Automated measurement of amylase isoenzymes by a double kinetic assay with "blocked" beta-2-chloro-4-nitrophenyl maltopentaoside as substrate and with wheat germ inhibitor.
We evaluated the enzymic mechanism by which 3-keto butylidene-beta-2-chloro-4-nitrophenyl maltopentaoside (3KB-G5-CNP) serves as a substrate for serum pancreatic (p-) and salivary (s-) amylases. In aliquots of the reaction mixture, three kinds of beta-2-chloro-4-nitrophenyl oligosaccharides (glucose, maltoside, and maltotrioside) were separated from the substrate by high-performance liquid chromatography. Both isoenzymes behaved nearly identically and produced almost the same products. We automated a double kinetic procedure for determining total (t-) and p-amylase with use of a selective inhibitor from wheat germ in a single channel on the Hitachi 7050 analyzer. Within- and between-run CVs were, respectively, 0.5% and 1.7% for t-amylase (240 U/L), and 0.7% and 2.3% for p-amylase (230 U/L). The test results varied linearly with concentrations up to approximately 2000 U/L for t- and p-amylase activities. p/s ratios varied from 0.2 to 5.0. Results correlated well with those obtained by the monoclonal inhibition method (r = 0.992).
Abe
A
A
Department of Laboratory Medicine, Gifu University School of Medicine, Japan.
Nishimura
T
T
Noma
A
A
Hamano
K
K
D000681:Amylases / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D006868:Hydrolysis; D007124:Immunoenzyme Techniques; D007527:Isoenzymes / Q000032:analysis*; D007700:Kinetics; D009844:Oligosaccharides / Q000378:metabolism*; D010179:Pancreas / Q000201:enzymology; D010940:Plant Proteins; D012463:Saliva / Q000201:enzymology; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D014361:Trypsin Inhibitors
D007527:Isoenzymes; D009844:Oligosaccharides; D010940:Plant Proteins; D014361:Trypsin Inhibitors; C051542:wheat germ trypsin inhibitor; C049162:2-chloro-4-nitrophenyl-maltopentaoside; D000681:Amylases