Production and certification of an enzyme reference material for pancreatic alpha-amylase (CRM 476).
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.
Gubern
G
G
Department de Bioquímica i Biologia Molecular, Universitat Autonòma de Barcelona, Spain.
Canalias
F
F
Gella
F J
FJ
Colinet
E
E
Profilis
C
C
Calam
D H
DH
Ceriotti
F
F
Dufaux
J
J
Hadjivassiliou
A G
AG
Lessinger
J M
JM
Lorentz
K
K
Vassault
A
A
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*
D007202:Indicators and Reagents; D000516:alpha-Amylases