Isopropylidine maltoheptosyl fructofuranoside, doubly blocked substrate for determination of endoamylase activity.

We synthesized o-(4,6-o-isopropylidene-alpha-D-glucopyranosyl)-(1----4)- [o-alpha-D-glucopyranosyl-(1----4])5-o-alpha-D-glucopyranosyl-(1----2)- alpha-D-fructofuranoside (IPG7F) and developed an assay for determining the activity of amylase in human serum and urine by using this substrate. Glucoamylase, alpha-glucosidase, and mannitol dehydrogenase are used as coupling enzymes. The coupled reactions are monitored by continuously measuring the oxidation rate of NADH. In this procedure, various substances in the test specimens do not interfere with the detection of amylase activity. Exactly one molecule of NADH is oxidized by one attack of amylase on the substrate, although four products can be produced in the reaction. The within-assay coefficient of variation (CV) ranged from 1.0% to 4.1% and the between-assay CV ranged from 2.6% to 5.3%. The results of our new assay correlate well with those of the amylase assay involving p-nitrophenol maltoheptaoside as substrate (r = 0.978) and with those of the amylase assay involving maltopentaose (r = 0.987).