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.

Oxidation of oxymyoglobin by poplar plastocyanins a and b.

Oxidation of oxymyoglobin [MbO2(Fe2+)] by isoplastocyanins a (PCa) and b (PCb) was experimentally investigated and the corresponding redox reaction was modeled using the physicochemical parameters of the isoforms to study the effect of the dimorphism. The kinetic curve of oxidation of MbO2 (Fe2+) by oxidized PCa [PCa(Cu2+)] and PCb [PCb(Cu2+)] and the pH-dependence of the rate constant kI were determined. In the range of pH 4.8-9.0, PCb reacts with higher k1, compared with PCa. For example, at pH 7.0, k1(PCb) = 4 x 10(2) M(-1)s(-1), whereas k1(PCa)= 2 x 10(2) M(-1)S(-1). The observed values of deltaE(0) for the reaction pairs Mb-PCa and Mb-PCb were -304 mV and -319 mV, respectively. The effect of the ionic strength (mu) on the rate of the electron transfer was also studied. It was found that: (i) the net charge Z1 of PCa and PCb fully corresponds to that calculated by their primary structures and Z2 of Mb corresponds to that calculated by its titration curve; (ii) the In k as function of mean square of mu was similar for both PCa and PCb; (iii) the curve of the reaction PCb <----(e(-1)) Mb (pH 7.0) wasshifted towards higher values of k, in agreement with the larger net negative charge of PCb; and (iv) the character of the electrostatic interactions remained unchanged by a replacement of PCa by PCb and by the change of pH from 7.0 to 4.8.

Some physicochemical peculiarities of poplar plastocyanins a and b.

The redox potentials of poplar plastocyanins a and b (PCa, PCb) were determined by spectrophotometric titrations of their reduced forms with [Fe(CN)6]3-. It was found that the two isoforms have the following millimolar extinction coefficients epsilon597 equilibrium constants Keq of one-electron exchange with [Fe(CN)6]4-/[Fe(CN)6]3-, and standard electron potentials E0: PCa: epsilon597 = (4.72 +/- 0.08) mM(-1) cm(-1), Keq = 0.133 +/- 0.009, E0' = (354 +/- 11) mV; PCb: epsilon597 = (5.23 +/- 0.16) mM(-1) cm(-1), Keq = 0.175 +/- 0.010, E0' = (363 +/- 12) mV. The pH dependence of the redox potential of PCb was studied too. It was found, that the value of E0' for PCb is constant in the pH range 6.5-9.5, but decreases in the range 4.8-6.5. On the whole, the dependence resembles that of PC from some well-known plant species, including poplar PCa. The changes of E0' in the pH-dependent region for poplar PCb, however, are smaller and are 13 mV per pH unit, whereas in the other well-known plant species the changes are about 50-60 mV per pH unit. It has been assumed that the weaker pH dependence of EO' of PCb accounts for some structural differences between PCa and PCb.

Plastocyanin microheterogeneity in Scenedesmus acutus MT8.

Two total plastocyanin (PC) fractions - loosely bound (lPC) and strongly bound (sPC) were extracted (84% and 16%, respectively) from the homogenate of Scenedesmus acutus MT8. Two-fold isolation-purification procedure including DE-52 chromatography separated IPC into a smaller oxidized [IPC (II)] and a larger reduced [IPC(I)] fractions, in contrast to sPC, where sPC(ll) greatly dominated over sPC(I). Analytical isoelectric focusing (IEF) separated IPC(II) into two main fractions only in the presence of 8 M urea, implying microheterogeneity. Preparative IEF in immobiline pH-gradient of 3.2-4.1 separated IPC(II) into two blue fractions - a more alkaline IPC(II) and a more acidic IPC"(II), which were probably stereoisomers. Their UV-Vis spectra exhibited rarely observed tryptophane (291.5 nm) and some differences at 270 and 287 nm. The exact molecular masses of apo-/holo-lPC (10131 Da/10194 Da) were determined by mass spectrometry. The number of -SH groups was determined from the mass difference between alkylated with 4-vinylpyridine (4-VP) and non-alkylated protein. Additionally, a simple procedure for simultaneous separation of both primary structure and stereoisomers of PC was developed.