A procedure is described for isolation of the pterin molybdenum cofactor, in the active molybdenum-containing state, starting from purified milk xanthine oxidase. The method depends on the use of anaerobic-glove-cabinet techniques and on working in aqueous solution, in the presence of 1 mM-Na2S2O4. SDS was used to denature the protein, followed by ion-exchange chromatography and gel filtration. The cofactor, obtained at concentrations up to 0.5-1.0 mM, was fully active in the nit-1 assay [Hawkes & Bray (1984) Biochem. J. 214, 481-493], with a specific activity of 22 nmol of NO2-/min per pg-atom of Mo (with 15% molybdate-dependence). The Mr, determined by gel filtration, was about 610, consistent with the structure proposed by Kramer, Johnson, Ribeiro, Millington & Rajagopalan [(1987) J. Biol. Chem. 262, 16357-16363]. At pH 5.9, under anaerobic conditions, the cofactor was stable for at least 300 h at 20-25 degrees C.
Research Article|October 15 1989
Isolation, in the intact state, of the pterin molybdenum cofactor from xanthine oxidase
Biochem J (1989) 263 (2): 477-483.
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J Deistung, R C Bray; Isolation, in the intact state, of the pterin molybdenum cofactor from xanthine oxidase. Biochem J 15 October 1989; 263 (2): 477–483. doi: https://doi.org/10.1042/bj2630477
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