Simple, high-yield purification of xanthine oxidase from bovine milk
Research output: Contribution to journal › Journal article › Research › peer-review
Xanthine oxidase, a commercially important enzyme with a wide area of application, was extracted from fresh milk, without added preservatives, using toluene and heat. The short purification procedure, with high yield, consisted of extraction, ammonium sulfate fractionation, and DEAE-Sepharose (fast flow) column chromatography. Xanthine oxidase was eluted as a single activity peak from the column using a buffer gradient. The purification fold, specific activity and yield for the purified xanthine oxidase were 328, 10.161 U/mg and 69%, respectively. The enzyme was concentrated by ultrafiltration, although 31% of the activity was lost during concentration, no change in specific activity was observed. Activity and protein gave coincident staining bands on native polyacrylamide gels. The intensity and the number of bands were dependent on the oxidative state(s) of the enzyme; reduction by 2-mercaptoethanol decreased the intensity of the slow-moving bands and increased the intensity of the fastest-moving band. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), two major bands (molecular masses of 152 and 131 kDa) were observed, accounting for > or = 95% of xanthine oxidase. Native- and SDS-PAGE showed that the purified xanthine oxidase becomes a heterodimer due to endogenous proteases.
Original language | English |
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Journal | Journal of Biochemical and Biophysical Methods |
Volume | 39 |
Issue number | 3 |
Pages (from-to) | 153-9 |
Number of pages | 6 |
ISSN | 0165-022X |
Publication status | Published - 1999 |
Externally published | Yes |
Bibliographical note
Keywords: Animals; Chemical Fractionation; Dialysis; Electrophoresis, Polyacrylamide Gel; Milk; Ultrafiltration; Xanthine Oxidase
ID: 12899130