Add time:09/07/2019         Source:sciencedirect.com

Publisher SummaryThis chapter discusses the assay for D-ribose-5-phosphate ketol isomerase and D-ribulose- 5-phosphate 3-epimerase. The assay for the isomerase is based upon the increase in absorbance observed at 290 nm, when ribose-5-phosphate ketol isomerase acts upon ribose 5-phosphate to form ribulose 5-phosphate. When the reaction has reached equilibrium, the addition of epimerase gives rise to a further increase in absorbance as xylulose 5-phosphate is formed. In the presence of an excess of the isomerase, the second rate of absorbance increase is proportional to the activity of the epimerase. In assay of ribose-5-phosphate ketol isomerase, the reaction mixture contains 1.78 ml 50 mM triethanolamine, HC1 buffer and 0.20 ml 100 mM ribose 5-phosphate (20 μmoles). It is placed in the cell compartment at 37° of a double-beam spectrophotometer connected to a recorder, and read against a control cuvette containing an equal volume of buffer. In the assay of ribulose-5-phosphate 3-epimerase, the assay mixture is set up as before, and 0.01 ml (2 units) of the isomerase is added. The absorbance increases rapidly at first and then levels off after 12–15 min. If small amounts of epimerase are present in the isomerase the recorder trace shows a slight upward slope. The calculation of isomerase and epimerase activity is presented. The isomerase assay has been used to follow the purification of the enzyme from spinach and other sources, in the determination of Michaelis constants.

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