Fluorescent Assays To Quantitate Enzymatic Activities Yielding as End Product an Aqueous-Insoluble Indigo-Blue Dye

Abstract

Hydrolytic enzymes acting upon indoxyl-derivatized substrates produce a water-insoluble indigo-blue dye. The generation of indigo dye works well in nonquantitative histochemical or diagnostic assays. For quantitative analyses however, the technique is unsuited. In this paper two fluorescent methods are described that permit quantitative measurement of sialidase/neuraminidase activity toward indoxyl-derivatized N-acetyl-neuraminic acid substrates. The first method is based upon the reaction of the sialic/ N-acetyl-neuraminic acid with pyridoxamine and Zn2+ to produce a fluorescent chelate. This method is not sialic acid-specific and could be used for the quantitation of alpha-oxo acids. The optimum conditions for sialic acid fluorescent chelation are described. The second method is based upon the fluorescence of the reaction intermediates, indoxyl- and indigo-white, by arresting their conversion to nonfluorescent indigo-blue. This method is suitable for measuring any enzymatic activity toward indoxyl-derivatized substrates. Enzyme kinetics derived for influenza viral neuraminidase using the two techniques are described in this paper.