Determination of alpha-amylase using a new blocked substrate (3-ketobutylidene beta-2-chloro-4-nitrophenyl-maltopentaoside).

A new substrate, 3-ketobutylidene beta-2-chloro-4-nitrophenylmaltopentaoside (3KB-CNPG5), was used for the determination of alpha-amylase (EC 3.2.1.1) in serum and urine. Under this alpha-amylase assay condition, 3KB-CNPG5 is resistant to glucoamylase and alpha-glucosidase, which are auxiliary enzymes, because the 4- and 6-positions of the non-reducing-end glucose residue are modified by the 3-ketobutylidene group. The assay using 3KB-CNPG5 for alpha-amylase activity is a highly sensitive method that uses 2-chloro-4-nitrophenol (CNP) as an aglycone, and is a stable method for determination of alpha-amylase activity in biological fluids.