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Direct injection of pigment–protein complexes and membrane fragments suspended in water from phototrophs to C18 HPLC

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Abstract

We discovered that pigments including carotenoids and (bacterio)chlorophylls in pigment–protein complexes, membrane fragments, and chlorosomes suspended in water could be injected directly into C18 HPLC and analyzed without any other treatments. We applied this method to LH1-RC and chromatophores of purple bacteria, chlorosomes of green sulfur bacteria, thylakoid membranes of cyanobacteria, and PSII and thylakoid membranes of spinach. HPLC elution profiles and pigment composition were the same as those of the conventional extraction method. The principle of this method might be that samples are first trapped on top of column, followed by the immediate extraction of the pigments with the HPLC eluent and their separation using the C18 column, as usual. In the conventional extraction method, pigments are first extracted with organic solvents, followed by evaporation of the solvents. The dried pigments are then dissolved in organic solvents and injected into C18 HPLC after filtration. The advantages of this method include the preventions of pigment isomerization and oxidation and the possibility of injecting all samples. Its drawbacks include the accumulation of denatured proteins at the top of column, causing increased HPLC pressure. The use of a guard column might solve this problem. Many factors, such as samples, column, and HPLC systems, may affect this method. Nevertheless, we think that some samples can be analyzed using this method.

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Acknowledgements

AstaP was a kind gift of Mr. H. Toyoshima and Dr. S. Kawasaki, Tokyo University of Agriculture. This work was supported in part by JSPS KAKENHI Grant Numbers JP16H04174 and JP18H05153 to S.O.

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Authors and Affiliations

  1. Department of Molecular Microbiology, Faculty of Science, Tokyo University of Agriculture, Sakuragaoka, Setagaya, Tokyo, 156-8502, Japan

    Shinichi Takaichi

  2. Faculty of Science, Ibaraki University, Mito, 310-8512, Japan

    Akira Okoshi & Seiu Otomo

  3. Faculty of Education and Integrated Arts and Sciences, Waseda University, Wakamatsu, Shinjuku, Tokyo, 162-8480, Japan

    Masahiro Misumi & Kintake Sonoike

  4. Department of Biochemistry, Kurume University School of Medicine, Kurume, Fukuoka, 830-0011, Japan

    Jiro Harada

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  1. Shinichi Takaichi
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  2. Akira Okoshi
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  3. Seiu Otomo
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  4. Masahiro Misumi
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  5. Kintake Sonoike
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  6. Jiro Harada
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Correspondence to Shinichi Takaichi.

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Takaichi, S., Okoshi, A., Otomo, S. et al. Direct injection of pigment–protein complexes and membrane fragments suspended in water from phototrophs to C18 HPLC. Photosynth Res 144, 101–107 (2020). https://doi.org/10.1007/s11120-020-00735-w

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  • DOI: https://doi.org/10.1007/s11120-020-00735-w

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