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Towards a more accurate future for chlorophyll a and b determinations: the inaccuracies of Daniel Arnon’s assay

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Abstract

A recent publication (Esteban in New Phytol 217:341–342, 2018) describes how the use and citation of the assay of chlorophylls a and b extracted in aqueous 80% acetone by Arnon (Plant Physiol 2:1–15, 1949) is increasing, even in journals with high impact factors. This is a very disconcerting situation: the assay is outdated because it relies on the seriously under-estimated extinction coefficients of Mackinney (J Biol Chem 140:315–322, 1941), and the assay of chlorophylls is one of the most important, and much reported, procedures in studies of photosynthesis and related plant biological fields. Using the assay has led to the accumulation of masses of inaccurate data and confusion during the resolution of some plant biological problems. A summary not only of an accurate assay of chlorophylls in aqueous 80% acetone but also of a long-known method to correct the data obtained by Arnon’s procedure (cf. Porra et al. in Biochim Biophys Acta 975:384–394, 1989) is briefly described below together with references to reliable assays in this and other solvents by other authors.

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References

  • Anderson JM (1986) Photoregulation of the composition, function and structure of thylakoid membranes. Ann Rev Plant Physiol 37:93–136

    Article  CAS  Google Scholar 

  • Andersson B, Akerlund HE, Albertsson PA (1976) Separation of subchloroplast membrane particles by counter-current distribution. Biochim Biophys Acta Bioenerg 423:122–132 https://doi.org/10.1016/0005-2728(76)90106-7

    Article  CAS  Google Scholar 

  • Arnon PI (1949) Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol 24:1–15

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Barros T, Kühlbrandt W (2009) Crystallisation, structure and function of plant light-harvesting complex II. Biochim Biophys Acta Bioenerg 1787:753–772. https://doi.org/10.1016/j.bbabio.2009.03.012

    Article  CAS  Google Scholar 

  • Butler PJG, Kühlbrandt W (1988) Determination of the aggregate size in detergent solution of the light-harvesting chlorophyll a/b -protein complex from chloroplast membranes. Proc Natl Acad Sci USA 85:3797–3801

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Comar CL, Zscheile FP (1942) Analysis of plant extracts for chlorophylls a and b by a photoelectric spectrophotometric method. Plant Physiol 17:198–209

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Esteban R, García-Plazaola JI, Hernández A, Fernández-Marín B (2018) On the recalcitrant use of Arnon’s method for chlorophyll determination. New Phytol 217:474–476. https://doi.org/10.1111/nph.14932

    Article  PubMed  Google Scholar 

  • Jeffrey SW, Humphrey GF (1975) New spectroscopic equations for determining Chlorophylls a, b, c1, c2 in higher plants, algae and natural phytoplankton. Biochem Physiol Pflanzen 167:191–194

    Article  CAS  Google Scholar 

  • Kühlbrandt W, Wang DN, Fujiyoshi Y (1994) Atomic model of plant light-harvesting complex by electron crystallography. Nature 367:614–621

    Article  PubMed  Google Scholar 

  • Lichtenthaler HK (1987) Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Methods Enzymol 148:350–386

    Article  CAS  Google Scholar 

  • Liu Z, Yan H, Wang K, Kuang T, Zhang J, Gui L, An X, Chang W (2004) Crystal structure of spinach major light-harvesting complex at 2.72 Å resolution. Nature 428:287–292

    Article  CAS  PubMed  Google Scholar 

  • Mackinney G (1941) Absorption of light by chlorophyll solutions. J Biol Chem 140:315–322

    CAS  Google Scholar 

  • Ogawa T, Shibata K (1965) A sensitive method for determining chlorophyll b in plant extracts.Photochem Photobiol 4:193–200

    Article  CAS  Google Scholar 

  • Porra RJ (1990a) The assay of Chlorophylls a and b converted to their respective magnesium-rhodochlorin derivatives by extraction from recalcitrant algal cells with aqueous alkaline methanol: prevention of allomerization with reductants. Biochim Biophys Acta 1015:493–502

    Article  CAS  Google Scholar 

  • Porra RJ (1990b) A simple method for extracting Chlorophylls from the recalcitrant alga, Nannochloris atomus, without formation of spectroscopically-different magnesium-rhodochlorin derivatives. Biochim Biophys Acta 1019:137–141

    Article  CAS  Google Scholar 

  • Porra RJ (1991) Recent advances and re-assessments in chlorophyll extraction and assay procedures for terrestrial, aquatic, and marine organisms, including recalcitrant algae. In: Scheer H (ed) Chlorophylls. CRC-Press, Boca Raton, pp 31–57

    Google Scholar 

  • Porra RJ (2002) The chequered history of the development and use of simultaneous equations for the accurate determination of chlorophylls a and b. Photosynth Res 73:149–156

    Article  CAS  PubMed  Google Scholar 

  • Porra RJ (2006) Spectrometric assays for plant, algal and bacterial chlorophylls. In: Grimm B, Porra R, Rüdiger W, Scheer H (eds) Chlorophylls and : biochemistry, biophysics, functions and applications. Advances in photosynthesis and respiration, vol 25. Springer, Dordrecht, pp 95–107. ISBN 1-4020-4515-8

    Google Scholar 

  • Porra RJ, Thompson WA, Kriedemann PE (1989) Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochim Biophys Acta 975:384–394

    Article  CAS  Google Scholar 

  • Ritchie R (2018) Measurement of chlorophylls a and b and bacteriochlorophyll a in organisms from hypereutrophic auxinic waters. J Appl Phycol. https://doi.org/10.1007/s10811-018-1431-4

    Article  Google Scholar 

  • Sane PV, Goodchild DJ, Park RB (1970) Characterization of chloroplast photosystems 1 and 2 separated by nondetergent method. Biochim Biophys Acta Bioenerg 216:162–178. https://doi.org/10.1016/0005-2728(70)90168-4

    Article  CAS  Google Scholar 

  • Smith JHC, Benitez A (1955) Chlorophylls: analysis in plant materials. In: Paech K, Tracey M (eds) Methods of plant analysis, vol 4. Springer, Berlin, pp 142–196

    Google Scholar 

  • Standfuss J, Terwisscha van Scheftinga AC, Lamborghini M, Kühlbrandt W (2005) Mechanisms of photoprotection and nonphotochemical quenching in pea light-harvesting complex at 2.5 Å resolution. EMBO J 24:919–928

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Thornber JP (1986) Biochemical characterization and structure of pigment-proteins of photosynthetic organism. In: Staehelin LA, Arntzen CJ (eds) Photosynthesis III: photosynthetic membranes and light-harvesting systems (new series), vol 19. Springer, Berlin, pp 98–142

    Chapter  Google Scholar 

  • Vernon LP (1960) Spectrophotometric determination of chlorophylls and pheophytins in plant extracts. Anal Chem 32:1144–1150

    Article  CAS  Google Scholar 

  • Wellburn AR (1994) The spectral determination of chlorophylls a and b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution. J Plant Physiol 144:307–313

    Article  CAS  Google Scholar 

  • Ziegler R, Egle K (1965) Zur quantitativen Analyse der Chloroplastenpigmente. I. Kinetische Überprüfung der spektrophotometrischen Chlorophyll-Bestimmung. Beitr Biol Pflanzen 41:11–37

    CAS  Google Scholar 

Download references

Acknowledgements

We thank Erhard Pfündel and John Evans for so promptly drawing our attention to the excellent publication of Esteban et al. (2018). We also thank Govindjee for reading this paper and urging us to submit it for immediate publication.

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Authors and Affiliations

  1. Formerly of CSIRO-Division of Plant Industry, P.O. Box 1600, Canberra, ACT, 2601, Australia

    Robert J. Porra

  2. Botanisches Institut, Ludwig-Maximilians Universität, München, Menzinger Str. 67, 80638, Munich, Germany

    Robert J. Porra & Hugo Scheer

Authors
  1. Robert J. Porra
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  2. Hugo Scheer
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Correspondence to Hugo Scheer.

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This research did not involve human or animal participants.

Additional information

Part of the data have been published before (Porra et al. 1989); this is an expansion emphasizing the use of the correct spectroscopic data for the assay.

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Porra, R.J., Scheer, H. Towards a more accurate future for chlorophyll a and b determinations: the inaccuracies of Daniel Arnon’s assay. Photosynth Res 140, 215–219 (2019). https://doi.org/10.1007/s11120-018-0579-8

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