A rapid, quantitative microplate assay for NAD-linked D-mannitol dehydrogenase.

A 96-well microtitre plate assay for NAD-linked D-mannitol dehydrogenase based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by reduced NAD is described. The assay allows rapid measurement of D-mannitol dehydrogenase in crude bacterial extracts derived by sonic disruption, in acetone permeabilized cells and in column eluates during enzyme purification. The absorbance of reaction mixtures in a microtitre plate is measured at 620 nm over a 3-4 min period using a programmable microplate reader. The rate of increase in absorbance is directly proportional to the amount of enzyme present and there is excellent correlation between activities derived using the microplate assay with those determined using conventional spectrophotometric methods.