A study on purified bovine erythrocuprein

Abstract

Erythrocuprein was isolated from bovine blood by precipitation with organic solvents and chromatography (Sephadex G-75 and DEAE-Sephadex A-50). The preparation was electrophoretically pure, had an absorption ratio A 259 nm A 680 nm = 31.5 and contained 2 atoms of copper and 2 atoms of zinc per mole. Preincubation at 45° in the presence of 1% sodium dodecyl sulphate resulted in a striking change of the electrophoretic properties of the protein: The molecular weights of the two bands observed were calculated to be 16 000 (monomeric species) and 64 000 (tetrameric species). Surprisingly, no dimeric form was obtained. However, the monomeric unit was obtained exclusively by preincubation with sodium dodecyl sulphate at 100° for 1 min. Upon cooling of erythrocuprein solutions to 77°K a marked increase of the absorption in the visible region was observed and the shoulder at 430 nm was more distinct. The EPR signal of Cu 2+ in the protein showed axial symmetry. Superhyperfine splittings were observed in the g ⊥ region, indicating possible coordination of the copper to one or more nitrogen atoms. Reductive titration with dithionite gave evidence of a transfer of two electrons per molecule or of one electron per Cu 2+. The optical rotatory dispersion and magnetic optical rotatory dispersion spectra, recorded from 220 to 600 nm, show only one trough (225 nm) or peak (around 225 nm), respectively. In the spectral range from 200 to 250 nm the circular dichroism (CD) spectrum shows only one negative Cotton effect at λ = 208 nm which is modified only slightly in sodium dodecyl sulphate or urea. From these spectral characteristics it is suggested that the helical content of erythrocuprein is low. It is demonstrated that magnetic circular dichroism is a tool for resolving overlapping CD and absorption bands in the region of the copper d → d transition.