Action pattern of human pancreatic alpha-amylase on maltoheptaose, a substrate for determining alpha-amylase in serum.
An enzymatic assay for the determination of alpha-amylase in serum was developed which employed a soluble substrate, maltoheptaose, and a coupled enzymatic indicator reaction consisting of alpha-glucosidase and the hexokinase-glucose-6-phosphate dehydrogenase system. We used high-performance liquid chromatography (HPLC) to establish the action pattern of maltoheptaose under the test conditions: (A) the action pattern of alpha-amylase, (B) that of the combined action of alpha-amylase and alpha-glucosidase. Conductive to this effect was: the availability of pure maltoheptaose and human pancreatic alpha-amylase; the development of an adequate procedure for sample pretreatment (partition chromatography on a mixed-bed ion exchange) and of an HPLC system for separation of substrate and reaction products without interference from by products of the assay (partition chromatography on a cation-exchange column with acetonitrile-water); and the use of a new, very sensitive refractometric detector revealing sugar amounts as low as 40 ng. We derived the following stoichiometric equations: (see formula index).
D000681:Amylases / Q000097:blood*; D055598:Chemical Phenomena; D002621:Chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004796:Clinical Enzyme Tests; D005936:Glucans / Q000378:metabolism*; D007700:Kinetics; D010179:Pancreas / Q000201:enzymology; D010195:Pancreatitis / Q000175:diagnosis*; D000516:alpha-Amylases / Q000097:blood*; D000520:alpha-Glucosidases / Q000097:blood
D005936:Glucans; C031146:maltoheptaose; D000681:Amylases; D000516:alpha-Amylases; D000520:alpha-Glucosidases