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Quantitative analysis of enzymatic assays using indoxyl-based substrates

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Abstract

Hydrolysis of indoxyl-based substrates by hydrolytic enzymes is a commonly used semiquantitative detection system that generates a water-insoluble indigo dye which is difficult to quantify. This work describes the quantitative analysis and enzyme kinetics for alkaline phosphatase (AP) and 5-bromo-4-chloro-3-indoxyl phosphate (BCIP) in solution obtained by applying known solubilization methodology from the textiles industry to the enzymatic product. This proposal is based on the reduction of the tetrahalo-indigo blue dye in a basic medium with the aim of generating its aqueous-soluble parent compound termed indigo white, which gives a rich yellow color in solution and is fluorescent. A quantitative ELISA (where a soluble end product is required) is accomplished for first time using BCIP as substrate.

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Acknowledgement

This work has been supported by the FICYT Project PC04-11.

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Authors and Affiliations

  1. Departamento de Química Física y Analítica, Universidad de Oviedo, 33006, Oviedo, Asturias, Spain

    Pablo Fanjul-Bolado, María Begoña González-García & Agustín Costa-García

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  1. Pablo Fanjul-Bolado
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  2. María Begoña González-García
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  3. Agustín Costa-García
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Correspondence to Agustín Costa-García.

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Fanjul-Bolado, P., González-García, M.B. & Costa-García, A. Quantitative analysis of enzymatic assays using indoxyl-based substrates. Anal Bioanal Chem 386, 1849–1854 (2006). https://doi.org/10.1007/s00216-006-0808-4

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  • DOI: https://doi.org/10.1007/s00216-006-0808-4

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