Rapid Determinationof a-Amylase Activityby Use of a New ChromogenicSubstrate

A new chromogenic substrate that is blocked at the nonreducing end, 4,6-benzylidene-a-D-4-nitrophenylmaltoheptaoside, is used to determine a-amylase (EC 3.2.1.1) activity in serum in a coupled assay with a-glucosidase (EC 3.2.1.20) and glucoamylase (EC 3.2.1.3) as auxiliary enzymes. The duration of the lag phase between 25 and 37#{176}C is <90 s, and the molar absorptivity of 4-nitrophenol is constant. The main cleavage product of the substrate by human pancreatic and salivary a-amylase is 4-nitrophenylmaltoside; in the presence of the auxiliary enzymes, >95% of hydrolyzed substrate is accounted for as 4-nitrophenol. The combined reagent is stable for at least 20 days at 2-8 #{176}C; precision is good, with CVs ranging from 1.7 to 3.3%; and the correlation of results with those by the 4-nitrophenylmaltoheptaoside method is excellent. Hepann (40 kilo-mt. units/L), ascorbic acid (2.8 mmol/L), bilirubin (430 pnloI/L), hemoglobin (170 /.Lmol/L),glucose (55 mmol/L), and triglycerides (11 mmol/L) do not interfere in the assay.