title: Production and certification of an enzyme reference material for pancreatic alpha-amylase (CRM 476).
abstract: We describe the preparation of a lyophilized material containing purified human pancreatic alpha-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 > 99% pure on polyacrylamide gel electrophoresis. Only trace amounts of lipase and lactate dehydrogenase were detected in the purified fraction. The purified pancreatic alpha-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 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 degrees C. The certified value for alpha-amylase catalytic concentration in the reconstituted 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 verify the comparability of results from laboratories, for intra-laboratory quality control or for calibration of alpha-amylase catalytic concentration measurements.
authors.affiliation: Department de Bioquímica i Biologia Molecular, Universitat Autonòma de Barcelona, Spain.
authors.forename: G
authors.initials: G
authors.lastname: Gubern
authors.forename: F
authors.initials: F
authors.lastname: Canalias
authors.forename: F J
authors.initials: FJ
authors.lastname: Gella
authors.forename: E
authors.initials: E
authors.lastname: Colinet
authors.forename: C
authors.initials: C
authors.lastname: Profilis
authors.forename: D H
authors.initials: DH
authors.lastname: Calam
authors.forename: F
authors.initials: F
authors.lastname: Ceriotti
authors.forename: J
authors.initials: J
authors.lastname: Dufaux
authors.forename: A G
authors.initials: AG
authors.lastname: Hadjivassiliou
authors.forename: J M
authors.initials: JM
authors.lastname: Lessinger
authors.forename: K
authors.initials: K
authors.lastname: Lorentz
authors.forename: A
authors.initials: A
authors.lastname: Vassault
mesh_terms: D002384:Catalysis; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D005612:Freeze Drying; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D010179:Pancreas / Q000737:chemistry / Q000201:enzymology*; D012015:Reference Standards; D013056:Spectrophotometry, Ultraviolet; D013997:Time Factors; D000516:alpha-Amylases / Q000737:chemistry / Q000302:isolation & purification*
chemical_list: D007202:Indicators and Reagents; D000516:alpha-Amylases