Separation of human salivary alpha-amylase isozymes by high-performance liquid chromatography with a continuous monitor system of the activity.
Human salivary alpha-amylase isozymes were rapidly separated from each other by high-performance liquid chromatography with a postcolumn assay. The eluate from the HPLC column was mixed continuously with an intramolecularly quenched fluorescent substrate, p-nitro-phenyl 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 delivered by a pump. The mixture was incubated in a reaction coil, and the fluorescence intensity was continuously measured by a fluorescence detector. The assay was based on the marked increase in fluorescence with the enzymatic cleavage of the glycosidic bond of the substrate that links the fluorogenic and quenching moieties.
Omichi
K
K
Department of Chemistry, Osaka University College of Science, Japan.
Ikenaka
T
T
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005470:Fluorometry; D006801:Humans; D007527:Isoenzymes / Q000302:isolation & purification*; D009578:Nitrobenzenes; D009844:Oligosaccharides; D012463:Saliva / Q000201:enzymology*; D000516:alpha-Amylases / Q000302:isolation & purification*
D007527:Isoenzymes; D009578:Nitrobenzenes; D009844:Oligosaccharides; C049264:4-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; D000516:alpha-Amylases