title: Novel affinity purification of xanthine oxidase from Arthrobacter M3.
abstract: 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 ∼35kDa and ∼100kDa, respectively. The desorption constant K(d) and the theoretical maximum absorption Q(max) on the affinity medium were 3.0μg/ml and 2.2mg/g medium in absorption analysis.
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China.
authors.forename: Yuran
authors.initials: Y
authors.lastname: Zhang
authors.forename: Yu
authors.initials: Y
authors.lastname: Xin
authors.forename: Hailin
authors.initials: H
authors.lastname: Yang
authors.forename: Ling
authors.initials: L
authors.lastname: Zhang
authors.forename: Xiaole
authors.initials: X
authors.lastname: Xia
authors.forename: Yanjun
authors.initials: Y
authors.lastname: Tong
authors.forename: Yi
authors.initials: Y
authors.lastname: Chen
authors.forename: Li
authors.initials: L
authors.lastname: Ma
authors.forename: Wu
authors.initials: W
authors.lastname: Wang
mesh_terms: D000327:Adsorption; D000595:Amino Acid Sequence; D000645:Ammonium Sulfate / Q000737:chemistry; D001173:Arthrobacter / Q000737:chemistry / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification*; D011232:Chemical Precipitation; D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D006147:Guanine / Q000737:chemistry; D008969:Molecular Sequence Data; D014969:Xanthine Oxidase / Q000737:chemistry / Q000302:isolation & purification*
chemical_list: D001426:Bacterial Proteins; D006147:Guanine; D014969:Xanthine Oxidase; D000645:Ammonium Sulfate