Production and certification of an enzyme reference material for creatine kinase isoenzyme 2 (CRM 608)

• Published in: Clinica chimica acta 1998-08, Vol.276 (1), p.35-52
• Main Authors: Gella, F.-Javier, Frey, Elena, Ceriotti, Ferruccio, Galán, Amparo, Hadjivassiliou, Anthony G, Hørder, Mogens, Lorentz, Klaus, Moss, Donald W, Schiele, Françoise, Canalias, Francesca
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Catalysis; Chromatography, Ion Exchange; Creatine Kinase - analysis; Electrophoresis, Polyacrylamide Gel; Enzyme activity; Enzyme Stability; Enzymes and enzyme inhibitors; Freeze Drying; Fundamental and applied biological sciences. Psychology; Humans; Isoenzymes; Kinetics; Myocardium - enzymology; Reference material; Reference Values; Standardisation; Transferases
• ISSN: 0009-8981; 1873-3492
• DOI: 10.1016/S0009-8981(98)00097-7

We describe the preparation of a lyophilized material containing purified human creatine kinase 2 (CK-MB), and the certification of its catalytic concentration. The material can be used to verify the comparability of results from different laboratories, for intra-laboratory quality control, or for calibration of the creatine kinase 2 catalytic concentration measurements. The enzyme was purified from human heart by ethanol precipitation and chromatography successively on DEAE–Sephacel and Blue-Sepharose. The purified enzyme had a specific activity of 998.4 U/mg and was >99% pure on polyacrylamide gel electrophoresis. The material was examined for several possible contaminating enzymes, which were found to be absent. The purified creatine kinase 2 had two subunits (B and M) with molecular masses of 43 650 and 41 700 g/mol, respectively, and an isoelectric point at pH 5.8. The material was prepared by diluting the purified creatine kinase 2 in a matrix containing 25 mmol/L PIPES buffer, pH 7.2, 2 mmol/L ADP, 5 mmol/L 2-mercaptoethanol, 154 mmol/L sodium chloride and 50 g/L human serum albumin, dispensing it into vials and freeze-drying. The batch was shown to be homogeneous. The loss of enzyme activity on storage at −20°C is predicted to be less than 0.18% per annum on the basis of accelerated degradation studies. The catalytic concentration of creatine kinase in samples of the reconstituted material is certified to be 67.2±1.8 U/L (1.12±0.03 μkat/L) when measured, at 30°C, by the Recommended Method of the International Federation of Clinical Chemistry.