Isolation and Identifi cation of Poplar Isoplastocyanins

A variety of methods have been developed for isolation and purifi cation of plastocyanin (PC) from both intact leaves and isolated chloroplasts (Freeman and Guss, 2001; Yokum, 1982). Briefl y, most of these methods utilize solvent extraction and precipitation with acetone or ammonium sulfate, followed by ion-exchange chromatography on DEAE cellulose, and DEAE Sephadex and/ or gel fi ltration. Total PC yields of 6 – 10 mg kg have been achieved, depending on the starting material (Freeman and Guss, 2001). Isolation of PC has been successfully realized by a method elsewhere described (Plesničar and Bendall, 1970). The modifi cation of this method is proved to be important for revealing PC dimorphism in poplar (Dimitrov, 2007). The respective procedure is briefl y described in Dimitrov et al. (1987) and Donchev and Dimitrov (1988). In the present paper we describe an improved variant of the method mentioned above (Dimitrov et al., 1987; Donchev and Dimitrov, 1988). The total PC yield is two times higher than that cited before (Freeman and Guss, 2001). Besides, together with the PCa(II) and PCb(II) fractions two additional PCa(I) and PCb(I) fractions are isolated. PCa(II) and PCb(II) show a higher degree of purity compared to our previous results (Dimitrov et al., 1987; Donchev and Dimitrov, 1988). The purity and the redox states of the fractions were determined by their specifi c UV-VIS absorption spectra. The identity of the protein fractions was found out by isoelectric focusing (IEF).