Figure 1.
Preparation and spectral properties of the phycobilisomes from the red macroalga Polysiphonia urceolata.
The phycobilisomes were prepared by sucrose step-gradient ultracentrifugation (a): a large fraction of phycobilisomes was situated within the boundary between 1.0 M and 1.5 M sucrose layers, and a small one was situated in the boundary between 1.5 M and 2.0 M sucrose layers; the former was used as samples for phycobiliprotein isolation and purification. Absorption (b solid line) and fluorescence emission (b dash dot line) spectra of the prepared intact phycobilisomes were recoded in pH 7.0 phosphate buffer at room temperature in which the phycobilisomes were excited at 498 nm.
Figure 2.
Isolation of the R-phycocyanin by gel filtration and spectral properties of the isolated R-phycocyanin.
(a) the chromatogram of the R-phycocyanin isolation from the dissociated phycobilisomes by the Sephadex G-150gel filtration and (b) the absorption (solid line) and second derivative (dash dot line) spectra of the obtained R-phycocyanin fraction. The gel column (3.9 cm×65 cm) was eluted with 50 mM phosphate buffer (pH 7.0) at 30 ml/h.
Figure 3.
Purification of the R-phycocyanin by ion exchange chromatography and spectral characteristics of the purified R-phycocyanin.
(a) The chromatogram of R-phycocyanin purification from the gel filtration fraction by ion exchange chromatography on DEAE-Sepharose Fast Flow, (b) absorption (solid line) and second derivative (dash dot line) spectra of the obtained R-phycocyanin fraction and (c) the fluorescence emission spectra of the R-phycocyanin fraction at room temperature when the fraction was excited at 495 nm (solid) and at 580 nm (dash dot). The ion exchange column (2.6 cm×10 cm) was eluted with the NaCl ion-strength gradient from 50 mM to 400 mM in 25 mM phosphate buffer (pH 7.0) at 30 ml/h.The spectra were recorded in pH 7.0 phosphate buffer.
Figure 4.
PAGE and IEF analysis of the R-phycocyanin purified by the chromatography on DEAE-Sepharose Fast Flow.
The native-PAGE of the purified R-PE (lane 1) and the R-PC (lane 2) purified by the ion exchange chromatography and the native-IEF (lane 3 and 4) of the R-PC prepared by the native-PAGE: the PAGE with a separating gel of 6.5% was performed in a neutral pH system, and the gel was stained with Coomassie Blue G-250 after it was imaged under UV-light at 365 nm; the native-IEF with a gel of 5.5% was performed in a pH range from 4.0 to 6.5, and the gel was visualized under UV-light at 365 nm (lane 3) and by staining with Coomassie Blue G-250 (lane 4).
Figure 5.
Spectral properties of the R-phycocyanin purified by the native-PAGE performed in a neutral pH system.
The absorption (a solid line) and second derivative (a dash dot line) spectra of the purified R-PC in was recorded in pH 7.0 phosphate buffer. The fluorescence excitation (b solid line) and emission (b dash line) spectra of the R-PC were recorded at room temperature in pH 7.0 phosphate buffer respectively on the examination at 640 nm and the excitation at 580 nm.
Figure 6.
Polypeptide analysis of the prepared R-phycocyanin by SDS-PAGE and denaturing IEF.
The SDS-PAGE (lane 1 and 2) had a gradient separating gel from 12% to 21% in pH 9.0 Tris-HCl buffer and the denaturing-IEF (lane 3 and 4) was a gel of 7% in a pH range from 3.0 to 10. The SDS-PAGE gel was examined under UV-light at 365 nm after it was stained with Zn(SO4)2 (lane 2) and then by staining with Coomassie Blue G-250 (lane 1). The denaturing-IEF gel was visualized under UV-light at 365 nm (lane 3) and by staining with Coomassie Blue G-250.
Figure 7.
Absorption spectra of the R-phycocyanin subunits.
The subunit with its pI of 6.4 (solid line) and the two subunits with their pIs of 5.4 and 5.4 (dash line) were obtained from the denaturing-IEF in a pH range from 3.0 to 10, and the spectra were measured by UV- microspectrometer (NanoDrop-1000).
Figure 8.
The SDS-PAGE of the R-PC subunits after the denaturing-IEF.
The SDS-PAGE with a separating gel of 16% was carried out in pH 8.8 Tris-HCl buffer, and then the gel was imaged by staining with Zn(SO4)2 (right) and silver (left), respectively.
Figure 9.
Mass spectra of the two β subunits.
The two β subunits, β22.6 (a) and β21.3 (b), from the 2D-PAGE were digested with Trypsin in gel and the peptides ware analyzed by MALDI-TOF/TOF mass spectrometry in a mass range from 500 Da to 3500 Da.