Purification, crystallization, and properties of plastocyanin from a green alga, Enteromorpha prolifera.

Abstract

Plastocyanin was extracted from a green alga, Enteromorpha prolifera, and purified to an electrophoretically homogeneous state. The ratio of plastocyanin to chlorophyll was 1 : 410. The protein was crystallized; this is the first time that algal plastocyanin has been crystallized. The oxidized form showed absorption maxima at 259, 277, 283.5, 460, 597, and 775 nm, and shoulders at around 253, 265, and 269 nm. The extinction coefficient at 597 nm was 4.7 mM-1.cm-1. The best A277/A597 ratio was 1.3. The maximum at 597 nm shifted to 592.5 nm at 77K. The midpoint redox potential of the plastocyanin was +0.369 volt at pH 7.0 and 26 degrees C. A one-electron change was involved. The molecular weight, estimated by gel filtration, was 12,000, though a minor component with a molecular weight of 22,000 was also observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point was at pH 4.1 for the oxidized form. The amino acid composition was: Asp15, Thr6, Ser4, Glu6, Pro5, Gly13, Ala12, Cys1, Val11, Met2, Ile7, Leu4, Tyr3, Phe4, Lys4, His2, Arg1, Trp1, giving a total of 101 residues. Enteromorpha plastocyanin was compared with other plastocyanins from various plants.