Rapid purification of photosystem I chlorophyll-binding proteins by differential centrifugation and vertical rotor.
Photosystem I (PSI), which consists of a core complex and light-harvesting complex I (LHCI), is an important multisubunit pigment-protein complex located in the photosynthetic membranes of cyanobacteria, algae and plants. In the present study, we described a rapid method for isolation and purification of PSI and its subfractions. For purification of PSI, crude PSI was first prepared by differential centrifugation, which was applicable on a large scale at low cost. Then PSI was purified by sucrose gradient ultracentrifugation in a vertical rotor to reduce the centrifugation time from more than 20 h when using a swinging bucket rotor to only 3 h. Similarly, for subfractionation of PSI into the core complex and light-harvesting complex I, sucrose gradient ultracentrifugation in a vertical rotor was also used and it took only 4 h to obtain the PSI core, LHCI-680, and LHCI-730 at the same time. The resulting preparations were characterized by sodium dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), absorption spectroscopy, and 77 K fluorescence spectroscopy. In addition, their pigment composition was analyzed by high-performance liquid chromatography and the results showed that each Lhca could bind 1.5-1.6 luteins, 1.0 Violaxanthins, and 0.8-1.1 beta-carotenes on average, demonstrating that fewer carotenoids were released than with the slower traditional centrifugation. These results showed that the rapid isolation procedure, based on differential centrifugation and sucrose gradient ultracentrifugation in a vertical rotor, was efficient, and it should significantly facilitate preparation and studies of plant PSI. Moreover, the vertical rotor, rather than the swinging bucket rotor, may be a good choice for isolation of some other proteins.
Qin
Xiaochun
X
Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing , 100093, P.R. China.
Wang
Kebin
K
Chen
Xiaobo
X
Qu
Yuangang
Y
Li
Liangbi
L
Kuang
Tingyun
T
D002499:Centrifugation, Density Gradient / Q000295:instrumentation / Q000379:methods; D005591:Chemical Fractionation / Q000379:methods; D045342:Light-Harvesting Protein Complexes / Q000737:chemistry / Q000302:isolation & purification*; D010100:Oxygen / Q000378:metabolism; D045331:Photosystem I Protein Complex / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D010860:Pigments, Biological / Q000032:analysis; D013053:Spectrophotometry; D018724:Spinacia oleracea / Q000737:chemistry
D045342:Light-Harvesting Protein Complexes; D045331:Photosystem I Protein Complex; D010860:Pigments, Biological; D010100:Oxygen