Novel affinity purification of xanthine oxidase from Arthrobacter M3

An affinity protocol for purification of xanthine oxidase (XOD) from Arthrobacter M3 was developed. The isolation procedure consisted of only three steps, ammonium sulfate precipitation, affinity extraction to exclude the major impurities, and the final refining procedure with DEAE ion-exchange chromatography for removal of minor contaminants. In this affinity preparation, guanine, an analogue of xanthine, was chosen as the affinity ligand, and was coupled with Sepharose 4B through spacers composed of epichlorohydrin and ethylenediamine. Crude protein has been run through ammonium sulfate precipitation and the affinity column, 99.1% of proteins were removed. After DEAE ion-exchange chromatography, the purity of the refined XOD was 97.5% by Native-PAGE analysis. The activity recovery of purified XOD (36.1%) was almost higher than that of other methods reported. Reducing SDS-PAGE analysis showed that the purified XOD (one band in Native-PAGE analysis) showed two polypeptides with the molecular weights ∼35 kDa and ∼100 kDa, respectively. The desorption constant Kd and the theoretical maximum absorption Qmax on the affinity medium were 3.0 μg/ml and 2.2 mg/g medium in absorption analysis.

► A novel affinity protocol for the purification of xanthine oxidase is developed. ► Only three steps successfully purified xanthine oxidase. ► The most important step of this protocol is affinity chromatography. ► The mechanism relies on the affinity interaction between ligand and the enzyme. ► This method has advantage of fewer steps, better recoveries, and higher purity.