全 文 ：Received 2 Apr. 2004 Accepted 27 Jul. 2004
Supported by the Knowledge Innovation Project of Institute of Botany, The Chinese Academy of Sciences, the Knowledge Innovation
Program of The Chinese Academy of Sciences (KSCX2-SW-307), the National Natural Science Foundation of China (60308004), and a grant
from Key Laboratory of Experimental Marine Biology at Institute of Oceanology, The Chinese Academy of Sciences.
* Author for correspondence. Tel: +86 (0)10 62591431-6229; Fax: +86 (0)10 82599636; E-mail: .
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Acta Botanica Sinica
植 物 学 报 2004, 46 (10): 1192-1199
Characterization of the Main Light-Harvesting Chlorophyll a/b-Protein
Complex of Green Alga, Bryopsis corticulans
CHEN Hui1*, SHEN Shi-Hua1, GONG Yan-Dao2, HE Jun-Fang3, WANG Guang-Ce4,
LI Liang-Bi1, KUANG Ting-Yun1
(1. Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany,
The Chinese Academy of Sciences, Beijing 100093, China;
2. Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China;
3. State Key Laboratory of Transient Optics and Technology, Xi’an Institute of Optics and Precision Mechanics,
The Chinese Academy of Sciences, Xi’an 710068, China;
4. Key Laboratory of Experimental Marine Biology, Institute of Oceanology, The Chinese Academy of
Sciences, Qingdao 266071, China)
Abstract: The main light-harvesting chlorophyll a/b -protein complex (LHCⅡ) has been isolated directly
from thylakoid membranes of shiphonous green alga, Bryopsis corticulans Setch. by using two consecutive
runs of anion exchange and gel-filtration chromatography. Monomeric and trimeric subcomplexes of LHC
Ⅱ were obtained by using sucrose gradient ultracentrifugation. Pigment analysis by reversed-phase high
performance liquid chromatography showed that chlorophyll a (Chl a), chlorophyll b (Chl b), neoxanthin,
violaxanthin and siphonaxanthin were involved in LHCⅡ from B. corticulans. The properties of electronic
transition of monomeric LHCⅡ showed similarities to those of trimeric LHCⅡ. Circular dichroism
spectroscopy showed that strong intramolecular interaction of excitonic dipoles between Chl a and
between Chl b exist in one LHCⅡ apoprotein, while the intermolecular interaction of these dipoles can be
intensified in the trimeric structure. The monomer has high efficient energy transfer from Chl b and
siphonaxanthin to Chl a similarly to that of the trimer. Our results suggest that in B. corticulans, LHCⅡ
monomer has high ordered pigment organization that play effective physiological function as the trimer,
and thus it might be also a functional organization existing in thylakoid membrane of B. corticulans.
Key words: light-harvesting protein complex; monomer; pigment composition; pigment interaction;
Bryopsis corticulans
Light harvesting of plants and green algae is carried out
by an array of light-harvesting chlorophyll (Chl) a/b com-
plexes (Simpson and Knoetzel, 1996). Understanding of light
harvesting function of the antenna requires recognition of
the organization of subunits and the knowledge of absorp-
tion/fluorescence energy levels. In higher plants, the main
light-harvesting chlorophyll a/b complex of photosystem
Ⅱ (LHCⅡ) is located mainly in oppressed regions of the
thylakoid membrane. The recent structural model shows
the positions of eight Chl a, six Chl b and three xantho-
phylls of lutein, neoxanthin and violaxanthin bound to the
complex polypeptides (Liu et al., 2004). The functional or-
ganization of LHCⅡ in thylakoids is so far believed to
have a trimeric structure, in which various permutations of
LHCⅡ apoproteins, which are encoded for by nuclear
genes (Green et al., 1991; Green et al., 1992), constitute
different trimeric subcomplexes (Spangfort and Andersson,
1989; Nussberger et al., 1993; Jackowski and Janson 1998).
Analysis of the chlorophyll organization in LHCⅡ trimers
have been carried out by absorption, linear dichroism, cir-
cular dichroism, fluorescence and polarized excitation spec-
tra (Hemelrijk et al., 1992; Kwa et al., 1992; Jennings et al.,
1993; Amerongen et al., 1994; Rogl and Kühlbrandt, 1999;
Jackowski and Pielucha 2001).
There has been evidence that LHCⅡ monomer exists
in vivo in thylakoid membrane (Kolubayev et al., 1986;
Garab et al., 1991; Bassi and Dianese, 1992). As many re-
searches focus on structure and function of LHC Ⅱ trimer,
the role of LHCⅡ monomer, which is the basic unit of
those trimers in thylakoid membrane, is still unknown, since
it was thought as a released product by treatment of deter-
gent in protein purification procedure. The monomers
CHEN Hui et al.: Characterization of the Main Light-Harvesting Chlorophyll a/b-Protein Complex of Green Alga,
Bryopsis corticulans 1193
released by treatment of trimers with detergents, proteoly-
sis and phospholipase have been studied and significant
differences of spectroscopic characteristics between mono-
mer and trimer have been observed (Nussberger et al., 1994).
Therefore the investigation of structure and function of
LHC Ⅱ monomer is of interest.
Bryopsis corticulans is a siphonous green alga grow-
ing in intertidal areas, where periodic changes of light ac-
cepted by the green alga during a cycle of tides may de-
mand that LHCⅡ of the alga operate with some mecha-
nism to adapt the drastically changing light. Therefore the
photosynthetic apparatus of the alga probably exhibit some
unique properties that clearly distinguish them from those
of higher plants. We obtained LHCⅡ monomers in large
proportion in purification of LHCⅡ from thylakoid mem-
branes of B. corticulans. In the present work, we investi-
gated the pigment organization and energy transfer of mo-
nomeric and trimeric LHCⅡ by combination of absorption,
circular dichroism and fluorescence spectroscopies. Com-
parison of the spectroscopic characteristics of the mono-
mer with those of the trimer provided evidence of
intramonomeric pigment interactions and intermonomeric
pigment interactions in LHCⅡ. These are important for a
further understanding of the relation between structure and
function of LHCⅡ.
1 Materials and Methods
1.1 Purification of LHCⅡ
Bryopsis corticulans Setch., a shiphonous green alga,
was collected in tideland near Qingdao in China. All the
following procedures were performed in dim light at 4 °C.
The whole plant of the green alga was homogenized
and osmotically broken in TSN buffer (10 mmol/L Tris-HCl,
pH 8.0, 200 mmol/L sucrose, 10 mmol/L NaCl) followed by
filtration through 8 layers of gauze and centrifuged at
10 000g for 10 min. The pellets were suspended in 10 mmol/
L Tricine-NaOH, pH 8.0 and the broken membrane frag-
ments were collected by a centrifugation at 12 000g for 10
min. For selective solubilization, the membrane fragments
were treated with 3% n-Octyl-b-D-glucopyranoside (OG)
in TMK buffer (20 mmol/L Tricine-NaOH, pH 8.0, 40 mmol/
L MgCl2, 80 mmol/L KCl) for 15 min at a chlorophyll con-
centration of 1 mg/mL. After a centrifugation at 180 000g to
remove unsolubilized membrane fragments, the superna-
tants were subjected to liquid chromatography.
Applied the supernatants to anion exchange chroma-
tography on Q Sepharose Fast Flow column (from
Amersham Biosciences) equilibrated with TMK buffer con-
taining 0.05% n-Dodycel-b-D-Maltoside (DM). Eluted the
column with same buffer at a linear gradient of 0.1-1.0 mol/
L NaCl. The LHCⅡ enriched fraction was pooled up to-
gether and concentrated with AMICON Centriprep-50 (50
kD cut off). Loaded the concentrated sample on Superose
12 column (from Amersham Biosciences) to implement gel-
filtration chromatography. Eluted isocratically the column
with TMK buffer containing 0.05% DM.
In order to separate LHCⅡ monomer from the other
oligomers of LHCⅡ, the LHCⅡ enriched fraction by gel-
filtration chromatography was loaded on top of linear su-
crose gradients prepared with 5%-25 % sucrose in TMK
buffer containing 0.05% DM. The gradients, onto which
were layered 1 mL of the LHCⅡsamples at a chlorophyll
concentration of 1 mg/mL, were centrifuged at 270 000g for
12 h with SW 40 rotor (from Beckman).
1.2 SDS-PAGE
Polypeptide composition was analyzed on 12% poly-
acrylamide gel containing 1% SDS and run in Laemmli sys-
tem (Laemmli, 1970). The gel was stained with silver nitrate.
1.3 Pigment analysis
Pigment analyses of samples extracted with 80% acetone
were performed by reversed-phase high performance liq-
uid chromatography (RP-HPLC) on Discovery C18 column
(4.6×250 mm I.D., from Supelco) according to the method
described by Thayer and Bjorkman (1990). The eluent was
composed of acetonitrile, methanol and ethyl acetate, as in
the original method (Thayer and Bjorkman, 1990) but the
program was modified as follows: from 0.0-14.5 min, aceto-
nitrile-methanol (85/15, V/V) was run isocratically, followed
by a 2 min linear gradient until the proportion of methanol-
ethyl acetate (68:32) was reached. This composition was
held for 15.5 min. All reversed phase HPLC was performed
at room temperature. The flow-rate was 1 mL/min. All sol-
vents were of HPLC grade and purchased from Fisher.
1.4 Analytical procedures
Protein concentration was determined by using BCA
Protein assay kit (from Pierce). Chlorophyll concentration
was determined according to Lichtenthaler’s method
(Lichtenthaler, 1987).
1.5 Spectroscopies
Room-temperature absorption spectrum was recorded
on an UVKON-943 double beam UV/VIS spectrophotom-
eter at a spectral bandwidth of 2 nm. Steady state fluores-
cence measurement was performed in a tiny testing tube.
The fluorescence emission spectra were recorded for the
fluorescence excitation at 436, 480 and 530 nm, respectively,
with an emission bandpass of 0.2 nm, in a Hitachi F-4500
fluorometer at 77 K. Chlorophyll content was adjusted to
15 mg/mL. Circular dichroism (CD) spectra were measured
Acta Botanica Sinica 植物学报 Vol.46 No.10 20041194
at room temperature with a Jasco-720 dichrograph using a
bandpass of 0.5 nm and were expressed in units of
absorbance.
2 Results
2.1 Purification of LHCⅡ
LHCⅡ has been isolated directly from thylakoid mem-
branes of B. corticulans by two consecutive runs of liquid
chromatography, namely anion exchange chromatography
on Q Sepharose Fast Flow column and gel-filtration chro-
matography on Superose 12 column. The polypeptide com-
positions of membrane extracts, LHCⅡ enriched fractions
respectively from Q Sepharose column and Superose 12
column are shown in Fig.1, indicating that the purification
method described in Materials and Methods was effective
for isolation of LHCⅡ from thylakoid membranes of B.
corticulans by a direct method, which is faster and readily
controlled. Comparison of polypeptide composition of LHC
Ⅱ preparation from Superose 12 column (Fig.1C) with that
from Q Sepharose column (Fig.1B) showed an improve-
ment of the purity of the LHCⅡ preparation by using
Superose 12 column chromatography. With two consecu-
tive runs of liquid chromatography, LHCⅡ has been
isolated directly from the thylakoid membrane in relatively
high purity.
The LHCⅡ was further purified, and monomeric and
trimeric subcomplexes of LHCⅡ were separated from the
LHCⅡ mixes, by 5%-25% sucrose density gradient
ultracentrifugation. Upon sucrose gradient centrifugation,
a monomer band, a trimer band and two bands of aggre-
gates were absolutely separated in the sucrose density gra-
dient (Fig.2). The analysis of polypeptide composition (Fig.
3) showed very high level of the LHCⅡ monomer and
trimer obtained in this study.
2.2 Pigment composition of LHC Ⅱ
The pigment composition of LHCⅡ was analyzed by
using reversed-phase HPLC method. The typical chromato-
grams of the pigment extracts from monomeric and trimeric
subcomplexes of LHCⅡ are shown in Fig.4. Chl a, Chl b,
neoxanthin, violaxanthin and siphonaxanthin were involved
both in monomer and trimer. The ratios of Chl a/Chl b in
monomer and trimer are 1.4 and 1.2, respectively (calculated
with the peak areas of Chl a and Chl b performed in chro-
matograms of reversed-phase HPLC), which are similar to
that of the native LHCⅡ reported in higher plants before
(Kühlbrandt et al., 1983).
Fig.1. Polypeptide compositions of thylakoid membrane of
Bryopsis corticulans (A), LHC II enriched fractions eluted from
Q Sepharose column (B) and LHC II enriched fraction eluted
from Superose 12 column (C). The fractions were resolved on
SDS-PAGE gel and were stained with silver nitrate.
Fig.2. Fractionation of LHC II sample eluted from Superose 12
column on 5% – 25% sucrose linear density gradient.
CHEN Hui et al.: Characterization of the Main Light-Harvesting Chlorophyll a/b-Protein Complex of Green Alga,
Bryopsis corticulans 1195
to former reports (Kolubayev et al., 1986; Jackowski and
Pielucha, 2001), a band at 663 nm (+) and a band at 679 nm
(-) combined into a split-like band, which is characteristic
for Chl a dipoles (Nussberger et al., 1994; Hobe Et al.,
1994), presented in CD spectrum of monomeric LHCⅡ.
2.4 Light harvesting and energy transfer
The antenna function of monomeric LHCⅡwas ana-
lyzed in terms of the fraction of blue light excitation (l ≤
500 nm), which effectively reaches the lowest energy levels
of Chl a. Both monomeric and trimeric LHCⅡ showed en-
ergy equilibration within the Chl pool, as tested by the
invariance of fluorescence emission spectra upon excited
at 436, 480 nm (Fig.7). Chl b–to-Chl a and Chl a-to-Chl a
energy transfers in monomer and trimer were revealed by
their fluorescence emission spectra (Fig.7), respectively. A
negligible difference at 654 nm cannot deny the similar shape
of fluorescence emission spectra between monomer and
trimer and indicate a high efficient energy transfer on a path
of Chl b - to -Chl a - to - Chl a. In addition, the fluorescence
Fig.3. Polypeptide compositions of LHC II monomer band (A)
and trimer band (B) obtained by sucrose density gradient
ultracentrifugation. The fractions were resolved on SDS-PAGE
gel and were stained with Coomassie brilliant blue. Molecular
weight standard shown on the left was from Amersham
Biosciences.
Fig.4. Reversed-phase HPLC chromatograms of pigments ex-
tracted from monomeric (A) and trimeric (B) LHC II, detecting at
440 nm. Peak identification: 1, neoxanthin; 2, violaxanthin; 3,
siphonaxanthin; 4, chlorophyll b; 5, chlorophyll a.
2.3 The organization of pigments bound to LHCⅡ pro-
tein
The absorption spectrum of the LHCⅡ monomer was
characterized and compared with that of the trimer (Fig.5).
Both two LHCⅡ subcomplexes show the main Chl a Qy
absorption band at 672 nm well separated from the Chl b
peak at 654 nm, which is characteristic of native LHCⅡ.
Gaussian deconvolution of the absorption spectrum reveals
that Ca 667 (lmax= 667 nm), Ca 677 (lmax= 677 nm) and Cb
653 (lmax= 653 nm) contributed to the absorptive character-
istics of monomer (Fig.5B). The absorption spectrum of the
trimer was also resolved into similar chlorophyll forms (Fig.
5C). The spectrum of LHCⅡ monomer after normalization
at its red maxima showed similarity of electronic transition
properties in Qy region to those of the trimer.
Three excitonic features, which were less significant in
LHCⅡ monomer spectrum, have previously been found in
LHCⅡ trimers, approximately at 648 nm (+), 478 nm (-) and
415 (+) nm (Hemelrijk et al., 1992; Hobe et al., 1994;
Nussberger et al., 1994). We detected a pronounced differ-
ence between CD spectra of monomeric and trimeric LHC
Ⅱ(Fig. 6). In Soret region, the monomer decreased the bands
at 472 nm (-) and 480 nm (+) in comparison with that from
the trimer. The negative band at 488 nm of monomer has
shifted by 2 nm towards to shorter wavelength. In contrast
Acta Botanica Sinica 植物学报 Vol.46 No.10 20041196
emission spectra excited at 530 nm of monomer and trimer
showed the occurrence of energy transfer from
siphonaxanthin to Chl a in both monomeric and trimeric
LHCⅡ.
3 Discussion
LHCⅡ has been purified for a great number of years.
The protocols of purification of LHCⅡ exist that usually
involved separation of BBY (Berthold et al., 1981) mem-
branes and isoelectric focusing (IEF) electrophoresis. In
this study we developed a new way by which LHCⅡ can
be isolated directly from thylakoid membranes via one step
detergent solubilization and liquid chromatography followed
by sucrose density centrifugation. It is therefore readily
controlled procedure so far for purification of LHCⅡ. The
application of column chromatography in purification of
LHCⅡ can be used to increase the yield of protein
preparation. It will be a positive way to resolve the bottle-
neck of protein yield to crystal growers.
In higher plants, eight chlorophylls (Chls) a, six Chls b,
two luteins and one neoxanthin have been identified and
accurately located within one monomeric LHCⅡ (Liu et
al., 2004). The xanthophylls have been intensively investi-
gated in the last decade. Reconstitution work has demon-
strated the obligatory requirement of two luteins for the
formation of a stable folded complex (Plumley and Schmidt,
1987). In contrast to these reports on higher plants (Berthold
et al., 1981; Plumley and Schmidt, 1987; Liu et al., 2004),
lutein has not been found in LHCⅡ of B. corticulans. This
indicates that lutein was not demanded by LHCⅡ reas-
sembly in B. corticulans, though it widely distributes in
LHCⅡ of higher plants and other green algae.
Fig.5. Absorption spectra of monomeric and trimeric LHC II. A.
Spectra of monomer (--------) and trimer (— — ), normalized to
their Chl a absorption maxima at 672 nm, respectively. B.
Gaussian deconvolution of absorption spectrum of the monomer.
C. Gaussian deconvolution of absorption spectrum of the trimer.
Solid line, absorption spectra; dotted line, Gaussian components;
dashed line, sum of Gaussian.
Fig.6. Circular dichroism (CD) spectra of monomeric (--------)
and trimeric (––––) LHC II from Bryopsis corticulans.
Fig.7. Low temperature (77K) fluorescence emission spectra of
monomeric (A) and trimeric (B) LHC II excited at 436, 480 and
530 nm, respectively.
CHEN Hui et al.: Characterization of the Main Light-Harvesting Chlorophyll a/b-Protein Complex of Green Alga,
Bryopsis corticulans 1197
Siphonaxanthin, which has absorption maximum at 531 nm,
was observed in LHCⅡ of B. corticulans (Fig.4).
The main antenna functions which are implemented by
Chls bound to LHCⅡ proteins are absorption of light en-
ergy and transfer of the energy to the Chls close to the
reaction center of PSⅡ. Thus organization of Chls in the
proteins is critical to functional performance of LHCⅡ.
The features of absorption of LHCⅡ monomer in Qy re-
gion are similar to those of the trimer. Though the spectra
differ marginally in detail, their overall appearance is rather
similar and all distinguishable peaks and shoulders are
present in the spectra of both LHCⅡ subcomplexes.
Deconvolution of the absorption spectra of monomeric and
trimeric LHCⅡ resolved two Chl a forms (Ca667, Ca677)
and one Chl b form (Cb653). Different Chl a forms were
ascribed to different molecular environments surrounding
Chl a in protein structure. Thus, the similarity of chloro-
phyll forms indicates the molecular organization of LHCⅡ
to be similar between monomer and trimer.
The CD spectra were provided to detect the organiza-
tion of pigments bound to the proteins (Fig.6). The fact
that all apparent peaks are seen in the case of monomer and
trimer can be also explained by the organizations of the
pigments in monomeric LHCⅡ was similar to that of trim-
eric LHCⅡ. This is different from the reports in higher
plants, since the signals around 472 nm and 480 nm were
reported missing in monomeric LHCⅡ (Hemelrijk et al.,
1992; Hobe et al., 1994; Nussberger et al., 1994). The nega-
tive band at 472 nm and positive band at 480 nm may origi-
nate from excitonic interactions involving Chl b. The bands
at 472 nm (-) and 480 nm (+), though weaker than that in
trimer, suggest the excitonic interaction between dipoles of
Chl b occurred in monomer, while it can be intensified sig-
nificantly in trimeric structure of LHCⅡ. Meanwhile, in Qy
region, the bands at 663 nm (+) and 679 nm (-) also demon-
strated that the excitonic interaction between Chl a mol-
ecules occurred in both monomeric and trimeric LHCⅡ.
These results indicate that the intramolecular interactions
between dipoles of Chl a molecules and between dipoles of
Chl b molecules occurred in LHCⅡ apoprotein and the
intermolecular interaction of those dipoles can be intensi-
fied in trimeric structure of LHCⅡ.
Comparison of fluorescence emission of LHCⅡ mono-
mer with that of the trimer excited at 430 nm, where Chl b
and xanthophylls absorb but Chl a absorption is nearly
absent, leads to mainly Chl a fluorescence, indicating effi-
cient singlet energy transfer in LHCⅡ monomer as that in
trimer. The fluorescence emission spectra excited respec-
tively at 436, 480 and 530 nm were similar in peak shape
after normalized to their respective fluorescence maxima
(Fig.7). This indicates that a highly efficient energy trans-
fer path of Chl b - to - Chl a - to - Chl a occurred in monomer.
However, the efficiency of energy transfer in the monomer
was different from that in trimer, since small shoulders were
seen at fluorescence emission spectra of monomer excited
at 480 and 436 nm, respectively. We tried to estimate the
efficiency of energy transfer from Chl b to Chl a and Chl a
to Chl a using 77 K fluorescence spectra of the purified
monomer (Fig.7A) and trimer (Fig.7B). The integrated areas
of both bands are then a measure for the relative amounts
of Chl a which gives results of that in monomer the fluores-
cence quantum excited at 480 nm and 436 nm yields for Chl
a 6% and 14%, respectively, less than that in trimer. These
results indicate that monomeric subcomplex of LHCⅡ can
compete with the trimer in absorption and transfer of light
energy in B. corticulans, though the abilities of energy
transfer from both Chl b to Chl a and Chl a to Chl a can be
increased slightly upon trimerization. The fluorescence
emission excited at 530 nm showed an occurrence of en-
ergy transfer from siphonaxanthin to Chl a in LHCⅡ of B.
corticulans (Fig.7), indicating an enhancement of absorp-
tion of blue-green light in the alga.
Previously, significant changes of pigment organization
and obvious Chl a fluorescence quenching were observed
in LHCⅡ monomers obtained by treatments of trimers with
detergents, proteolysis and phospholipase (Nussberger et
al., 1994) and light-induced monomer from trimer (Garab et
al., 2002). These were supposed that lipids surrounded tri-
mer were destructed and Chl a partly lost from the proteins
(Nussberger et al., 1994; Garab et al., 2002). The LHCⅡ
monomer we obtained in this work is supposed to have
intact structure as it exists in vivo, since the characteristic
of the monomer is much different from those of the
artificialized monomers (Nussberger et al., 1994; Garab et
al., 2002). In this work, we have investigated the organiza-
tion and function of monomeric LHCⅡ in comparison with
those of the trimeric LHCⅡ and found evidence for which
the LHCⅡ monomer has high level organization of pig-
ments engaging it to play effective physiological function
as the trimer. The protein patterns of monomeric and trim-
eric LHCⅡ were resolved on SDS-PAGE (Fig.3), showing
an apoprotein with estimated molecular weight of 31 kD is
dominantly present in two LHCⅡ subcomplexes. This in-
dicates that 31 kD protein is mainly responsible for con-
struction and function of LHCⅡ of B. corticulans. Based
on the results that monomeric LHCⅡ has similar perfor-
mance on absorption and transfer of light energy as those
of the trimeric LHCⅡ, we concluded that this 31 kD
Acta Botanica Sinica 植物学报 Vol.46 No.10 20041198
protein with its bound pigments acts as light-harvesting
antenna in B. corticulans. It is suggested that blue-green
light-harvesting antenna system using siphonaxanthin can
be considered to be the most ancient antenna system in
green plants (Yoshii et al., 2003). Therefore, this
siphonaxanthin-Chl a/b-protein of 31 kD might be an evo-
lutionary relic of LHCⅡ in green plants, indicating that
ancient antenna may function without defined structures.
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