucose oxidase [29]. The assay developed for Staphylococcus aureus DNA ligase utilizes an AE (acridinium ester)-labelled nicked DNA substrate.…”

Section: Introductionmentioning

confidence: 99%

Staphylococcus aureus DNA ligase: characterization of its kinetics of catalysis and development of a high-throughput screening compatible chemiluminescent hybridization protection assay

Gul

Brown²,

May

et al. 2004

Biochemical Journal

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DNA ligases are key enzymes involved in the repair and replication of DNA. Prokaryotic DNA ligases uniquely use NAD+ as the adenylate donor during catalysis, whereas eukaryotic enzymes use ATP. This difference in substrate specificity makes the bacterial enzymes potential targets for therapeutic intervention. We have developed a homogeneous chemiluminescence-based hybridization protection assay for Staphylococcus aureus DNA ligase that uses novel acridinium ester technology and demonstrate that it is an alternative to the commonly used radiometric assays for ligases. The assay has been used to determine a number of kinetic constants for S. aureus DNA ligase catalysis. These included the K(m) values for NAD+ (2.75+/-0.1 microM) and the acridinium-ester-labelled DNA substrate (2.5+/-0.2 nM). A study of the pH-dependencies of kcat, K(m) and kcat/K(m) has revealed values of kinetically influential ionizations within the enzyme-substrate complexes (kcat) and free enzyme (kcat/K(m)). In each case, the curves were shown to be composed of one kinetically influential ionization, for k(cat), pK(a)=6.6+/-0.1 and kcat/K(m), pK(a)=7.1+/-0.1. Inhibition characteristics of the enzyme against two Escherichia coli DNA ligase inhibitors have also been determined with IC50 values for these being 3.30+/-0.86 microM for doxorubicin and 1.40+/-0.07 microM for chloroquine diphosphate. The assay has also been successfully miniaturized to a sufficiently low volume to allow it to be utilized in a high-throughput screen (384-well format; 20 microl reaction volume), enabling the assay to be used in screening campaigns against libraries of compounds to discover leads for further drug development.

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Section: Introductionmentioning

confidence: 99%

Staphylococcus aureus DNA ligase: characterization of its kinetics of catalysis and development of a high-throughput screening compatible chemiluminescent hybridization protection assay

Gul

Brown²,

May

et al. 2004

Biochemical Journal

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“…The linked assay method is widely used to assay for glucose oxidase activity and is based on the release of hydrogen peroxide which is quantified by colorimetric methods in the presence of horseradish peroxidase [7]. Some workers have used a wide range of different reducing compounds as substrates for peroxidase to increase the assay sensitivity [9][10][11]. The reactions catalyzed by peroxidase in the literature are based on colorimetry, chemiluminescence, fluorescence, and amperometric measurements.…”

mentioning

confidence: 99%

Assay for glucose oxidase from Aspergillus niger and Penicillium amagasakiense by Fourier transform infrared spectroscopy

Karmali

Teixeira

et al. 2004

Analytical Biochemistry

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“…Instead, we observe a slow rise of the chemiluminescent signal. By comparison, Brown et al (10), using an acridinium ester to detect glucose oxidase-generated peroxide, obtained a plateau level of signal within less than 10 min. The fact that in our system the chemiluminescent signal with peroxide alone takes time to reach the plateau level is probably a major contributing factor to the slow rise of the signal from glucose oxidase.…”

Section: Glucose Oxidasementioning

confidence: 99%

“…Recently, Brown et al (10) used an acridinium ester to detect glucose oxidase-generated peroxide. They report a sensitivity of 180 amol for glucose oxidase.…”

Section: Alternative Systems For Detection Of Peroxidementioning

confidence: 99%

Chemiluminescent determination of hydrogen peroxide with 9-acridinecarbonylimidazole and use in measurement of glucose oxidase and alkaline phosphatase activity

2000

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Several activated derivatives of 9-acridinecarboxylic acid were prepared in order to investigate their utility for detection of hydrogen peroxide. One of these derivatives, 9-acridinecarbonylimidazole (I), is especially stable and is a useful reagent for measuring, by chemiluminescence, the activity of a number of enzymes that directly produce peroxide, including glucose oxidase. Other enzymes can also be assayed if an appropriate intermediate substrate exists that ultimately produces hydrogen peroxide after being acted upon by the enzyme. 5-Bromo-4-chloro-3-indolyl phosphate (BCIP) and 3-indolyl phosphate (3-IP) are such substrates for alkaline phosphatase. The detection limits for both of these enzymes are in the 1-10 amol range. Other enzymes that can potentially be assayed using I include oxidases, hydrolases and dehydrogenases. Negative assays for compounds that consume or bind peroxide such as reducing agents, antioxidants, catalases and peroxidases are also feasible.

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Employment of a Phenoxy-Substituted Acridinium Ester as a Long-Lived Chemiluminescent Indicator of Glucose Oxidase Activity and Its Application in an Alkaline Phosphatase Amplification Cascade Immunoassay

Cited by 23 publications

References 7 publications

Staphylococcus aureus DNA ligase: characterization of its kinetics of catalysis and development of a high-throughput screening compatible chemiluminescent hybridization protection assay

Staphylococcus aureus DNA ligase: characterization of its kinetics of catalysis and development of a high-throughput screening compatible chemiluminescent hybridization protection assay

Assay for glucose oxidase from Aspergillus niger and Penicillium amagasakiense by Fourier transform infrared spectroscopy

Chemiluminescent determination of hydrogen peroxide with 9-acridinecarbonylimidazole and use in measurement of glucose oxidase and alkaline phosphatase activity

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