Add time:09/24/2019         Source:sciencedirect.com

Publisher SummaryThis chapter describes the enzymatic method for the determination of D-ribulose. D-ribulose has been determined by the cysteine–carbazole method; however, this is not specific. By comparison, the high degree of specificity of the ribitol dehydrogenase from Aerobacter aerogenes allows the estimation of D-ribulose in the presence of other sugars. The enzymatic method is based on the principle that ribitol dehydrogenase catalyzes the reversible oxidation of ribitol to D-ribulose in the presence of diphosphopyridine nucleotide (DPN). The apparent equilibrium constant for this reaction is 7.17 ×10–3 at pH 8.0 and 28°C. With excess reduced diphosphopyridine nucleotide (DPNH), the D-ribulose is virtually quantitatively converted to ribitol with the simultaneous oxidation of an equivalent amount of DPNH. For the stability of the solutions, the DPNH solution should be prepared freshly each week and stored in a frozen state. The tris buffer is stable indefinitely at 4°C. The RDH solution keeps for longer than a month at 3°C. Repeated freezing and thawing leads to considerable loss of activity. The reaction proceeds stoichiometrically. For each mole of D-ribulose reduced, one mole of DPNH is oxidized to DPN.

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