Liquid chromatographic assay of the relative activities of serum pancreatic and salivary alpha-amylase using reductively pyridylaminated maltopentaose as a fluorescent substrate.
A method for the high-performance liquid chromatographic assay of the relative activities of serum pancreatic and salivary alpha-amylase has been developed, using maltopentaose reductively aminated with 2-aminopyridine as a fluorescent substrate. Both enzymes showed similar modes of action, cleaving the second and the third (from the non-reducing terminal) interglycosidic linkages of this substrate. However, the relative ease of cleavage of these two sites by the pancreatic enzyme was significantly different from that by the salivary enzyme. Therefore, determination of the molar ratio of the cleavage products by HPLC could lead to estimation of the activity ratio of these enzymes. The optimum chromatographic conditions for HPLC were as follows: column, LiChrosorb RP-18 (Merck, 7 microns, 250 X 4 mm I.D.); column temperature, ambient; eluent, 0.01% orthophosphoric acid-acetonitrile (4:1, v/v) containing 2.4 mM sodium laurylsulphate; flow-rate, 1.0 ml/min; wavelengths for fluorimetric detection, 320 nm (excitation)/400 nm (emission). The problem of interference by serum alpha-glucosidase was solved by specific inhibition with tris (hydroxymethyl) aminomethane and erythritol. The data obtained by the proposed method correlated well with those produced by the conventional method based on electrophoresis.
D000631:Aminopyridines; D002851:Chromatography, High Pressure Liquid; D005456:Fluorescent Dyes; D005959:Glucosidases / Q000037:antagonists & inhibitors; D006801:Humans; D009844:Oligosaccharides; D010179:Pancreas / Q000201:enzymology*; D012463:Saliva / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D000516:alpha-Amylases / Q000097:blood*
D000631:Aminopyridines; D005456:Fluorescent Dyes; D009844:Oligosaccharides; C019193:maltopentaose; D005959:Glucosidases; D000516:alpha-Amylases