Colorimetric microtiter plate assay of glucose and fructose by enzyme-linked formazan production: applicability to the measurement of fructosyl transferase activity in higher plants.
A rapid, enzyme-linked colorimetric assay, for the sequential determination of nanomole quantities of glucose and fructose in the same sample, has been developed for the measurement of fructosyl transferase activity in plant extracts. The assay extends the conventional dehydrogenase-linked assay for these sugars by utilizing the intermediary electron carrier, phenazine methosulfate, to couple NADP reduction to the production of a formazan dye from the tetrazolium salt, thiazolyl blue, in a form suitable for measurement using a microtiter plate reader. When the microtiter plate assay was used to measure the activities of yeast invertase and sucrose:sucrose fructosyl transferase from Lolium temulentum, results obtained were very similar to results obtained using the conventional procedure. The rapidity, small scale, and ease of execution of the method offers considerable advantages over the conventional hexose assay and is particularly suitable for screening of large numbers of small samples, exploiting both the speed of the microtiter plate reader and the facility of for microcomputer processing of data. The potential of this method for use with other enzyme systems and other metabolites is discussed.
Plant and Cell Biology Department, Welsh Plant Breeding Station, Aberystwyth, Wales B, United Kingdom.
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D003124:Colorimetry / Q000379:methods*; D005562:Formazans; D005632:Fructose / Q000032:analysis*; D005947:Glucose / Q000032:analysis*; D006026:Glycoside Hydrolases / Q000032:analysis; D006602:Hexosyltransferases / Q000032:analysis; D010944:Plants / Q000201:enzymology; D013778:Tetrazolium Salts; D013844:Thiazoles; D043324:beta-Fructofuranosidase; D001426:Bacterial Proteins
D005562:Formazans; D005632:Fructose; D005947:Glucose; D006026:Glycoside Hydrolases; D006602:Hexosyltransferases; D013778:Tetrazolium Salts; D013844:Thiazoles; D043324:beta-Fructofuranosidase; C021451:inulosucrase; D001426:Bacterial Proteins; C022616:thiazolyl blue