Bathocuproine Sulfonate Research Articles

The effects of copper depletion on structural aspects of cytochrome c oxidase.

The removal of copper from beef heart cytochrome c oxidase by either dialysis against potassium cyanide or by treatment with bathocuproine sulfonate produced changes in the enzyme which are indicative of a spin state transition. In the Soret region of the CD spectrum copper depletion of the enzyme caused a significant decrease in amplitude in combination with a red shift of the peak maximum for oxidized samples, while reduced copper-depleted samples exhibited decreased amplitude and a blue shift of the peak maximum. In the magnetic CD spectra of oxidized copper-depleted samples the peak at 420 nm was shifted to lower wave-length along with a significant increase in amplitude. In reduced samples the peak at 446 nm exhibited a slight red shift concomitant with a substantial decrease in amplitude. The conformational changes indicated by the CD and magnetic CD spectra when copper is removed from the enzyme were supported by the EPR spectra of the NO complex of the reduced copper-depleted enzyme. The removal of copper from cytochrome c oxidase caused the NO complex to exhibit a 3-line splitting pattern of gz in the EPR spectrum instead of the 9 lines seen in the NO complex of the native enzyme. When [15N]NO was used, a 2-line pattern was seen at gz when copper was removed from the enzyme. The changes in the CD and magnetic CD spectra and in the EPR spectra of the NO derivatives of cytochrome c oxidase can be explained by the rearrangement of the axial ligands to iron in cytochrome a3 as a result of copper depletion. These results emphasize the close structural interdependence of the metallic components of this enzyme.

Detection of copper on polyacrylamide gels

A color test for the localization of copper on polyacrylamide gels is described. The test is based upon the quenching of fluorescence of bathocuproine sulfonate by Cu 1+ and is sensitive to 0.1 nmol of free or protein-bound copper. There is no false positive reaction with 10 nmol of hemeprotein, free Fe 3+, Fe 2+, Co 2+, or Mn 2+.

Reversibility of the cyanide inhibition of electron transport in spinach chloroplast thylakoids

The prior treatment of thylakoids with cyanide (30 mM) was shown to inhibit plastocyanin-dependent electron transport reactions. We find that cyanide inhibition of electron flow from either water or diaminodurene to methyl viologen, but not from water to ferricyanide, is partially reversed when the thylakoids are collected by centrifugation and resuspended in a cyanide-free medium. However, methyl viologen reduction in thylakoids pretreated with cyanide is sensitive to cyanide (∼1 mM) added to the reaction mixtures, whereas that in control thylakoids is unaffected. The cyanide must be added in the dark. Electron transport to methyl viologen in chloroplasts pretreated with cyanide is also sensitive to inhibition by EDTA and bathocuproine sulfonate. Thus, KCN inhibition of electron transport in thylakoids is partially reversible. Moreover, the accessibility of plastocyanin to various reagents is probably altered by the KCN treatment.

Noninvolvement of Copper in the l-Tryptophan 2,3-Dioxygenase Reaction

Abstract l-Tryptophan 2,3-dioxygenase, a hemoprotein, purified to near homogeneity from cells of Pseudomonas fluorescens, has been shown to contain only trace amounts of copper. During the purification procedure, the heme content of the enzyme preparation increased in parallel with the specific catalytic activity, whereas that of copper decreased. The level of copper present in the highly purified enzyme preparations was about one-tenth that of the heme, indicating that the copper found was adventitious. The inhibitory effect of copper-chelating agents such as sodium diethyldithiocarbamate and bathocuproine sulfonate on the enzyme activity was not due to their specific chelating action but was attributable to their unforeseen properties as a hydrogen peroxidetrapping reagent and a nonspecific inhibitor, respectively. The results are thus incompatible with the view that l-tryptophan 2,3-dioxygenase is a copper-hemoprotein.

Spectrophotometric study of the reaction of 3-(2-pyridyl)-5,6-bis(4-phenylsulfonic acid)-1,2,4-triazine with iron and copper

A modified procedure is presented for the sequential determination of copper and iron in a single aqueous aliquot. Copper is measured specifically as the bathocuproine sulfonate complex, then the iron color is formed in the same solution as the 3-(2-pyridyl)-5,6-bis(4-phenylsulfonic acid)-1,2,4-triazine complex. This simple procedure for the determination of two biologically related trace metals can be applied for serum analysis.