Purification and properties of a pink copper protein from human erythrocytes.

A previously undetected pink copper protein has been isolated from human erythrocytes and purified by chromatographic procedures. The purified protein sedimented as a single symmetrical peak in the ultracentrifuge but minor impurities were detected by polyacrylamide disc electrophoresis. The apparent molecular weight was calculated to be approximately 32,000 by both Bio-Gel P-60 chromatography and sedimentation velocity centrifugation. The presence of cupric ions was established by reaction with 2,2’biquinoline and diethyldithiocarbamate. Electron paramagnetic resonance spectroscopy indicated the presence of cupric ion in an environment of tetragonal symmetry. The visible spectrum, with absorbance maxima located at 372, 503, and 7’60 mp, disappeared upon reduction of the protein with sodium dithionite or cysteine. These spectral properties are similar to those of a number of the amine oxidases and the fluorescence spectra of the oxidized and reduced forms suggested that the protein contains an additional chromophoric group, possibly pyridoxal. Studies of the reaction of the protein with phenylhydrazine and sodium diethyldithiocarbamate further suggested the close proximity of this group and the cupric ion. The pink copper protein exhibited no activity in assays for amine oxidases, &aminolevulinic acid dehydrase, lipoyl dehydrogenase, glutathione peroxidase, glutathione reductase, dichloroindophenol reductase, or porphobilinogen deaminase. The pink copper protein was distinguished from erythrocuprein by its spectral, electrophoretic, and chromatographic properties and by its inability to catalyze the dismutation of the superoxide anion.