Isolation and identification of poplar isoplastocyanins

An improved four-stage isolation and purification procedure for preparing poplar isoplastocyanins is described in detail. Absorbance (UV-VIS) spectroscopy and isoelectric focusing (IEF) are used to determine the protein purity and identity. The present procedure increases twice the total plastocyanin (PC) yield. Four PC isoform fractions are consecutively isolated at the third chromatographic step: oxidized PCa(II) and PCb(II) and reduced PCb(I) and PCa(I). PCa(II) and PCb(II) obtained at the fourth chromatographic step are highly purified PC isoforms which show the purity index (p.i.) A278/A597 < or = 0.85. Isoelectric points (pl values) of the PC isoforms are found to be at pH 3.92 +/- 0.04 for PCa and at pH 3.85 +/- 0.02 for PCb. The results of appropriate biological experiments that include the highly purified poplar PC isoforms could give answers to the questions about the physiological significance of PC dimorphism for photosynthesis.