PRODUCTION AND CERTIFICATION OF AN ENZYME REFERENCE MATERIAL FOR PANCREATIC ALPHA-AMYLASE (CRM-476)

We describe the preparation of a lyophilized material containing purif ied human pancreatic a-amylase and the certification of its catalytic concentration. The enzyme was purified from human pancreas by ammonium sulphate precipitation and chromatography successively on DEAE-Sephac el, CM-Sepharose and Sephadex G-75. The purified enzyme had a specific activity of 529 kU/g protein and was >99% pure on polyacrylamide gel electrophoresis. Only trace amounts of lipase and lactate dehydrogenas e were detected in the purified fraction. The purified pancreatic alph a-amylase had a molar mass of 57 500 g/mol and an isoelectric point at 7.1. The material was prepared by diluting the purified alpha-amylase in a matrix containing PIPES buffer 25 mmol/l, pH 7.0, sodium chlorid e 50 mmol/l, calcium chloride 1.5 mmol/l, EDTA 0.5 mmol/l and human se rum albumin 30 g/l, dispensing in ampoules and freeze-drying. The ampo ules were homogeneous and the yearly loss of activity on the basis of accelerated degradation studies was less than 0.01% at -20 degrees C. The certified value for alpha-amylase catalytic concentration in the r econstituted reference material is 555 U/l +/- 11 U/l when measured by the specified method at 37 degrees C. The material can be used to ver ify the comparability of results from different laboratories, for intr a-laboratory quality control or for calibration of alpha-amylase catal ytic concentration measurements.