Action pattern of human pancreatic and salivary alpha-amylase on 1,4-alpha-D-nitrophenylmaltooligosaccharides. 1,4-alpha-D-nitrophenylmaltooligosaccharides as substrates of alpha-amylse, I.
High performance liquid chromatography (HPLC) was used to monitor the purity of the substrates and to establish the patterns of hydrolysis of ortho- and para-nitrophenylmaltooligosaccharides (2-7 glucose residues) catalysed by human pancreatic and salivary alpha-amylase. Separation of the reaction products from the remaining substrate was performed on a TSK-G-2000 PW or a RP18 column. By measuring the quantitative distribution of products, and assuming a 5-subsite model for the active site of alpha-amylase, differential activities for the hydrolysis of the different glycosidic bonds in the 2 series of substrates were deduced. A highly sensitive coupled continuous assay system is based on the formation of phenyloligosaccharides with 1-4 glucose residues by the action of the amylase under test, coupled to hydrolysis of these products by yeast alpha-glucosidase. The most suitable test substrates were shown to be para-nitrophenyl-alpha-D-maltotetraoside and -pentaoside. Direct production of nitrophenol from ortho-nitrophenyl-alpha-D-maltotrioside is recommended for the measurement of the alpha-amylase activity of pancreatic and salivary gland secretions and extracts.
D000681:Amylases / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D009844:Oligosaccharides / Q000378:metabolism*; D010179:Pancreas / Q000201:enzymology*; D012463:Saliva / Q000201:enzymology*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D000516:alpha-Amylases / Q000032:analysis* / Q000378:metabolism; D000520:alpha-Glucosidases
D009844:Oligosaccharides; D000681:Amylases; D000516:alpha-Amylases; D000520:alpha-Glucosidases