Single step affinity Chromatographic purification of human α-amylase from aspirated duodenal juice and its application in the measurement of pancreatic α-amylase synthesis rates in man

Human α-amylase was purified from aspirated duodenal juice to electrophoretic homogeneity in a single step by affinity chromatography with the competitive inhibitor acarbose (IC50 = 1.22 μmol/l) as ligand. Duodenal juice was applied to an agarose resin to which acarbose had been coupled covalently via a 1.9 nm spacer group. Pure α-amylase, eluted with free acarbose, had a molecular mass of 55 000, and isoelectrofocussing revealed the presence of six isozymes with pI values of 7.3, 6.8, 6.7, 6.5, 6.4 and 6.3, all of which possessed amylase activity based on positive starch/iodine staining. The potential usefulness of this one-step purification procedure in the measurement of pancreatic α-amylase synthesis rates was evaluated in two control patients with non-pancreatic disease. Aspirated duodenal juice was obtained during a pulse/continuous intravenous 4 h infusion of [14C]leucine together with secretin and pancreozymin, and α-amylase purified using our protocol. Pancreatic α-amylase synthesis rates were determined from the rate of incorporation of [14C]leucine into α-amylase; values of 4.4 and 5.1 h were obtained for the two control patients. © 1989.