Skip to main content
SpringerLink
Log in

Phosphatidylglycerol molecular species of photosynthetic membranes analyzed by high-performance liquid chromatography: Theoretical considerations

  • Methods
  • Published:
Lipids

Abstract

A reversed-phase high-performance liquid chromatography technique was developed to separate, identify, and quantify individual phsphatidylglycerol (PG) molecular species in thylakoid membranes isolated from higher plant leaves. PG was first separated by thin-layer chromatography; then the dinitrobenzoyl derivatives of diacylglycerols produced after phospholipase C hydrolysis of PG were separated by a C18 reversed-phase column and detected at 254 nm. A linear response of the detector was observed in the range of 0.025 to 12 nmol of PG molecular species. It was established that there was an excellent correlation (r=0.996) between the carbon and double-bond number in the aliphatic residues and the relative retention time of dinitrobenzoyl derivatives. A new equivalent carbon number value (ECN*) which takes into consideration the number ofcis-(n c ) andtrans-(n t ) double bonds per molecular species was defined as ECN*=CN-2n c -n t′ where CN is the number of carbon atoms in the aliphatic residues. The logarithm of the retention time increased linearily as a function of ECN* value. However, in this type of correlation, it may happen that two molecular species of PG having distinct relative retention times has the same ECN* value. In this case, the two molecular species can be identified by the linear correlation (r=1) existing between the reciprocal of the relative retention time and the number of double bonds (0≤n≤3) in the separate 18:n/Δ3-trans-hexadecenoic acid [16:1(3t) and 18:n/16:0 molecular species series. The advantages of this method are good separation, short elution time, quantitative precision, and predictable retention times of PG molecular species from chloroplast membranes. The method has been used routinely to identify the ten PG molecular species of thylakoid membranes in squash, potato, lettuce, and spinach leaf: 18:3/16:1(3t), 18:3/16:0, 18:2/16:1(3t), 18:2/16:0, 18:1/16:1(3t), 18:1/16:0, 18:0/16:1(3t), 18:0/16:0, 16:0/16:1(3t), and 16:0/16:0.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

DAG:

diacylglycerol

DNB:

dinitrobenzoyl

ECN:

equivalent carbon number

HPLC:

high-performance liquid chromatography

PG:

phosphatidylglycerol

PLC:

phospholipase C

RRT:

relative retention time

TLC:

thin-layer chromatography

16:1(3t):

Δ3-trans-hexadecenoic acid

References

  1. Rawyler, A., and Siegethaler, P.A. (1980) Rapid Analysis of membrane Lipids Using a Combination of Thin-Layer Chromatography and Scanning of Photographic Negatives,J. Biochem. Biophys. Methods 2, 271–281.

    Article  PubMed  CAS  Google Scholar 

  2. Dorne, A.J., Joyard, J., and Douce, R. (1990) Do Thylakoids Really Contain Phosphatidylcholine?Proc. Natl. Acad. Sci. (USA) 87, 71–74.

    Article  CAS  Google Scholar 

  3. Siegenthaler, P.A., Rawyler, A., and Smutny, J. (1989) The Phospholipid Population Which Sustains the Uncoupled Non/Cyclic Electron Flow Activity Is Localized in the Inner Monolayer of the Thylakoid Membrane,Biochim. Biophys. Acta 975, 104–111.

    CAS  Google Scholar 

  4. Haverkate, F., de Gier, J., and van Deenen, L.L.M. (1964) The Occurrence of Δ3-Trans-Hexadecenoic Acid in Phosphatidylglycerol from Spinach Leaves,Experientia 20, 511–512.

    Article  PubMed  CAS  Google Scholar 

  5. Roughan, P.G. (1985) Phosphatidylglycerol and Chilling Sensitivity in Plants,Plant Physiol., 77, 740–746.

    PubMed  CAS  Google Scholar 

  6. Dubacq, J.P., and Trémolières, A. (1983) Occurrence and Function of Phosphatidylglycerol Containing Δ3-Trans-Hexadecenoic Acid in Photosynthetic Lamellae,Physiol. Vég. 21, 293–312.

    CAS  Google Scholar 

  7. Siegenthaler, P.A., and Giroud, C. (1986) Transveral Distribution of Phospholipids in Prothylakoid and Thylakoid Membranes from Oat,FEBS Lett. 201, 215–220.

    Article  CAS  Google Scholar 

  8. Murata, N., Sato, N., Takahashi, N., and Hamazaki, Y. (1982) Composition and Positional Distributions of Fatty Acids in Phospholipids from Leaves of Chilling-Sensitive and Chilling-Resistant Plants,Plant & Cell Physiol. 23, 1071–1079.

    CAS  Google Scholar 

  9. Lynch, V.D., and Thompson, Jr. G.A. (1984) Chloroplast Phospholipid Molecular Species Alterations During Low Temperature Acclimation inDunaliella Plant Physiol. 74, 198–203.

    PubMed  CAS  Google Scholar 

  10. Lynch, D.V., Gundersen, R.E., and Thomspon, Jr. (1983) Separation of Galactolipid Molecular Species by High-Performance Liquid Chromatography,Plant Physiol. 72, 903–905.

    Article  PubMed  CAS  Google Scholar 

  11. Haverkate, F., and van Deenen, L.L.M. (1965) Isolation and Chemical Characterization of Phosphatidylglycerol from Spinach Leaves,Biochim. Biophys. Acta 106, 78–92.

    PubMed  CAS  Google Scholar 

  12. Murata, N. (1983) Molecular Species Composition of Phosphatidylglycerols from Chilling-Sensitive and Chilling-Resistant Plants,Plant & Cell Physiol. 24, 81–86.

    CAS  Google Scholar 

  13. Kito, M., Takamura, H., Narita, H., and Urade, R. (1985) A Sensitive Method for Quantitative Analysis of Phospholipid Molecular Species by High-Performance Liquid Chromatography,J. Biochem. 98, 327–331.

    PubMed  CAS  Google Scholar 

  14. Takamura, H., Narita, H., Urade, R., and Kito, M. (1986) Quantitative Analysis of Polyenoic Phospholipid Molecular Species by High-Performance Liquid Chromatography,Lipids 21, 356–361.

    PubMed  CAS  Google Scholar 

  15. Bruisma, J. (1961) A Comment on the Spectrophotometric Determination of Chlorophyll,Biochim. Biophys. Acta 850, 264–274.

    Google Scholar 

  16. Chapman, D.J., and Barber, J. (1987) Polar Lipids of Chloroplast Membranes, inMethods in Enzymology (Packer, L., and Douce, R., eds.) Vol. 148, pp. 294–319, Academic Press, Inc., San Diego.

    Google Scholar 

  17. Browse, J., and Somerville, C. (1991) Glycerolipid Synthesis: Biochemistry and Regulation,Annu. Rev. Plant Physiol. Plant Molec. Biol. 42, 467–506.

    Article  CAS  Google Scholar 

  18. Nishihara, M., Yokota, K., and Kito, M. (1980) Lipid Molecular Species Composition of Thylakoid Membranes,Biochim. Biophys. Acta 617, 12–19.

    PubMed  CAS  Google Scholar 

  19. Sekiya, J., Nagai, K., and Shimose, N. (1990) Molecular Species of Phosphatidylglycerol from Leaves of VariousCitrus Species,Agric. Biol. Chem., 54, 2777–2782.

    CAS  Google Scholar 

  20. Christie, W.W. (1987)High-Performance Liquid Chromatography and Lipids, A Practical Guide, Pergamon Press, Oxford.

    Google Scholar 

  21. Bevel, L., Smutny, J., and Siegenthaler, P.A. (1995) Lipid Dependence of Protein Kinase Activities in Spinach Chloroplast Envelope Membranes, inPlant Lipid Metabolism Kader, J.C., and Mazliak, P., eds.), pp. 173–175, Kluwer Academic Publishers, Dordrecht.

    Google Scholar 

  22. Kerwin, J.L., Tuininga, A.R., and Ericsson, L.H. (1994) Identification of Molecular Species of Glycerophospholipids and Sphingomyelin Using Electrospray Mass Spectrometry,J. Lipid Res. 35, 1102–1114.

    PubMed  CAS  Google Scholar 

  23. Kim, H.-Y., Wang, T.-C.L., and Ma, Y.-C. (1994) Liquid Chromatography/Mass Spectrometry of Phospholipids Using Electrospray Ionization,Anal. Chem. 66, 3977–3982.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Laboratoire de Physiologie Végétale, Université de Neuchâtel, Rue Emile Argand 13, rue CH-2007, Neuchâtel, Switzerland

    Yinong Xu & Paul-André Siegenthaler

Authors
  1. Yinong Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Paul-André Siegenthaler
    View author publications

    You can also search for this author in PubMed Google Scholar

About this article

Cite this article

Xu, Y., Siegenthaler, PA. Phosphatidylglycerol molecular species of photosynthetic membranes analyzed by high-performance liquid chromatography: Theoretical considerations. Lipids 31, 223–229 (1996). https://doi.org/10.1007/BF02522624

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02522624

Keywords

Search

Navigation