Enzymatic, fluorometric assay of alpha-amylase in serum.
An enzymatic, fluorometric method for kinetic assay of serum alpha-amylase (1,4alpha-D-glucan 4-glucanohydrolase, EC 3.2.1.1) is described. A soluble starch is used as substrate, in tris(hydroxymethyl)methylamine buffer. All measurements are made in Pyrex cuvettes at 37 degrees C, with a reaction volume of 1.16 ml. The assay is based on the following reaction sequence: (see article) The rate of appearance of fluorescence of NADH (lambdaex = 365 nm, lambdaem = 460 nm), developed in the indicator reaction (4), is measured and equated to the activity of alpha-amylase in serum. A calibration plot of the change of fluorescence per min vs. enzyme concentration shows a good proportionality in the range of 0.50-5.0 kU/liter.
D000681:Amylases / Q000097:blood*; D005954:Glucosephosphate Dehydrogenase; D005959:Glucosidases; D006593:Hexokinase; D006801:Humans; D008722:Methods; D008832:Microchemistry; D013050:Spectrometry, Fluorescence
D005954:Glucosephosphate Dehydrogenase; D006593:Hexokinase; D000681:Amylases; D005959:Glucosidases