IDENTIFICATION OF TOPAQUINONE, AS ILLUSTRATED FOR PIG-KIDNEY DIAMINE OXIDASE AND ESCHERICHIA-COLI AMINE OXIDASE

Pig kidney diamine oxidase was purified to homogeneity. The reaction p roduct of the cofactor with p-nitrophenylhydrazine (pNPH) was liberate d with pronase treatment and purified, H-1 NMR, uv/vis, and electrospr ay tandem mass spectroscopy revealed it to be a dipeptide with the seq uence topaquinone-pNPH and aspartate. No heterogeneity was observed, i ndicating that no intramolecular cyclization of the quinone moiety occ urs in the time span of the isolation and of the measurements. Similar results were obtained with the more widely applicable reagent, phenyl hydrazine, 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 t he identification of protein-integrated quinone cofactors. (C) 1995 Ac ademic Press, Inc.