Abstract
We describe the preparation of a lyophilized material containing purified human pancreatic α-amylase and the certification of its catalytic concentration. The enzyme was purified from human pancreas by ammonium sulphate precipitation and chromatography successively on DEAE-Sephacel, CM-Sepharose and Sephadex G-75. The purified enzyme had a specific activity of 52.9 kU/g protein and was 299% pure on polyacrylamidegel electrophoresis. Only trace amounts of lipase and lactate dehydrogenase were detected in the purified fraction. The purified pancreatic α-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 α-amylase in a matrix containing PIPES buffer 25 mmol/l, pH 7.0, sodium chloride 50 mmol/l, calcium chloride 1.5 mmol/l, EDTA 0.5 mmol/l and human serum albumin 30 g/l, dispensing in ampoules and freeze-drying. The ampoules were homogeneous and the yearly loss of activity on the basis of accelerated degradation studies was less than 0.01% at -20°C. The certified value for α-amylase catalytic concentration in the reconstituted reference material is 555 U/l ± 11 U/l when measured by the specified method at 37°C. The material can be used to verify the comparability of results from different laboratories, for intra-laboratory quality control or for calibration of α-amylase catalytic concentration measurements.
Original language | English |
---|---|
Pages (from-to) | 145-162 |
Journal | Clinica Chimica Acta |
Volume | 251 |
DOIs | |
Publication status | Published - 30 Jul 1996 |
Keywords
- Enzyme activity
- Refeence material
- Standardization