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Isolation and Spectrophotometric Characterization o f hydrocarbon side chain enhances the solubility of chlorophyll in
Photosynthetic Pigments nonpolar solvents. The only difference between chlorophyll a
and b is the substituent on position 3 (ring 3): chlorophyll a has a
RODNEY F BOYER methyl group, whereas b has an aldehyde. The chlorophylls are
biosynthesized by condensation of ~-aminolevulinic acid in a
Department of Chemistry process similar to the synthesis of heme in mammals. In the
Hope College living plant cell, the chlorophylls serve as the primary photo-
Holland, MI49423-3698, USA synthetic pigments. They absorb light in the blue (450 nm) and
red regions (650-700 nm). Absorption of light, which excites an
electron in the chlorophyll molecule, provides energy for
Introduction initiation of the photosynthetic process producing NADPH and
When students in the biochemistry laboratory are introduced to ATP to be used for carbohydrate biosynthesis by the plant.
the extraction and characterization of biomolecules, the tissue of The second group of plant pigments, the carotenoids, can be
choice is most often of animal origin. Plant products are seldom divided into two different types, (1) the carotenes which contain
used; therefore, students rarely gain knowledge and appreci- only carbon and hydrogen, and (2) the xanthophylls which
ation of the unique biomolecules present in the plant kingdom. contain carbon, hydrogen and oxygen atoms in the form of
In order to fill in this gap, we have designed an experiment in hydroxyl or epoxide functional groups. The structures for several
which students isolate a mixture of pigments by solvent major carotenoids are shown in Fig 2. Note that they all contain
extraction of plant tissue, separate some of the components by 40 carbon atoms and, because of the extensive conjugation,
chromatography and identify them by visible region spectro- would be expected to be highly colored. Most carotenoids are
photometry. Students who complete this experiment gain knowl- yellow, red or orange, but some are green, pink and even black.
edge and technical skills in biomolecule extraction, chromato- Many of the bright colors found in flower petals are due to the
graphic methods, spectrophotometric techniques and plant presence of carotenoids (although some are due to antho-
pigment structure. Students will follow procedures which are cyanins). The yellow colors of fall foliage are due primarily to
typical for the general extraction and characterization of lipids. preferential destruction of the green chlorophylls, revealing the
However, unlike most lipids (especially those in animals), the carotenoid color. 6
plant pigments are highly colored and may be characterized and The percentage composition of carotenoids in plants varies
quantified by visible spectrophotometry. with growing conditions. The average weight % ranges are: 13-
carotene, 25-40%; lutein, 40-60%; violaxanthin, 10-20%; and
Theory neoxanthin, 5-13%. 2 The carotenoids are biosynthesized by
Many of the colors associated with higher plants (green leaves in condensation of acetylCoA through the mevalonic acid pathway.
the spring and summer, yellow or red leaves in the fall, the The physiological function of the carotenoids is not completely
orange color of carrots, some colors in flower petals) are due to understood; however, two probable functions have been
the presence of pigment molecules. Review articles on plant described. First, the carotenoids, particularly the xanthophylls,
pigments have been recently written by Goodwin 1 and Lichten- may participate in light adsorption for photosynthesis. It has
thaler. 2 General information on photosynthetic pigments can been shown that illuminated xanthophylls are able to transfer
also be obtained from the standard biochemistry texts by excitation energy directly to chlorophyll a. Secondly, the
Mathews and van Holde, 3 Stryer, 4 and Voet and Voet. 5 carotenes may inhibit the photo-oxidative destruction of chloro-
The major photosynthetic pigments of higher plants can be phyll, a process which consumes oxygen.
divided into two groups, the chlorophylls and the carotenoids.
Both types of pigments are present in the subcellular organelles
called chloroplasts, where they are bound to proteins in the
thylakoids, the photochemically active photosynthetic biomem-
branes. Intact pigment-protein complexes, which are held
together by weak, noncovalent bonds, have been isolated from F-Carotene
chloroplasts and characterized b~( polyacrylamide gel electro-
phoresis and isoelectric focussing/The pigments are released in
a protein-free form by grinding plant tissue in solvents such as
acetone, methanol or hexane. Since the chlorophylls and the
carotenoids are readily soluble in organic solvents, they are
classified biochemically as lipids.
The most abundant plant pigments are chlorophyll a and Lutein
chlorophyll b which occur in a ratio (a:b) of approximately 3:1.
As is shown in Fig 1, the chlorophylls possess a porphyrin ring
with a coordinated magnesium atom at its center, a fused, 5- ~ ~ ~ T ~ ~ ~ o OH
membered ring and a C20 phytyl side chain. This nonpolar,
HO
HzC=CH H C~ H2CffiCH H H-C-O
"3~c#. H~C~ceH
H H
a
Violaxanthin
o -oc.. -'Lo OH
CLN3H~I Offi~-OCH3
Chlorophyll a Chlorophyll b Neo~mnthln
Figure 1 Structures of chlorophyll a and chlorophyll b (From Figure 2 Structures of major carotenoids: B-carotene, lutein,
Moore7) violaxanthin and neoxanthin (From Lichtenthaler2)
l.(a:
[~.9"
B.B~
u :
~_~.~. -~_~Z:- \ ~" o
11
aoo.o ~o.o 4so.o sso.o
~Q. 4 Wavelength (nm)
of a mixture of chlorophylls a and b, f3-carotene, lutein, In this paper we describe the simulation of radioactive tracer
neoxanthin, and violaxanthin. Assume that the solvent is the incorporation and distribution. The simulation can be used in
same as used in this experiment. isolation, and makes a useful introduction to the topic of
(3) Repeat Question 2 (above) except substitute paper chroma- metabolic analysis by using labelled precursors. However, most
tography with petroleum ether:acetone (9:1) as developing benefit will be derived if an actual experiment is carried out in
solvent. conjunction with the simulation. We therefore present a brief
(4) How does acetone disrupt the plant pigments from their description of a suitable experimental system which we have
natural state as pigment-protein complexes in the chloroplasts? found reliable.
(5) What extraction solvent system would you use if you wanted
to isolate the intact protein-pigment complexes? Describe Experimental System
experimental techniques which could be used to determine the The experiment involves photosynthetic incorporation of radio-
molecular weight of each protein-pigment complex. active CO2 by algal cells. Strains of Chlorella or Chlamydomonas
are probably most useful, although any photosynthetically
References competent alga could be used. Details of suitable strains, media
tGoodwin, T W (1983) 'Introduction to Plant Biochemistry', Second and growth conditions can be found in references 3-5.
Edition, Pergamon Press, Oxford, pp 96-103 The experiment requires access to a scintillation counter and
2Lichtenthaler, H K (1987) in 'Methods in Enzymology', Academic facilities to work with 14COa. The labelling apparatus is simple to
Press, New York, vol 148, pp 350-382 construct, and modifications may be incorporated to suit
3Mathews, C and van Holde, K (1990) 'Biochemistry', Benjamin- materials available. A key safety point is that release of 14CO2 to
Cummings, Redwood City, CA, pp 643-669 the atmosphere should be prevented. A suitable apparatus is
4Stryer, L (1988) 'Biochemisty', Third Edition, W H Freeman, New shown in Fig 1.
York, pp 517-533
5Voet, D and Voet, J (1990) 'Biochemistry', John Wiley and Sons, New
York, pp 586-594
[
6Goodwin, T W (1958) Biochem J 68, 503-511
7Moore, T C (1981) 'Research Experiences in Plant Physiology - - A
U c
Laboratory Manual', Second Edition, Springer-Verlag, New York, pp
35-45 E
B I O C H E M I C A L E D U C A T I O N 18(4) 1990