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 graphite electrode type RW0 (Ringsdorff-Werke GmbH) was modified by adsorption of a phenoxazine derivative (prepared by reaction of Nile blue with terephthaloyl chloride). Glucose-6-phosphate dehydrogenase (0.5 mg) and polyoxyethylene glycol - NAD+ conjugate (1 mg) (cf. Bueckmann, Biocatalysis, 1987, 1, 173) was mixed with phosphate buffer solution and applied to the electrode surface. A cellulose dialysis membrane (Nephrophan, mol. wt. cut-off 12,000 to 15,000, Filmfabrik Wolfen, Germany) was tightened around the electrode to retain the macromolecules. The enzyme electrode was conditioned in buffer solution for 20 min before use. A flow injection manifold, with electrochemical cell, potentiostat and recorder was used with the prepared electrode. The calibration graph for concentration. of glucose 6-phosphate from 10 µM to 10 mM was slightly non-rectilinear. The electrode life was a few days.