Extraction of xanthine oxidase from milk by counter-current distribution in an aqueous two-phase system

• Published in: Journal of Chromatography A 1991, Vol.558 (2), p.357-367
• Main Authors: Ortin, Aurora, López-Pérez, Manuel J., Muiño Blanco, María T., Cebrian Perez, José A.
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; centrifugal counter-current distribution; electrophoresis; extraction; Food industries; Fundamental and applied biological sciences. Psychology; General aspects; Methods of analysis, processing and quality control, regulation, standards; milk; xanthine oxidase
• ISSN: 0021-9673
• DOI: 10.1016/0021-9673(91)80003-Y

By comparing the partitioning of total milk protein with that of pure xanthine oxidase, it was established that a two-phase system containing 7% (w/w) Dextran T 500 and 5% (w/w) polyethylene glycol 6000 in the range of 5–25 m M sodium phosphate is suitable for the extraction of the enzyme from milk. In this system, the bulk of milk protein partitioned in the upper phrase, whereas the pure enzyme showed a preferential affinity for the lower phase. The enzyme appears also to be released from the membrane of the milk fat globules as a consequence of the phase partitioning itself, as the enzyme activity (undetectable in the boud form) increased when successive partitionings of milk were carried out. After 57 transfers, centrifugal counter-current distribution (CCCD) of milk in this two-phase system allowed the separation of the xanthine oxidase from the bulk of milk protein. The enzyme thus isolated was mostly resolved as an unique peak, identified densitometrically after sodium dodecyl sulphate polyacrylamide gel electrophoresis. The results obtained show that CCCD of milk in a two-phase system is an useful procedure to achieve the release and isolation of xanthine oxidase from milk.