Abstract: The assay involves the use of alkanal monooxygenase (FMN-linked) - oxidoreductase co-immobilized on CNBr-activated Sepharose 4B (0.1 g in a 6 cm x 2.5 mm coil). Reagent streams consisted of phosphate buffer solution (pH 7.5) containing (A) 1 µM-glucose-6-phosphate dehydrogenase (I), 0.1 mM NADP, 0.1 mM glucose 6-phosphate (II) and 0.1 mM dithiothreitol and (B) 1 µM-flavine mononucleotide, decaldehyde (10 ppm) and Triton X-100 (1 ppm). Bioluminescence was detected at 490 nm. For determination of II, carrier stream (A) contained II instead of I, carrier stream (B) was unchanged and the serial glass coils contained immobilized I and alkanal monooxygenase (FMN-linked) - oxidoreductase (0.1 g of each). Analogous methods were used to assay malate dehydrogenase (III) and determine L-malate (IV). Detection limits were 15 fmol of I, 10 nM-II, 30 fmol of III and 1 µM-IV; the coefficient of variation (n = 5) were 5%.

Abstract: A improved setup for a previously described capillary electrophoresis system combined with an online enzymatic assay (cf. J. Chromatogr. A, 1994, 662, 275) was evaluated for glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. The old configuration involved a post-column reactor with a press-driven substrate flow; the new setup involved an electrically controlled substrate flow. The post-column reactor was coupled to the separation capillary by a liquid junction. Two different setups are described: (i) a high-voltage supply connected between the injector and the outlet of the reaction capillary; and (ii) two high-voltage supplies connected between the injector and the Tee and the Tee and the outlet of the reaction capillary, respectively (details given). The setup allowed capillaries of the same diameter to be used for the separation and the post-column reaction, which meant that dilution of solutes was minimized resulting in better detection sensitivity. Also, stopped-flow procedures could be carried out allowing enhanced reaction product formation. The method offers a high degree of freedom in optimizing the separation and enzymatic reaction conditions individually. Results are discussed.