Optimized conditions for determining activity concentration of alpha-amylase in serum, with 1,4-alpha-D-4-nitrophenylmaltoheptaoside as substrate.
We describe a method for measuring the catalytic activity of alpha-amylase (EC 3.2.1.1) in serum and urine, by use of a defined substrate: 1,4-alpha, D-4-nitrophenyl maltoheptaoside. We use a phosphate buffer of pH 7.10, containing chloride as activator and alpha-glucosidase (EC 3.2.1.20) as the auxiliary enzyme. After a lag phase of 4 min at 25 degrees C or 30 degrees C, or 3 min at 37 degrees C, the increase of absorption of 4-nitrophenol is measured at 410 nm or 405 nm. The pH value of the assay mixture is a compromise between optimum pH for the alpha-amylase reaction, shortest possible lag phase, and an acceptable absorptivity of 4-nitrophenol. Because the dissociation of 4-nitrophenol depends strongly on pH and temperature, we determined its absorptivity with various combinations of these variables in the assay. Heparin-treated plasma can be used, but not EDTA, fluoride, or citrate. Lipemia, hemoglobin less than or equal to mumol/L, bilirubin less than or equal to 170 mumol/L, glucose less than or equal to 100 mmol/L, and ascorbic acid less than or equal to 1 mmol/L of sample do not interfere in the assay.
D002021:Buffers; D005069:Evaluation Studies as Topic; D005960:Glucosides / Q000378:metabolism*; D006027:Glycosides / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000097:blood; D010179:Pancreas / Q000201:enzymology; D012463:Saliva / Q000201:enzymology; D013053:Spectrophotometry; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors; D000516:alpha-Amylases / Q000097:blood* / Q000652:urine
D002021:Buffers; D005960:Glucosides; D006027:Glycosides; D007527:Isoenzymes; C036167:4-nitrophenyl maltoheptaoside; D000516:alpha-Amylases