Tromethamine hydrochloride → 4-Nitrophenol

Participants

Reaction Steps

A p-nitrophenylacetate stock solution is made by dissolving p-nitrophenylacetate in dimethylsulfoxide (DMSO) to constitute a 0.1 M solution. Before assay, a sample of the stock solution is diluted 100-fold in 50 mM sodium acetate pH 5.0 to make a 1 mM solution. A 100 μl volume of 1 mM p-nitrophenylacetate is mixed with each dilution of the enzyme and then incubated at 25° C. for 10 minutes. Substrate alone, enzyme alone, and buffer alone are run as controls. p-Nitrophenol standard solutions of 0.25, 0.2, 0.1, 0.05, and 0.02 mM are prepared by diluting a 10 mM stock solution in 50 mM sodium acetate pH 5.0. At 10 minutes, 50 μl of 1.0 M Tris-HCl pH 8.0 buffer is added to each well (including samples, substrate control, enzyme control, reagent control, and standards), mixed, and the absorbance at 405 nm immediately measured on e.g. a SPECTRAMAX™ 340 PC plate reader (Molecular Devices, Sunnyvale, Calif., USA). One unit of acetyl xylan esterase activity is defined as the amount of enzyme capable of releasing 1 μmole of p-nitrophenolate anion per minute at pH 5, 25° C.

Provenance & Evidence