Quantitative analysis of enzymatic assays using indoxyl-based substrates.
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.
Fanjul-Bolado
Pablo
P
Departamento de Química Física y Analítica, Universidad de Oviedo, 33006, Oviedo, Asturias, Spain.
González-García
María Begoña
MB
Costa-García
Agustín
A
D000469:Alkaline Phosphatase / Q000032:analysis* / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D006868:Hydrolysis; D007211:Indoles / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D015394:Molecular Structure; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D007211:Indoles; C035455:5-bromo-4-chloro-3-indoxyl phosphate; C034082:indoxyl; D000469:Alkaline Phosphatase