Rapid determination of alpha-amylase activity by use of a new chromogenic substrate.
A new chromogenic substrate that is blocked at the nonreducing end, 4,6-benzylidene-alpha-D-4-nitrophenylmaltoheptaoside, is used to determine alpha-amylase (EC 3.2.1.1) activity in serum in a coupled assay with alpha-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 degrees C is less than 90 s, and the molar absorptivity of 4-nitrophenol is constant. The main cleavage product of the substrate by human pancreatic and salivary alpha-amylase is 4-nitrophenylmaltoside; in the presence of the auxiliary enzymes, greater than 95% of hydrolyzed substrate is accounted for as 4-nitrophenol. The combined reagent is stable for at least 20 days at 2-8 degrees 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. Heparin (40 kilo-int. units/L), ascorbic acid (2.8 mmol/L), bilirubin (430 mumol/L), hemoglobin (170 mumol/L), glucose (55 mmol/L), and triglycerides (11 mmol/L) do not interfere in the assay.
D005087:Glucan 1,4-alpha-Glucosidase / Q000378:metabolism; D005960:Glucosides; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009596:Nitrophenols; D010179:Pancreas / Q000201:enzymology; D012463:Saliva / Q000201:enzymology; D000516:alpha-Amylases / Q000097:blood*; D000520:alpha-Glucosidases / Q000378:metabolism
D005960:Glucosides; D009596:Nitrophenols; C036988:4-nitrophenylmaltoside; D000516:alpha-Amylases; D000520:alpha-Glucosidases; D005087:Glucan 1,4-alpha-Glucosidase; C024836:4-nitrophenol