Radial diffusion assay of NAD- and NADP-dependent oxidoreductases

With a radial diffusion assay we measured the activity of six NAD- and NADP-dependent oxidoreductases: alcohol, glucose-6-phosphate, hydroxysteroid, lactate and malate dehydrogenases, and glutathione reductase. The enzyme was allowed to diffuse for 24h in an agarose gel in which the substrate was incorporated, then reacted the pyridine nucleotide coenzyme. The size of an enzyme diffusion zone could be made visible by the change of the fluorescence of the coenzyme against the background when the coenzyme was either oxidized or reduced. The procedure for each enzyme is reported. The results indicate that this new technique may be applicable to all NAD- and NADP-dependent enzymes. Because of its simplicity and potentiality for screening many samples, we think this method has applications in the clinical laboratory and in nutrition studies.