Part 4 Results

Separation of Fractions - After solubilization with DDM, PSII was partially removed by nickel affinity chromatography and the remaining fraction was subjected to sucrose density centrifugation. Three Chl-containing bands were obtained (F1, F2, and F3), and absorption spectroscopy identified the top fraction (F1) as LHC proteins having an absorption maximum at 673 nm and a shoulder at 653 nm due to Chl b. Fig. 1a shows strong absorption in the 450-500 nm range, in part due to Chl b (peaking at 476 nm) and in part due to the presence of carotenoids in the F1 fraction. F2 (Fig. 1a) is characterized by the lack of Chl b absorption and a red shift of the long wavelength absorption peak to 676 nm compared with F1 (Fig. 1a). In contrast the densest fraction (F3) showed some Chl b absorption at 653 nm and 467 nm and had maximum long wavelength absorption at 680 nm. Low temperature fluorescence was measured at 77 K for each band. The emission profile peaking at 681 nm for the top band is consistent with it containing free Lhc proteins. The middle fraction (F2) showed a broad emission spectrum peaking at 686 nm but having significant contribution beyond 700 nm. This broad, low temperature fluorescence spectrum is indicative of a mixture of PSII (emission .700 nm) and PSI (emission .700 nm). However, the fluorescence spectrum of F3 peaks at 715 nm, which implies that it is highly enriched in PSI. Reverse phase HPLC pigment analyses of the F3 sample gave a Chl a/b ratio of .4.5. Protein Characterization of the Isolated Fractions-To characterize further each fraction of the sucrose density gradient SDS-PAGE and immunoblotting analyses were conducted. As shown in Fig. 2a, the top fraction (F1) was enriched in polypeptides, mainly LHCII but also LHCI as confirmed by immunoblotting (see Fig. 2b). On the other hand, none of the PSI antibodies raised to the proteins of the reaction center core of PSI cross-reacted with proteins in this fraction. According to SDS-PAGE and immunoblotting, F2 contained a range of proteins consistent with it being a mixture of PSI and PSII core proteins, while the LHC protein level was generally lower in this fraction compared with F1. F3 was distinguished by having no cross-reactions with antibodies raised to PSII (D1-antibody) or to LHCII antibodies raised against Lhcb1 and Lhcb2 (see Fig. 2b). On the other hand the SDS-PAGE profile and immunoblotting showed that F3 contains the PSI core subunits, PsaA/B, PsaC, PsaD, PsaE, and PsaF. Moreover polypeptides having apparent molecular masses in the range 22-30 kDa were present in F3. Immunoblotting showed these to be proteins of LHCI, and in particular Lhca3 was identified by a specific antibody response. The protein analysis shown in Fig. 2 seems to indicate that the dense fraction (F3) separated by sucrose density gradient centrifugation consists of a supercomplex composed of PSI and its associated light harvesting Lhca proteins. To dissociate the Lhca proteins from the PSI reaction center core we treated the F3 fraction with 0.6% zwittergent 16 plus 1% DDM, and separated the solubilized products by sucrose density centrifugation. Despite the relatively strong solubilizing conditions used, we were unable to remove all the Lhca subunits from the PSI reaction center core. Those that were removed formed a band at the top of the sucrose gradient, and SDS-PAGE analyses coupled with immunoblotting showed that several different types of Lhca proteins were present (see Fig. 3a inset), including Lhca3 (Western blot, not shown). The absorption spectrum of this LHCI fraction is shown in Fig. 3a, and its emission in Fig. 3b. Reverse phase HPLC analyses gave a Chl a/b ratio of 2 for this LHCI fraction. Both spectra contrast with the corresponding spectra obtained with the F1 fraction shown in Fig. 1a in that they have long wavelength maxima that are blue-shifted. Moreover, the lower level of Chl b is evident in the absorption spectrum of the Lhca proteins in comparison to F1 (compare Figs. 1a and 3a). The differences between this LHCI fraction and fraction F1 are consistent with the presence of a significant level of LHCII proteins in the latter.