Photosynthesis Research

, Volume 73, Issue 1–3, pp 149–156

The chequered history of the development and use of simultaneous equations for the accurate determination of chlorophylls a and b

  • Robert J. Porra
Article

Abstract

Over the last half century, the most frequently used assay for chlorophylls in higher plants and green algae, the Arnon assay [Arnon DI (1949) Plant Physiol 24: 1–15], employed simultaneous equations for determining the concentrations of chlorophylls a and b in aqueous 80% acetone extracts of chlorophyllous plant and algal materials. These equations, however, were developed using extinction coefficients for chlorophylls a and b derived from early inaccurate spectrophotometric data. Thus, Arnon's equations give inaccurate chlorophyll a and b determinations and, therefore, inaccurate chlorophyll a/b ratios, which are always low. This paper describes how the ratios are increasingly and alarmingly low as the proportion of chlorophyll a increases. Accurate extinction coefficients for chlorophylls a and b, and the more reliable simultaneous equations derived from them, have been published subsequently by many research groups; these new post-Arnon equations, however, have been ignored by many researchers. This Minireview records the history of the development of accurate simultaneous equations and some difficulties and anomalies arising from the retention of Arnon's seriously flawed equations.

absorption spectroscopy accurate chlorophyll a and b determinations algebraic correction method for Arnon's Chl determinations chlorophyll a/b ratios Daniel Arnon LHC II light-harvesting complex of photosytem II G. Mackinney magnesium atomic absorption spectroscopy molar and specific extinction coefficients Richard Willstätter 

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References

  1. Anderson JM (1986) Photoregulation of the composition, function, and structure of thylakoid membranes. Annu Rev Plant Physiol 37: 93–136CrossRefGoogle Scholar
  2. Andersson B, Åkerlund H-E and Albertsson P-Å (1976) Separation of sub-chloroplast membrane particles by counter-current distribution. Biochim Biophys Acta 423: 122–132PubMedCrossRefGoogle Scholar
  3. Arnon DI (1949) Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol 24: 1–15PubMedGoogle Scholar
  4. Böger P (1964) Das Strukturproteid aus Chloroplasten einzelliger Grünalgen und seiner Beziehung zum Chlorophyll. Flora 154: 174–211Google Scholar
  5. Butler PJG and Kühlbrandt W (1988) Determination of the aggregate size in detergent solution of the light-harvesting chlorophyll a/b-protein complex from chloroplast membranes. Proc Nat Acad Sci USA 85: 3797–3801PubMedCrossRefGoogle Scholar
  6. Comar CL and Zscheile FP (1942) Analysis of plant extracts for chlorophyll a and b by a photoelectric spectrophotometric method. Plant Physiol 17: 198–209PubMedGoogle Scholar
  7. Delaporte N and Laval-Martin D (1971a) Analyse spectrophotometrique des chlorophylles et des pheophytines a et b en milieu hydroacetonique. I. Determination extinctions molaires. Anal Chem Acta 55: 415–424CrossRefGoogle Scholar
  8. Delaporte N and Laval-Martin D (1971b) Analyse spectrophotometrique des chlorophylles et des pheophytines a et b en milieu hydroacetonique. II. Methode Cinetique de dosage. Anal Chem Acta 55: 425–435CrossRefGoogle Scholar
  9. Inskeep WP and Bloom PR (1985) Extinction coefficients of chlorophyll a and b in N,N-dimethylformamide and 80% acetone. Plant Physiol 77: 483–485PubMedCrossRefGoogle Scholar
  10. Kühlbrandt W and Wang DN (1991) Three-dimensional structure of plant light-harvesting complex determined by electron crystallography. Nature (London) 350: 130–134CrossRefGoogle Scholar
  11. Kühlbrandt W, Wang DN and Fujiyoshi Y (1994) Atomic model of plant light-harvesting complex by electron crystallography. Nature (London) 367: 614–621CrossRefGoogle Scholar
  12. Lichtenthaler HK (1987) Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Meth Enzymol 148: 350–382CrossRefGoogle Scholar
  13. Mackinney G (1941) Absorption of light by chlorophyll solutions. J Biol Chem 140: 315–322Google Scholar
  14. Ogawa T and Shibata K (1965) A sensitive method for determining chlorophyll b in plant extracts. Photochem Photobiol 4: 193–200Google Scholar
  15. 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: 493–502CrossRefGoogle Scholar
  16. 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: 137–141CrossRefGoogle Scholar
  17. 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, pp 31–57. CRC Press, Boca Raton, FloridaGoogle Scholar
  18. Porra RJ and Grimme LH (1974) A new procedure for the determination of chlorophylls a and b and its application to normal and regreening Chlorella. Anal Biochem 57: 255–267PubMedCrossRefGoogle Scholar
  19. Porra RJ, Thompson WA and 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 spectrometry. Biochim Biophys Acta 975: 384–394Google Scholar
  20. Rabinowitch E (1945) Photosynthesis and Related Processes, Vol 1, pp 399–432. Interscience Publishers, New YorkGoogle Scholar
  21. Rogl, H and Kühlbrandt W (1999) Mutant trimers of lightharvesting complex II exhibit altered pigment content and spectroscopic features. Biochemistry 38: 16214–16222PubMedCrossRefGoogle Scholar
  22. Sane PV, Goodchild DJ and Park RB (1970) Characterization of chloroplast Photosystems 1 and 2 separated by a non-detergent method. Biochim Biophys Acta 216: 162–178PubMedCrossRefGoogle Scholar
  23. Seybold A and Egle K (1938) Zur Chromatographischen Methode der Blattpigmente. Planta 29: 114–118CrossRefGoogle Scholar
  24. Smith JHC and Benitez A (1955) Chlorophylls: analysis in plant materials. In: Paech K and Tracey MV (eds) Modern Methods of Plant Analysis. Vol IV, pp 142–196. Springer-Verlag, BerlinGoogle Scholar
  25. Strain HH and Svec WA (1966) Extraction separation, estimation and isolation of the chlorophylls. In: Vernon LP and Svec GR (eds) The Chlorophylls, pp 21–66. Academic Press, New YorkGoogle Scholar
  26. Talbot MFJ and Sauer K (1997) Spectrofluorimetric method for determination of large chlorophyll a/b ratios. Photosynth Res 53: 73–79CrossRefGoogle Scholar
  27. Thompson EW and Preston RD (1967) Proteins in the cell walls of some green algae. Nature (London) 213: 684–685CrossRefGoogle Scholar
  28. Thompson EW and Preston RD (1968) Evidence for a structural role of protein in algal cell walls. J Exp Bot 19: 690–697Google Scholar
  29. Thornber JP (1986) Biochemical characterization and structure of pigment-proteins of a photosynthetic organism. In: Staehelin LA and Arntzen CJ (eds) Encyclopedia of Plant Physiology 'Photosynthesis III', Vol 19, pp 98–142. Springer-Verlag, BerlinGoogle Scholar
  30. Vernon LP (1960) Spectrophotometric determination of chlorophylls and pheophytins in plant extracts. Anal Chem 32: 1144–1150CrossRefGoogle Scholar
  31. 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: 307–313Google Scholar
  32. Willstätter R and Stoll A (1913) Untersuchungen über Chlorophyll: Methoden und Ergebnisse, pp 53–125. Julius Springer-Verlag, BerlinGoogle Scholar
  33. Ziegler R and Egle K (1965) Zur quantitativen Analyse der Chloroplastenpigmente. I. Kritische Ñberprüfung der spektralphotometrischen Chlorophyll-Bestimmung. Beitr Biol Pflanzen 41: 11–37Google Scholar

Copyright information

© Kluwer Academic Publishers 2002

Authors and Affiliations

  • Robert J. Porra
    • 1
    • 2
  1. 1.Division of Plant IndustryCommonwealth Scientific and Industrial Research OrganizationCanberraAustralia
  2. 2.Botanisches InstitutLudwig-Maximilians-Universität MünchenMünchenGermany

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