Professional Documents
Culture Documents
PLASTIDS
[34]
glycerol and MGDG substrates containing specific fatty acids. Thus, the
extent of the observable galactosylation reactions in any tissue preparation depends upon the molecular species of diacylglycerol and MGDG
available in the preparation. The type and amount of substrate available in
the plant preparation may be altered by both biotic and abiotic factors,
Factors which may alter the rate of galactolipid biosynthesis and the
nature (molecular species) of the galactolipids formed include (1) the temperature of growth of plant tissue,~4 (2) herbicide treatment of plant material, 13 (3) addition of drugs to the assay mixture, ~5etc. The light intensity
during growth of the plant appears to have little effect on galactolipid
metabolism. H
14j. p. Williams, M. U. Khan, and K. Mitchell, in "Structure, Function and Metabolism of
Plant Lipids" (P.-A. Siegenthaler and W. Eichenberger, eds.), p. 123. Elsevier, Amsterdam, 1984.
1~N. W. Lem and P. K. Stumpf, Plant Physiol. 75, 700 (1984).
[34] ChlorolShylls a n d C a r o t e n o i d s : P i g m e n t s of
Photosynthetic Biomembranes
B y HARTMUT K . LICHTENTHALER
Introduction
The photosynthetic pigments chlorophylls and carotenoids, together
with sterols, prenylquinones, and prenols, belong to the group of isoprenoid plant lipids, which in 1976 were named prenyl lipids by Goodwin ~
and Lichtenthaler. 2 Carotenoids as tetraterpenoids are simple or pure
prenyl lipids, the carbon skeleton of which is made up solely of isoprenoid
units. Chlorophyll a and b are mixed prenyllipids: they possess an isoprenoid phytyl chain which is bound to a nonisoprenoid porphyfin ring system. The possession of the phytyl side chain, which is esterified to the
carboxyl group of the ring, gives the chlorophylls their lipid character.
Because of their biogenetic relationship (isopentenoid pathway), chlorophylls and carotenoids are also referred to as prenyl pigments.
1 T. W. Goodwin, in "Lipids and Lipid Polymers in Higher Plants" (M. Tevini and H. K.
Lichtenthaler, eds.), p. 29. Springer-Verlag, Berlin and New York, 1977.
2 H. K. Lichtenthaler, in "Lipids and Lipid Polymers in Higher Plants" (M. Tevini and
H. K. Lichtenthaler, eds.), p. 231. Springer-Verlag, Berlin and New York, 1977.
[34]
351
Chlorophylls
The chlorophylls of higher plants, ferns, mosses, and green algae, as
well as of the prokaryotic organism Prochloron, consist of chlorophyll a
as the major pigment and of chlorophyll b as an accessory pigment. Both
chlorophylls are genuine components of the photosynthetic membranes
and occur in a ratio (a/b) of approximately 3 to 12-5 Growth conditions
and environmental factors can modify this a/b ratio. High-light and sunexposed plants (high-light chloroplasts) exhibit a/b ratios of 3.2 to 4,
whereas shade plants (low-light chloroplasts) possess lower values for the
a/b ratio (e.g., 2.5 to 2.9). 4-6 In older literature minor amounts of an
additional chlorophyll (a') were found. 7 This is a C-10 epimer of chlorophyll a and is now regarded in the form of the a' dimer as identical
with the reaction center chlorophyll P-700 of the photosynthetic photosystem 1.8
Carotenoids
The group of primary plant carotenoids can be divided (1) into the
oxygen-free carotenes and (2) into the xanthophylls, which contain oxygen in different forms, such as one or several hydroxy or epoxy groups.
There are two types of isomers on each oxidation level, the a-carotene
derivatives (one e-ionone and one/3-ionone ring) and the/3-carotene derivatives (2/3-ionone rings). Introduction of hydroxy and epoxy functions
into a-carotene (ionone rings) gives rise to lutein and lutein epoxide./3Carotene is the precursor of the xanthophylls zeaxanthin, violaxanthin,
and antheraxanthin. The carotenoids of green, photosynthetically active
plant tissue, which are needed for photosynthetic function, are classified
as primary carotenoids, whereas those of red fruits and flowers have been
termed secondary carotenoids.
The carotenoids of functional chloroplasts include/3-carotene, lutein,
violaxanthin, and neoxanthin as major and regular components of the
photochemically active thylakoids of chloroplasts of higher plants and
3 R. Willst~itter and A. Stoll, "Untersuchungen tiber Chlorophyll." Springer-Verlag,
Berlin, 1913.
4 A. Seybold and K. Egle, Planta 26, 491 (1970).
5 H. K. Lichtenthaler, C. Buschmann, M. D611, H.-J. Fietz, T. Bach, U. Koze|, and U.
Rahmsdorf, Photosynth. Res. 2, 115 (1981).
6 H. K. Lichtenthaler, G. Kuhn, U. Prenzel, C. Buschmann, and D. Meier, Z. Naturforsch., C: Biosci. 37C, 464 (1982).
7 H. H. Strain, B. T. Cope, and W. A. Svec, this series, Vol. 23, p. 452.
8 T. Watanabe, M. Kobayashi, A. Hongu, M. Nakazato, T. Hiyama, and N. Murata, FEBS
Lett. 191, 252 (1985).
352
PLASTIDS
[34]
d-Carotene
i-Corotene
H O ~ d ~ ~ ~ ~ V ~
0H Zeoxonthin
OH
Lutein
Violoxanthin
HO
.~:]~
HO
OH
Neoxanthln
OH
green algae (Fig. 1). Depending on growth conditions and stress factors,
the percentage composition of carotenoids (weight %) can vary within the
ranges: /3-carotene 25-40% lutein, 40-57%, violaxanthin, 9-20%, and
neoxanthin, 5-13%. The xanthophylls antheraxanthin, luteinepoxid, and
zeaxanthin are regular but minor carotenoid components. In green algae
a-carotene shows up as an additional carotene to/3-carotene. Under highlight conditions the xanthophyll violaxanthin (two epoxy, two hydroxy
groups) can by deepoxidized via antheraxanthin to zeaxanthin, the corresponding deepoxy derivative, which exhibits a chromatographic mobility
similar to lutein. Though this xanthophyll cycle is well established, 9-11 its
9 A.
l0 D.
H.
" K.
Hager, in "Pigments in Plants" (F. C, Czygan, ed.), p. 57. Fischer, Stuttgart, 1980.
Siefermann-Harms, in "Lipids and Lipid Polymers in Higher Plants" (M. Tevini and
K. Lichtenthaler, ds.), p. 218. Springer-Verlag, Berlin and New York, 1977.
Grumbach, Z. Naturforsch., C: Biosci. 38C, 393 (1983).
[34]
353
Chlorophyll-Carotenoid Proteins
Much of the present research work on photosynthetic pigments concerns isolation and characterization of pigment proteins. The main features of their isolation and pigment content will therefore be treated here.
12 H. A. W. Schneider, Ber. Dtsch. Bot. Ges. 88, 83 (1975).
13 W. Riidiger and J. Benz, in "Chloroplast Biogenesis" (R. J. Ellis, ed.), p. 225. Cambridge
Univ. Press, London and New York, 1984.
14 B. H. Davies, in "Chemistry and Biochemistry of Plant Pigments" (T. W. Goodwin, ed.),
2nd ed., Vol. 2, p. 38. Academic Press, New York, 1976.
354
PLASTIDS
[34]
A
I1. n
~-~
C
",,I
0..'
12.
It.
U
IZ
IIII
IIII
.10
",-'r
-J-~
,<
t.2
"1-.I
t.
rQ
IX.
'..,It
o~
fit.
(.2
"1-
..a
o-"1
it
ffl
.13
<
I~igrolion
(~
(1981).
,6 j . p. M a r k w e l l , J. P. Thornber, and R. T. B o g g s , Proc. Natl. Acad. Sci. U.S.A. 76, 1223
(1979).
[34]
355
TABLE I
PRENYL PIGMENT RATIOS AND PERCENTAGE COMPOSITION OF CAROTENOIDS
IN PIGMENT PROTEINS
Protein
Chloroplasts
CPIa
CPI
CPa
LCHPI_3
FP
4-7
5-6
12
9-21
7-9
9
3-8
3-5
4-8
1.1-1.3
3-5
60-180
2.6
3
14
56
24
9
5
6
70
15
4
6
5
75
13
5
4
3
Pigment ratios
Chlorophyll a / b
3.2
4
12
Percentage carotenoid composition c
fl-Carotene
30
Lutein
45
Neoxanthin
6
Violaxanthin
11
Others
8
a + b/x + cb
a/c b
1-2
56-65
21-29
4-5
6
24
37
10
20
9
(Fig. 2), one obtains the following: (1) the P700-containing chlorophyll
a/fl-carotene proteins CPI and CPIa of photosystem I, (2) CPa, the presumable reaction center of photosystem II, which is also a chlorophyll
356
PLASTIDS
[34]
[34]
357
other problem which is not yet fully resolved is the quantitative extraction
of chlorophylls and carotenoids from the pigment proteins on the polyacrylamide gels. Repetitive extraction of the material seems to be the
method of choice.
Isolation and Chromatography of Pigments
Extraction
All prenyl pigments, chlorophylls and carotenoids, are fat-soluble
compounds that can be extracted from water-containing living plant tissue
by organic solvents (grinding with quartz sand or use of a homogenizer)
such as acetone, methanol, or ethanol, which can take up water. Though
aqueous solutions of these solvents are also suitable (and may sometimes
be preferable) for extraction, their water content should not exceed 5 or
10%. The widely used method to extract isolated chloroplasts by 80%
aqueous acetone3~ does not fully extract the less polar pigment chlorophyll a and the polyene/3-carotene. An addition step of extraction with
100% acetone is needed to guarantee complete extraction. By addition of
petroleum hydrocarbon (or hexane) and water the pigments are transferred to the water-free epiphase, and concentrated in a rotary evaporator
to a certain standard volume. In a water-free organic solvent, pigments
can be stored longer than in aqueous solution.
Freeze-dried plant material can be extracted directly with hexane or
petroleum hydrocarbon; this, however, will mainly remove fl-carotene
and some chlorophyll a. Addition of small amounts of a polar solvent
(acetone) to the extracting hydrocarbon solvent will fully extract the chlorophylls and carotenoids. Since carotenoids and chlorophylls are extremely light sensitive and easily become photobleached, the extraction
and concentration procedures should be performed in dim light. For correct pigment values the quantitative determination of chlorophylls and
total carotenoids in the whole leaf extract should be carried out immediately after preparation of the extract. If storage of the extract is required,
it should be done in concentrated form and under nitrogen.
Chromatographic Procedures
Several procedures have been developed for the separation of the
photosynthetic pigments including column (CC), paper (PC), and thinlayer chromatography (TLC) and also high-pressure liquid chromatogra3t D. I. Arnon, Plant Physiol. 24, 1 (1949).
358
PLASTIDS
[34]
-111-rain
_.
.c_
"-.r" 0
II
>,,
--"
",.
!
start
detection:
1.50 n m
FIG. 3. High-performance liquid chromatography of a leaf pigment extract (reversedphase HPLC). Column: 25 cm, Nucleosil 5 C-8; solvent: 9% water in methanol; flow rate:
1.3 ml/min, na, Neoxanthin a.
[34]
359
(l
mln
start
detection:/,10 nm
FIG. 4. Reversed-phase HPLC of leaf pigments including pheophytins. Column: 12.5 cm,
Nucleosil 7 C-18; solvent: methanol; flow rate: 1.8 ml/min.
360
PLASTIDS
[34]
{'I
I'
350.0
450.0
550.0
650.0
750.0
Wavelength (nm)
FIG. 5. Absorption spectrum of freshly isolated chlorophyll a (solid line) and chlorophyll
b (broken line) in diethyl ether (pure solvent). The positions of the absorption maxima are
given in Table II.
vents assayed (see Table II). The shortest wavelength for hmax in the blue
and red is found for both chlorophylls in water-free diethyl ether. The two
major absorption maxima in the red and blue of both chlorophylls shift to
longer wavelengths with increasing polarity and/or water content of the
solvent. The shift at hmax red is less pronounced than that at hmax in the
blue region and it is larger for chlorophyll b than for chlorophyll a. The
wavelengths of the much smaller, minor absorption maxima also undergo
this shift.
The specific absorption coefficients are highest in dried (water-free)
diethyl ether and decrease via diethyl ether (pure solvent), water-saturated diethyl ether, to acetone (100 ---> 80%), ethanol (95%), and methanol
(100 ~ 90%) (Table II). The absorption in the red and blue maxima is
highest in freshly isolated chlorophylls and also decreases in older solutions, particularly in those which are light exposed. Quantitative chlorophyll determinations should therefore be carried out immediately after
extraction. In the case of chlorophyll a the decrease in absorbance in the
blue is stronger than in the red region, which is documented by a continu-
[34]
361
1
o
III
0.500 T
0.500
//i
3SQ.O
450.0
550.0
6S0.0
750.0
Wavelength (rim)
FIG. 6. Absorption spectrum of freshly isolated chlorophyll a (solid line) and chlorophyll
b (broken line) in aqueous ethanol (95%), The wavelengths of the absorption maxima are
given in Table II.
ous decrease of the values for the ratio of absorption coefficients of the
blue and the red maximum from 1.28 to 0.96 (Table IV). For chlorophyll b
the decrease is about the same in the blue and in the red region, as seen
from the ratio of the absorption coefficients, which remains about the
same with values around 2.7 (Table IV).
With increasing polarity and water content of the solvent, the absorption maxima in the blue and red not only become smaller, but at the same
time broader. Parallel to the broadening of the red maximum of chlorophyll a there occurs a considerable increase in the absorption coefficients
of the minor maxima at about 614 to 618 nm and 576 to 582 nm. Due to the
shift in the wavelengths, the specific absorption coefficients of chlorophyll a and b at 470 nm, the reference point of simultaneous carotenoid
determination in a whole leaf pigment extract, rise continuously with
increasing polarity and water content of the solvent. In ethanol (95%)
and in methanol (100 and 90%), the two minor absorption maxima (in
the range of 576 to 582 nm and 531 to 537 nm) do not show up (see also
Fig. 6).
362
PLASTIDS
[34]
TABLE II
SPECIFIC ABSORPTION COEFFICIENTS FOR CHLOROPHYLL a AND b REDETERMINED a
Diethyl ether
(water free)
(nm)
(sp. coeff.)
Diethyl ether
(pure solvent)
(nm)
(sp. coeff.)
Diethyl ether
(H20 saturated)
(nm)
Acetone
(100%)
(sp. coeff.)
(nm)
(sp. coeff.)
Chlorophyll a
660
101.9
660.6
101c
661.6
98.46
661.6
92.45
641.8
614.4
576.2
531.0
470
460
450
428.0
408.8
15.20
15.7
8.7
4.67
1.3
1.5
3.6
130.4
83.15
642.2
614.8
576.6
531.6
470
460
450
428.4
409.4
15.0
15.88
8.80
4.48
1.43
1.71
3.87
127.21
82.83
643.2
615.8
578.2
532.6
470
460
450
429.4
410.0
15.31
16.07
8.86
4.19
1.38
1.79
5.47
120.96
81.22
644.8
616.0
579.0
534.2
470
460
450
429.6
410.6
19.25
17.25
9.92
4.34
1.90
2.44
7.40
112.36
83.18
Chlorophyll b
660
641.8
4.7
62.3
660.6
642.2
6.0
62.0~
661.6
643.2
7.20
58.29
661.6
644.8
9.38
51.64
593.2
470
460
450
452.2
428.2
11.57
33.12
122.17
162.3
171.33
60.13
593.6
470
460
450
452.6
428.4
12.35
35.87
121.70
158.51
167.87
62.09
594.2
470
460
450
453.8
430.2
12.02
48.05
128.88
139.70
156.69
58.57
595.8
470
460
450
455.8
--
12.11
63.14
133.86
122.08
145.36
--
By use of a UV-260 Shimadzu UV-visible recording spectrometer, which allows autoabsorption maxima of the pigments, sp. coeff., Specific absorption coefficient.
b Wavelengths (nm) of the two major absorption maxima in the red and blue specThe solvents used for the determination of the absorption coefficients were of purest
stadt, FRG, No. 929; diethyl ether (pure solvent), diethyl ether extra pure DAB7 qualresidue analysis, Merck No. 12; ethanol (95%), ethanol for fluorometry, Merck No.
water to 95%; methanol, methanol z.A. (analytical grade), Merck No. 6009, or methac Accepted from Smith and Benitez 49 (for a light path of I cm).
[34]
363
663.2
646.8
618.2
582.4
537.0
470
460
450
431.2
412.6
663.2
646.8
597.6
470
460
450
459.0
(sp. coeff.)
Ethanol,
95% (v/v)
(aqueous sol.)
Methanol,
90% (v/v)
(aqueous sol.)
Methanol
(100%)
(nm)
(sp. coeff.)
(nm)
(sp. coeff.)
(nm)
(sp. coeff.)
86.3
20.49
18.15
10.35
4.10
1.82
2.87
11.62
95.82
78.76
664.2
648.6
84.60
25.06
19.14
665.2
652.4
79.24
35.52
18.68
665,2
652.4
618.2
78.95
35.36
18.84
11.20
49.18
12.80
85.02
134.14
94.89
134.50
664.2
648.6
617.8
.
.
470
460
450
431.6
414.4
600.6
470
460
450
463.6
.
.
617.6
.
.
2.13
4.32
18.42
83.89
76.60
16.0
41.2
11.81
97.64
103.32
69.95
107.45
.
.
470
460
450
431.8
--
.
.
1.63
6.11
28.80
77.05
--
470
460
450
431.8
1.91
5.94
26.37
75.97
417.6
--
665.2
652.4
21.28
38.87
12.74
104.96
87.48
63.07
105.36
665.2
652.4
19.84
35.97
11.78
95.15
82.03
58.83
99.51
602.6
470
460
450
469.2
.
.
602.8
470
460
450
469.2
matic recording and printing of wavelength and absorbance in the major and minor
tral region are in boldface, those of the minor absorption maxima (italics).
obtainable quality: Diethyl ether (water free), diethyl ether dried GR, Merck, Darmity, Merck No. 926; acetone (100%), acetone extra pure, Merck No. 13, and acetone for
973, or ethanol absolute, DABs quality, Roth Karlsruhe, FRG, No. 5054 diluted with
nol dried, Merck No. 6012.
364
PLASTIDS
[34]
T h e r e exist various equations from different authors for different solvents for the determination o f chlorophylls in leaf pigment extracts (see
reviews o f Szestak 42 and Holden43). The disadvantage of these equations
is that the pigment values obtained in one solvent are not comparable with
those o f another. The discrepancies are particularly large in the values for
the ratio o f chlorophyll a/b. The cause o f this is that the equations are
based on rather early determinations of the specific absorption coefficients at times where the resolution o f spectrophotometers was not as
good as today. The widely used equations given by Arnon 31 for 80%
acetone are still based on the specific absorption coefficients of Mackinn e y / 4 Though the equations o f A r n o n may still be used for estimating the
total chlorophyll a + b content, they can never be applied to determine
the ratio o f chlorophyll a/b o f a pigment extract. Several attempts have
been made to correct and modify the equations for acetone (I00 and 80%),
e.g., by Vernon, 45 Ziegler and Egle, 46 and Wintermans and de Mots. 47 Yet
there remained differences in the wavelength position of the red peak
maxima and in the relative absorption o f chlorophyll a at hrn~ of chlorophyll b and vice versa. The same holds true for the determination of
chlorophylls in diethyl ether solutions. The original absorption coefficients o f C o m a r and Zscheile, 48 were redetermined by Smith and Benitez, 49 and their data were confirmed by Falk, 5 who applied a different
technique o f magnesium determination. The equations given by Smith
and Benitez 49 for diethyl ether,
Ca = 10.1A662- 1.01A644
Cb = 1 6 . 4 A ~ - 2.57.4662
[34]
365
More recently, in establishing equations for the simultaneous determination of total carotenoids, together with chlorophyll a and b, of leaf extracts in different solvents 51 we reinvestigated and confirmed the absorption coefficients of Smith and Benitez and adjusted the equations for a
Unicam SP 8000 spectrophotometer:
Ca = 10.05A662 - 0.766A644
Cb = 16.37A644 - 3.14A662
The differences found by authors for the same solvent are mainly due
to (1) the resolution properties of the spectrophotometers applied in the
red spectral region (which will determine the position of hmax of the red
absorption maxima of chlorophyll a/b and the relative absorption at hmax
of the other chlorophyll), (2) the accuracy with which a particular wavelength can be chosen for the absorbancy readings, and (3) the purity of the
solvents: slight differences in the content of water or organic material of
the solvent will largely influence the refractive index as well as height and
broadness of the maxima.
By applying a spectrophotometer of the new generation, which exhibits automatic baseline correction and can be programmed for peak
picking, automatic readings, and printing out of absorbancy readings at
certain wavelengths, the absorption coefficients were again redetermined
using pure solvents of different sources (cf. footnote of Table II). These
new spectrophotometers guarantee a better selection of wavelengths (or
parts of wavelengths) and absorbancy readings. The position of hmaxfor
chlorophyll a and b in diethyl ether, as determined by repetitive recording
of spectra (in a UV-260 Shimadzu spectrometer) of freshly isolated chlorophyll from several plants (with five aliquots per isolation) are at shorter
wavelengths (660.6 and 642.2 nm) than so far reported using other instruments (662 and 644 nm). The new equations for diethyl ether (pure solvent) for the determination of chlorophylls a and b in a UV-260 spectrophotometer are as follows:
Ca = 10.05A660.6 - 0.97A642.2
Cb = 16.36A642.2 -' 2.43A660.6
Based on the redetermined specific absorption coefficients in various
other solvents (Table II), equations are given for the simultaneous determination of chlorophyll a and b in a pigment extract solution (Table III).
The validity of the equations was cross-checked by dissolving the same
amount of plant extract in each of the solvents. The advantage and progress of the new equations is that the pigment values obtained in one
5, H. K. Lichtenthaler and A. R. Wellburn, Biochem. Soc. Trans. 603, 591 (1983).
366
PLASTIDS
[34]
TABLE III
EQUATIONS FOR THE DETERMINATIONS OF THE CONCENTRATIONS OF CHLOROPHYLL a
(Ca), CHLOROPHYLL b (Cb), OF TOTAL CHLOROPHYLLS (Ca+b)AND OF TOTAL
CAROTENOIDS (Cx+c) IN LEAF PIGMENT EXTRACTS FOR SOLVENTS OF DIFFERENT
POLARITY AND WATER CONTENT a
205
Diethyl
Ca
Cb
Cab
213
Diethyl ether (water saturated):
Ca
= 10.36A66L6- 1.28A643.2
Cb = 17.49A643.2- 2.72A66L6
Ca+b = 7.64A66L6 + 16.21A643.z
C~+~ = 1000A470- 1 . 3 8 C a - 48.05Cb
211
Ethanol, 95% (v/v):
C,
= 1 3 . 3 6 A ~ , 2 - 5.19A64s.6
Cb = 27.43As4s.6 - 8.12A~.2
Co+b = 5.24A~.2 + 22.24A~.6
C~+c = 1000A470 - 2.13C~ - 97.64Cb
Methanol,
Ca =
Cb =
Ca+b =
Cx+c =
90% (v/v):
16.82A665.2- 9.28A652.4
36.92A652.4- 16.54A665.2
0.28A665.2 + 27.64A652.4
1000A47o- 1.91Ca- 95.15Cb
225
209
a The equations are based on the redetermined specific absorption coefficients listed in
Table II. The pigment concentrations obtained by inserting the measured absorbance
values are micrograms per milliliter plant extract solution.
Spectral Characteristics
of Carotenoids
General Remarks
All chloroplast carotenoids exhibit a typical absorption spectrum
w h i c h is c h a r a c t e r i z e d b y t h r e e a b s o r p t i o n m a x i m a ( v i o l a x a n t h i n , n e o x -
[34]
.Q
o
.Q
<
367
0.500
0.500
'\
0
300.0
-~-~
350.0
450.0
0
550.0
Wavelength (nm)
FIG. 7. Absorption spectrum of the major leaf carotenoids in diethyl ether. The wavelengths at the absorption maxima are given in Table V. /3-Carotene (--), lutein ( - - - ) ,
violaxanthin (.-.), and neoxanthin (-'..).
368
PLASTIDS
[34]
.--.
j:
fll
t
-s
.:
"
t
?
.-
",~
I
t
t
"
:'
~:
to
,<
IX.
6606 0
460
wovelength
[nm]
FIG. 8. Photoacoustic spectra ~3 of leaf pigments as measured on a silica gel plate after
HPTLC of leaf pigments. 34 Chlorophyll a ( ~ ) , chlorophyll b (....), fl-carotene (- - - -).
(Spectra taken by E. Nagel, Karlsmhe.)
SpecO~cAbsorption Coefficients
The molecular weights of carotenoids differ due to the differential
oxygen content (B-carotene, 536.85, lutein, 568.85, violaxanthin, 600.85,
neoxanthin, 600.85). The specific absorption coefficients for the individual carotenoids found in literature vary considerably. Most of them lie in
[34]
369
the range of 2400 to almost 2600 for a 1% solution and a light path of 1 cm
(A I~
1 cm values). To avoid the problem of the proper coefficient, it was
recommended to apply for the sum of leaf carotenoids 54 and for all individual carotenoids 55 an ,11
AI~cm of 2500 at their main absorption maximum.
This is still the best approach today. The specific absorption coefficients
also vary depending on the type of solvent and its water content. Per
.41%
definition the .,1
~c m of 2500 for the individual carotenoids is determined
here with diethyl ether (pure solvent) as a reference basis.
370
PLASTIDS
[34]
TABLE IV
RATIOS OF THE SPECIFIC ABSORPTION COEFFICIENTS AT ~.maxIN THE BLUE TO hmax IN
THE RED FOR CHLOROPHYLL a AND b AND AVERAGE ABSORPTION COEFFICIENTS Alc~m
AT 470 nm FOR TOTAL LEAF CAROTENOIDS (XANTHOPHYLLS PLUS CAROTENES, X + C) IN
SOLVENTS OF DIFFERENT POLARITY AND WATER CONTENT
Ratio of the
specific coefficients
at 7~m~xblue to hmaxred
Solvent
Chlorophyll a
Chlorophyll b
Total
carotenoids (x + c):
A lcm at 470 nma
Diethyl ether
(water free)
Diethyl ether
(pure solvent)
Diethyl ether
(water saturated)
Acetone
(pure solven0
Acetone, 80% (v/v)
(aqueous solution)
Ethanol, 95% (v/v)
(aqueous solution)
Methanol
(pure solvent)
Methanol, 90% (v/v)
(aqueous solution)
1.28
2.75
2130
1.26
2.71
2050b
1.23
2.69
2110
1.22
2.81
2140
1.11
2.73
1980
0.99
2.61
2100
0.97
2.71
2210
0.96
2.66
2250
a The mean values listed are based on the relative carotenoid composition of green leaf
tissue from several plants (e.g., radish, spinach, tobacco, maize, oat, and wheat).
b This value consists of the relative absorption of the individual leaf carotenoids (/3carotene, lutein, violaxanthin, and neoxanthin) at 470 nm as compared to the absorption maximum. An absorption coefficient Alcm
1~ of 2500 in diethyl ether at the main
absorption maximum (between 436 and 450 nm) was assumed for all individual leaf
carotenoids.
c h l o r o p h y l l a a n d b s o l u t i o n s , a n d t h e w a v e l e n g t h d e v i a t i o n at ~kmax a n d
t h e shift at 470 n m b e a c c o u n t e d f o r ( s e e a d j u s t m e n t b e l o w ) . F u r t h e r
requirements are clear solutions and a correct measurement of the baseline.
T h e r e c o r d i n g o f t h e full a b s o r p t i o n s p e c t r u m o f t h e p i g m e n t e x t r a c t
s o l u t i o n b e t w e e n 750 a n d 450 n m is n e c e s s a r y to b e a b l e to j u d g e f r o m t h e
shape of the spectrum whether one measures a clear pigment solution. In
fully clear pigment extract solutions of normal green leaves the absorb e n c y r e a d i n g s at 750 n m s h o u l d b e z e r o , a n d in t h e r a n g e o f 550 to 510 n m
[34]
CHLOROPHYLLSAND CAROTENOIDS
371
they should be very low and amount to far less than 10% of the absorbancy readings at the red absorption maximum of the extract (in the range
of 660-665 nm). If this is not the case, a centrifugation step (caution: use
closed flasks to avoid evaporation of the solvent) has to be applied.
Strictly sticking to another rule is essential for correct pigment values:
after the recording of the full spectrum the absorbancy readings need to be
performed by setting the proper wavelengths (absorption maxima of chlorophyll a and b as well as 470 nm) on the spectrometer and by marking the
height of the absorbancy by a line with the help of the spectrometer pen.
The measurement of the absorbancy on the recording paper, after the
spectrum had been recorded, by trying to visually determine the proper
wavelengths, is to be avoided. It results in errors up to 20 or 25% of the
chlorophyll b and total carotenoid amounts as well as of the ratio of
chlorophyll a/b.
The determination of the concentration of x + c in the total pigment
extract is the fastest and therefore best method and should always be
applied as a reference before other methods, HPLC or TLC, which allow
determination of individual carotenoids, are applied. TLC with subsequent elution and spectrophotometric determination of separated carotenoids always yields lower values (for B-carotene about - 5 % and for
xanthophylls up to - 13%). The sum of carotenoids determined by evaluation of the individual carotenoid peaks obtained by HPLC often does not
add up to the x + c content obtained by the direct measurement of the leaf
extracts or exceeds it to a large extent, depending on where the baseline is
drawn on the H P L C scans. It is therefore recommended to proof the
values for individual carotenoids obtained by HPLC with the TLC technique 34 mentioned above (see Chapter 2).
The total sum of carotenoids can also be determined in a pigment
extract together with pheophytin a and b, provided that the absorbancy
readings are performed within 10 min after pheophytinization (e.g., with
HC1). A longer standing in acid solution will progressively destroy carotenoids. The x + concentrations obtained using this method (see equations in Table VII) represent a close but only a first approximation. Since
the spectra of pheophytins change with the acid content, one should stick
to the standard conditions (see The Pheophytins a and b, p. 373).
Al%
1 cm o f
The A [~m values at 470 nm given in Table IV and applied in the equations for Cx+c in Table III are calculated from the relative absorbance of
the individual carotenoids as compared to their absorbance at the main
absorption maximum. For the latter a common absorption coefficient of
372
PLASTIDS
[34]
EL
0
Z
>
.J
0
z
,..1
O
0
e~
>
1.-,
a~
0
E
..g
0
,.J
0
@
N
m
,..1
>
[34]
373
374
PLASTIDS
[34]
nizer should therefore be kept very short (20 to 30 sec) and lie far below 1
min. It is also recommended to perform the extraction with solvents
which have been stored in the cold room (e.g., at 3 to 5).
Once a pigment extract has been prepared, the chlorophyll content
should be determined immediately thereafter. The level of pheophytin a
and b will increase with increasing storage time of the extract. This is
particularly the case in organic solvents which are miscible with water
such as acetone, methanol, or ethanol. In cases where the storage of a
pigment extract is required, it should only be done in a water-free organic
solvent, i.e., petroleum hydrocarbon (e.g., bp 40 to 70) to minimize
pheophytinization. This can be accomplished in a separatory funnel by
transferring the pigments of the aqueous extraction solvent, adding petroleum hydrocarbon and water, into the organic hydrocarbon epiphase. The
aqueous acetone (methanol or ethanol) hypophase should contain at least
50% water to guarantee a quantitative transfer of the more polar pigments
(chlorophyll b and xanthophylls) into the water-free epiphase also.
5s A.
[34]
375
Equations in 80% acetone (transformation with oxalic acid) were determined by Vernon45:
20.15A663 - 5.87A655
Cph b = 31.90A655 - 13.40A666
Cpha+b = 6.75A663 + 26.03A655
Cpha ~-
376
PLASTIDS
[34]
Z
ga
t~
I~ ~
t~
0
u~
>
,.d
0
r/)
~l ~ ~i~ ,~ ~l ~ l ~ l
zz
~z~
;0 <
~ r.,.)
e~
~1~ " ~
~l ~
~qeq
r--
m
0
r~
e~
<
6.
m
~g
e q o o
,0
[34]
377
TABLE VII
EQUATIONS FOR THE DETERMINATION OF THE CONCENTRATIONS OF PHEOPHYTIN a (Cph a),
PHEOPHYTIN b (Cphb), TOTAL PHEOPHYTINS (Cph a + b), AND OF TOTAL CAROTENO1DS (Cx + c) IN
LEAF PIGMENT EXTRACTS, IN WHICH THE CHLOROPHYLLS a AND b HAD BEEN COMPLETELY
TRANSFORMED TO PHEOPHYTINS a
Diethyl e t h e r (pure solvent) b
Cph,
= 17.87A666.6- 3.58A654.2
Cphb
= 24.62A6s4.2 - 8.46A666.6
Cpha + b = 9.41A666.6 + 21.03Aas4.2
Cx + c = 1000A470 - 4.39A666.6 - 6.44A654.2
A c e t o n e , 80% ( a q u e o u s , v / v )
Cph a
~--- 22.42A66L4 -- 6.81A653.4
Cphb
= 40.17A653.4 - 18.58A665.4
Cph,,+b = 3.84A665.4 + 33.36A653.4
C, + ,. --- 1000A470 - 4.28A~5.4 - 4.78.4653.4
164
174
Diethyl e t h e r ( w a t e r saturated) c
Cpha
= 18.22A666.6- 3.15A654.0
Cphb
= 32.33A654 - 10.14At~6.6
Cph~+b = 7.08A666.6 + 29.18A654
Cx+,, = 1000A47o - 7.09A666.6 - 4.51A654
M e t h a n o l (pure solvent)
Cph a
= 43.77A654.2 - 33.40Ae476
Cphb
= 50.71A647.6 - 36.32A654.2
Cpha+b = 7.45A654.2 + 17.31A6476
C~+,
= I000A470 - 1.53A654.2 - 1.57A647.6
160
135
E t h a n o l , 95% (v/v)
Cpna
= 42.41A666.2
Cphb
= 55.67A654
Cph~+b = 32.39A654
C~ + c = 1000A470 -
- 23.28A654
- 45.53A6622
- 3.12A662.2
4.32A662.2 - 7.30A654
Methanol,
Cpho
Cph b
Cph, + b
Cr + ~
90% ( a q u e o u s , v / v )
= 306.8A655.2 - 294-2A6s3.4
~ 442.2A653.4 - 429.4A655.~
=
148A653.4 -- 122.6A655.2
= 1000/147o - 2.66,4655.2 - 6.38,46.53.4
160
126
Cph ~ + b = 23. IA654
A c e t o n e (pure solvent)
Cpha
= 516.7A653.4
Cob b
= 732.5A652.6
Cpha + b = 321.3A652.6
C~ + c = 1000A470 -
- 501.2A652.6
-- 725. IA~3.4
- 208.4A653.4
2.02A653.4 - 5.97A652.6
148
Cpha + b = 22.3A653
378
PLASTIDS
[34]
[34]
379
1
0
U
C
q
0.500
0.500
iI
<
/ :,
qL~~!ili!l
so.o
4so.o
sso.o
6so.o
7so.o
Wavelength (nm)
FIG. 9. Absorption spectra of freshly isolated pheophytin a (solid line) and pheophytin b
(broken line) in diethyl ether. The positions of the absorption maxima are given in Table VI.
In acetone (100%), methanol (100 and 90%), and ethanol (95%) the red
absorption maximum of chlorophyll a, however, shifts to shorter wavelengths upon pheophytinization with HCI (see Tables II and VI). The blue
maximum only shifts to ca. 418 nm with a considerable increase in the
absorption in the place of 411 nm in acid-free solution. By increasing
addition of water there is a change in the spectral characteristics of
pheophytin a with the appearance of the proper spectra of the acid-free
pheophytin. Correspondingly the coefficient ratio (hrnax blue to ~r,x red) is
much higher for pheophytin a in acid solution than under neutral conditions (see Table VIII). The differential spectral behavior of pheophytin a
in acid and neutral solution is a particular property and has nothing to do
with incomplete pheophytinization. By addition of one drop of HCI to a
neutral solution of isolated pheophytin a, one changes the absorption
spectrum to that of pheophytin a in the presence of acids (see Fig. 10).
For pheophytin b a shift to shorter wavelength only occurs in 100%
methanol. In the other solvents one obtains even in the presence of some
HC1 spectra which are fairly similar in wavelengths and absorption values
to those of the acid-free solvent solution. This indicates that the spectrum
of pheophytin b - - w i t h the exception of pure methanol solutions--is less
sensitive to changes in an acid milieu than that of pheophytin a.
380
PLASTIDS
[34]
TABLE VIII
RATIOS OF THE SPECIFIC ABSORPTION COEFFICIENTS a AT )~rnax
IN THE BLUE TO )Lrn~ IN THE RED SPECTRAL REGION FOR
PHEOPHYTIN a AND b
Pheophytin a
Pheophytin b
2.10
5.02
2.08
5.03
2.36
5.07
4.25 (2.15)
5.10 (4.91)
3.33 (2.09)
4.31 (4.99)
4.09 (2.08)
4.91 (4.72)
3.85 (2.12)
4.03 (4.57)
[34]
CHLOROPHYLLSAND CAROTENOIDS
381
1.0'
417!!
!t li
ethanol 95%
--
,. li
ii
,~ Ii
HCt
5%
........
HCI
25%
os
/i
/.."
'
666.6
/-
as0
662-
~g0
/J':
~ "sg0
" "696 ....
7s0
wovelength (nm )
FIG. 10. Absorption spectrum of pheophytin a in aqueous (95%) ethanol (solid line) and
after addition of one droplet (-0.005 ml) of a 5 % ( - - - ) and of one droplet of a 25% HCI
solution (' "").
"
aCa + b'Cb
bCb + a' Ca
A470n m = a"Ca + b"Cb + kCx+c
a
Amax b
=
=
(1)
(2)
(3)
382
PLASTIDS
[34]
(4)
(5)
Ca = [(b/Z)Amaxa] - [(b'/z)Amaxb]
Cb = [(a/Z)Amax b] - [(a'/Z)Amaxa]
where
z = ab - a ' b '
=
=
=
=
aEa/lO0
aE470/lO0
bEb/lO0
bE470~ 100
Ea = % a b s o r b a n c e of chl a
E470 = % a b s o r b a n c e o f chl
Eb = % a b s o r b a n c e of chl b
E470 = % a b s o r b a n c e of chl
at hmax chl b
a at 470 nm
at hmx chl a
b at 470 nm
B y inserting the recalculated values for a ' and b' in Eqs. (4) and (5)
one obtains the n e w equations for Ca and Cb, which are adjusted to the
particular s p e c t r o p h o t o m e t e r .
T h e equation to determine the concentration of total carotenoids Cx+c
(/zg/ml) is then obtained using the redetermined a" and b" values:
Cx+c = (1000A470 - a"Ca - b"Cb/O.1 AlUm o f x + c
A recalculation o f the A 1~
cm for x + c at 470 requires isolation o f the
total leaf carotenoids (e.g., b y T L C 33) in diethyl ether t,-,tal%l
c m for x + c at
hm~, = 2450) and m e a s u r e m e n t o f the relative proportion o f their absorption at 470 n m as c o m p a r e d to hmax o f x + c. The "1
A 1~
~cm value in diethyl
ether (2450) is used as reference.
Acknowledgments
The establishment of the absorption coefficients was made possible by the kindness of
Shimadzu Germany, Diisseldorf (Messrs. Steinfeld and Baumann), who for several weeks
provided a new UV-260 UV-visible two-beam spectrometer; this is gratefully acknowledged. My particular thanks are due to Dr. Claus Buschmann for writing the programme for
the HP 85 and to my son, Stefan Lichtenthaler, for programming the Apple IIe computer and
doing most of the calculating and cross-checking. Finally I would like to thank Mr. E. Nagel
for measuring the photoacoustic spectra of pigments as well as Mrs. U. Prenzel and U.
Sieber for excellent assistance.