On the Interaction of the Electron Acceptor 2,6-Dichlorophenolindophenol with Bovine Milk Xanthine Oxidase

Abstract

Abstract The electron acceptor 2,6-dichlorophenolindophenol (DCI) forms a spectrophotometrically and kinetically detectable complex with bovine milk xanthine oxidase. The visible absorption spectrum of complexed DCI exhibits an absorption maximum at 660 nm at pH 7.8 (e = 34.2 x 103 m-1 cm-1), a marked bathochromic shift from the visible absorption maximum at 608 nm characteristic of the free dye (e = 21.0 x 103 m-1 cm-1. The bound dye can be partially removed from the enzyme by prolonged dialysis (24 hours), and totally removed by gel filtration, indicating a reversible rather than a covalent interaction. From the DCI concentration dependence of the spectral change, the stoichiometry of the interaction is 2.08 DCI-binding sites per molecule of xanthine oxidase (assuming a molar extinction coefficient for the enzyme of 66 x 103 m-1 cm-1 at 450 nm); and the dissociation constant of the DCI-enzyme complex is 1.3 x 10-6 m. The spectral shift characteristic of DCI-xanthine oxidase interaction is blocked by 2 mm arsenite, by 2 mm p-hydroxymercuribenzoate, by the competitive inhibitor 2,4-diamino-6-hydroxy-s-triazine, 1.5 mm, and by the stoichiometric xanthine oxidase inhibitor, 4,6-dihydroxypyrazolo[3,4-d]pyrimidine (4,6-diHPP), at levels equimolar with those of the enzyme. With the latter inhibitor, the DCI spectral shift, although not present initially, develops concomitantly with reoxidation of the reduced enzyme molybdenum of the 4,6-diHPP-xanthine oxidase complex. In contrast to the results seen with the other xanthine oxidase inhibitors, the DCI-xanthine oxidase interaction is not blocked by 16 mm sodium cyanide. In kinetic studies, DCI, in addition to acting as an electron acceptor, is also a noncompetitive inhibitor of the enzyme, with a Ki of 2.8 x 10-6 m. Prior incubation with DCI protects the enzyme from inhibition by arsenite, but not from inhibition by cyanide. On the basis of the spectrophotometric and kinetic data, it is proposed that the DCI-binding site is the molybdenum or molybdenum-sulfur component of the internal electron transport chain of the enzyme.