Isolation, in the intact state, of the pterin molybdenum cofactor from xanthine oxidase.
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.
Deistung
J
J
School of Chemistry and Molecular Sciences, University of Sussex, Brighton, U.K.
Bray
R C
RC
D000818:Animals; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003067:Coenzymes; D004355:Drug Stability; D006863:Hydrogen-Ion Concentration; D008667:Metalloproteins / Q000302:isolation & purification*; D008892:Milk / Q000201:enzymology; D008970:Molecular Weight; D000090006:Molybdenum Cofactors; D011189:Potassium Chloride; D011489:Protein Denaturation; D011621:Pteridines / Q000302:isolation & purification*; D012967:Sodium Dodecyl Sulfate; D012995:Solubility; D012996:Solutions; D014969:Xanthine Oxidase / Q000032:analysis*
D003067:Coenzymes; D008667:Metalloproteins; D000090006:Molybdenum Cofactors; D011189:Potassium Chloride; D011621:Pteridines; D012967:Sodium Dodecyl Sulfate; D012996:Solutions; D014969:Xanthine Oxidase; C022261:molybdopterin