Quantitative analysis of enzymatic assays using indoxyl-based substrates

Fanjul-Bolado, Pablo; González-García, María Begoña; Costa-García, Agustín
December 2006
Analytical & Bioanalytical Chemistry;Dec2006, Vol. 386 Issue 6, p1849
Academic Journal
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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