Differential rate assay of human pancreatic and salivary alpha-amylases in serum using two coupled enzymes.

p-Nitrophenyl O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl-(1----4)-O-alpha- D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranosyl-(1----4)-alpha-D-glucopyranoside (FG5P) is hydrolyzed by human pancreatic a-amylase (HPA) or salivary alpha-amylase (HSA) to O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D- glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-D-glucose (FG3) and p-nitrophenyl alpha-maltoside or to O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl-(1----4)-O-alpha- D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-D-glucose (FG4) and p-nitrophenyl alpha-glucoside. The use of alpha-D-glucosidase (maltase) [EC 3.2.1.20] of Saccharomyces carlsbergensis and oligo-1,6-glucosidase (isomaltase) [EC 3.2.1.10] of bakers' yeast as coupled enzymes differentiates between the two reactions, because alpha-D-glucosidase liberates p-nitrophenol from both p-nitrophenyl alpha-glucoside and p-nitrophenyl alpha-maltoside, but oligo-1,6-glucosidase liberates it only from p-nitrophenyl alpha-glucoside. HPA produces more FG4 and p-nitrophenyl alpha-glucoside than HSA. Taking advantage of the differences in the action of the two amylases and in the substrate specificity of the coupled enzymes, we have developed a new colorimetric differential rate assay of alpha-amylases in human serum.