Determination of α-amylase using 4- O-β- d-galactopyranosylmaltotetraose (Gal-G4) as a substrate

A new substrate, 4- O-β- d-galactopyranosylmaltotetraose (Gal-G4) is applied for the determination of α-amylase in serum and urine in a coupled assay with α-glucosidase (EC 3.2.1.20), glucokinase (EC 2.7.1.2) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) as auxiliary enzymes. Gal-G4 having a 4-position of the non-reducing-end glucose residue modified by a β-galactopyranose group is resistant for degradation by α-glucosidase as auxiliary enzyme. Moreover, this substrate is hydrolyzed at just one position by α-amylase in serum and urine. More than 99% of the products generated from Gal-G4 by α-amylase are identified 4- O-β- d-galactopyranosylmaltose (Gal-G2), maltose, respectively. Glucose and maltose do not interfere the value of α-amylase activity at least up to 0.056 mmol/l (1 g/dl) glucose and 0.027 mmol/l (1 g/dl) maltose, respectively. We are now carrying out this work under the authority of The Enzyme committee of Japanese Society of Clinical Chemistry (JSCC) as a standard method for determination of α-amylase in clinical chemistry.