A study on purified apo-erythrocuprein

Abstract

Apo-erythrocuprein was prepared by passing freshly isolated bovine erythrocuprein through an EDTA-equilibrated Sephadex G-25 column at pH 3.8 for 8–10 h. The apoprotein proved to be homogeneous and metal free as examined by several physicochemical methods. The fine structure of the ultraviolet absorption spectrum was very distinct. The molar coefficient of absorption at 259 nm was only about one third ( ϵ 259 = 3670) of the corresponding value of the native protein ( ϵ 259 = 9840). The apoprotein was electrophoretically homogeneous and displayed one symmetrical boundary as determined by sedimentation velocity measurements. The s 20, w value was 3.0 S. Absolutely no Zn 2+ and Cu 2+ were detected either with atomic absorption spectroscopy or, in the case of Cu 2+, using electron spin resonance (EPR). The apoprotein was fairly stable in the pH range of 2.5–9.5, provided the exposure to the more extreme pH values did not exceed 5 min. From magnetic circular dichroism (MCD) data it was concluded that the protein contained no tryptophan. Furthermore, MCD measurements were found to be an elegant method for the detection of 2 moles of tyrosine per mole of apo-erythrocuprein. The reconstitution of erythrocuprein by incubating the apoprotein together with Cu 2+ and/or Zn 2+ was successful under both aerobic and anaerobic conditions. However, the quality of the aerobic reconstituted erythrocuprein was not fully satisfactory as shown by absorption spectra in the visible region, EPR and enzyme assay. From optical titrations of the apoprotein using Cu 2+ and Zn 2+ it was concluded that Cu 2+ is able to produce both a profound increase of ultraviolet absorption and a change of the absorption profile, while Zn 2+ was less active. However, Zn 2+ was able to cause a shift of the visible absorption maximum by 50 nm. From enzymic studies using the reduction of cytochrome c by O 2 − it was concluded that 4 metal ions, two of which must be Cu 2+, are required for optimal superoxide dismutase activity. No measurable differences between the native and the reconstituted protein were apparent at all if the reconstitution was performed anaerobically using a second Sephadex G-25 column which contained Cu 2+ and Zn 2+ in the upper section.