Photosynthesis Research

, Volume 90, Issue 3, pp 195–204 | Cite as

Rapid purification of photosystem I chlorophyll-binding proteins by differential centrifugation and vertical rotor

  • Xiaochun Qin
  • Kebin Wang
  • Xiaobo Chen
  • Yuangang Qu
  • Liangbi Li
  • Tingyun Kuang
Regular Paper

Abstract

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 β-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.

Keywords

Differential centrifugation Light-harvesting complex I (LHCI) LHCI-680 LHCI-730 Photosystem I Purification Vertical rotor 

Abbreviations

a.u.

Arbitrary units

Chl

Chlorophyll

DDM

Dodecyl-β-d-maltoside

HPLC

High-performance liquid chromatography

LHCI

Light-harvesting complex I

LHCII

Light-harvesting complex II

PSI

Photosystem I

PSII

Photosystem II

SDS-PAGE

Sodium dodecyl-sulfate-polyacrylamide gel electrophoresis

zw 3-16

3-(N,N-dimethylpalmitylammonio)propanesulfonate

Notes

Acknowledgment

This work was supported by the National Natural Science Foundation of China (39890390).

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Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  • Xiaochun Qin
    • 1
    • 2
  • Kebin Wang
    • 1
  • Xiaobo Chen
    • 1
  • Yuangang Qu
    • 1
  • Liangbi Li
    • 1
  • Tingyun Kuang
    • 1
    • 3
  1. 1.Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of BotanyChinese Academy of SciencesBeijing P.R. China
  2. 2.Graduate University of Chinese Academy of SciencesBeijingP.R. China
  3. 3.College of Life ScienceCapital Normal UniversityBeijingP.R. China

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