Purification and Some Properties of Spinach Plastocyanin

Abstract

1. Plastocyanin, a non-autooxidizable copper protein, has been isolated from spinach leaves and extensively purified by means of ammonium sulfate fractionation and column chromatography using diethylaminoethyl cellulose.
2. Electrophoretic analysis indicated an acidic nature of the protein, with an isoelectric point below pH 4.0. The molecular weight was calculated to be 21, 000 from the values for the sedimentation and diffusion constants.
3. Oxidized plastocyanin shows three absorption bands in the visible and far red regions at 460, 597 and 770mμ, which completely disappear on reduction. The absorption spectrum in the ultraviolet region was characterized by the presence of vibrational fine structure bands due to the amino acid constituents of the protein.
4. Plastocyanin shows a constant oxidation-reduction potential of 0.37 volt. between pH 5.4 and 9.9. At more acidic pH below pH 5.4, it increases at a rate of 0.06 volt. per pH.
5. Plastocyanin contains 0.58 per cent of copper, which gives a minimal molecular weight of 10, 800. The blue color of the oxidized protein is found to. be due to the copper in a cupric combination with the protein moiety.
6. The amino acid composition of plastocyanin was determined. It was found that the protein contains glycine, glutamic and aspartic acids in a large amount, but lacks arginine and tryptophan. A little amount of carbohydrate was also detected in the acid hydrolyzate of the protein.
This paper is dedicated to Prof. Hiroshi Tamiya to celebrate his 60th birthday, the 5th January, 1963.
The authors wish to express their cordial thanks to Dr. K. Iwai of this department, for his kind help in conducting the amino acid analysis.