Add time:09/05/2019         Source:sciencedirect.com

Publisher SummaryFor the coupled assay at 340 nm of transketolase with α-glycerophosphate dehydrogenase and triosephosphate isomerase, or with glyceraldehydes phosphate dehydrogenase, as auxiliary enzymes, a mixture of D-ribose 5-phosphate and D-xylulose 5-phosphate is needed. A mixture of D-ribose 5-phosphate and D-ribulose 5-phosphate is employed for the assay of D-ribulose-5-phosphate 3-epimerase by coupling it to the transketolase reaction. A simple procedure is described in the chapter for preparing mixtures of the pentose phosphates for the assay of the above two enzymes. Although the commercial purifed epimerase is specified, it need not be free of the isomerase, and a crude concentrate of the two enzymes from spleen, muscle, or liver, may be used provided that transketolase is absent. The ultrafiltration is carried out, at or near, 37°, to prevent the composition of the equilibrium mixture changing after the removal from the spectrophotometer. The procedure may also be used with a 20 mM solution of ribose 5-phosphate. At this higher concentration, however, further spectral changes of the isomerase product occur, and may take place before the epimerase is added, obscuring the progress of the enzymic reaction. Ribose 5-phosphate is measured, without interference from the ketopentose phosphates, by the colorimetric phloroglucinol method. Ribulose 5-phosphate is measured in ribose 5-phosphate-ribulose 5-phosphate mixtures with cysteine-carbazole; a heating time of 2 hr at 37° is used to develop the color. Xylulose 5-phosphate is measured, either alone or in conjunction with ribulose 5-phosphate by the spectrophotometric method described in the chapter. Spectrophotometric determination of D-Xylulose 5-Phosphate and D-Ribulose 5-Phosphate uses α-glycerophosphate dehydrogenase and triose phosphate isomerase to measure the glyceraldehyde 3-phosphate formed. One molecule of ketopentose phosphate causes the oxidation of 1 molecule of NADH.

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