title: Identification of topaquinone, as illustrated for pig kidney diamine oxidase and Escherichia coli amine oxidase.
abstract: Pig kidney diamine oxidase was purified to homogeneity. The reaction product of the cofactor with p-nitrophenylhydrazine (pNPH) was liberated with pronase treatment and purified. 1H NMR, uv/vis, and electrospray tandem mass spectroscopy revealed it to be a dipeptide with the sequence topaquinone-pNPH and aspartate. No heterogeneity was observed, indicating that no intramolecular cyclization of the quinone moiety occurs in the time span of the isolation and of the measurements. Similar results were obtained with the more widely applicable reagent, phenylhydrazine, and using the aromatic amine oxidase from Escherichia coli. From the amount and ease with which the dipeptide could be isolated, the procedure used here is more convenient than the existing one for the identification of protein-integrated quinone cofactors.
authors.affiliation: Kluyver Laboratory of Biotechnology, Delft University of Technology, The Netherlands.
authors.forename: V
authors.initials: V
authors.lastname: Steinebach
authors.forename: B W
authors.initials: BW
authors.lastname: Groen
authors.forename: S S
authors.initials: SS
authors.lastname: Wijmenga
authors.forename: W M
authors.initials: WM
authors.lastname: Niessen
authors.forename: J A
authors.initials: JA
authors.lastname: Jongejan
authors.forename: J A
authors.initials: JA
authors.lastname: Duine
mesh_terms: D006631:Amine Oxidase (Copper-Containing) / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D000818:Animals; D016384:Consensus Sequence; D004295:Dihydroxyphenylalanine / Q000031:analogs & derivatives* / Q000032:analysis; D004926:Escherichia coli / Q000201:enzymology*; D007202:Indicators and Reagents; D007668:Kidney / Q000201:enzymology*; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D015394:Molecular Structure; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000302:isolation & purification*; D010659:Phenylhydrazines; D013053:Spectrophotometry / Q000379:methods; D013552:Swine
chemical_list: D007202:Indicators and Reagents; D010659:Phenylhydrazines; C005307:4-nitrophenylhydrazine; D004295:Dihydroxyphenylalanine; C067626:6-hydroxydopa quinone; D006631:Amine Oxidase (Copper-Containing); D000587:Oxidoreductases Acting on CH-NH Group Donors