全 文 ：Received 20 Oct. 2003 Accepted 7 Jun. 2004
Supported by the State Key Basic Research and Development Plan of China (G1998010100) and Experimental Marine Biological Laboratory
(EMBL) at Institute of Oceanology, The Chinese Academy of Sciences.
* Author for correspondence. Fax: +86 (0)10 82599636; E-mail: .
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (8): 915-920
Isolation of the Main Light-harvesting Chlorophyll a/b-protein Complex
from Thylakoid Membranes of Marine Alga, Bryopsis corticulans
by a Direct Method
CHEN Hui1*, SHEN Shi-Hua1, WANG Guang-Ce2, 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. Key Laboratory of Experimental Marine Biology Institute of Oceanology, The Chinese
Academy of Sciences, Qingdao 266071, China)
Abstract: The main chlorophyll a/b light-harvesting complex (LHCⅡ) has been isolated directly from
thylakoid membranes of marine green alga (Bryopsis corticulans Setch.) by two consecutive runs of anion
exchange and gel-filtration chromatography. LHCⅡ proteins in the membrane extracts treated with 3%
n-Octyl-b-D-glucopyranoside (OG) obtained specific binding ability on Q Sepharose column, and thus were
isolated from the thylakoid membranes in a highly selective fraction. The monomeric, trimeric and
oligomeric subcomplexes of LHCⅡ have been obtained by fractionation of the LHCⅡ mixes with sucrose
density gradient ultracentrifugation. The SDS-PAGE analysis of peptide composition and absorption
spectrum showed that LHCⅡ monomers, trimers and oligomers prepared through this work were intact
and in high purity. Our report is the first to show that it is possible to purify LHCⅡ directly from thylakoid
membranes without extensively biochemical purification.
Key words: photosynthesis; light-harvesting complex; purification; high performance liquid chroma-
tography
In green algae and higher plants, light energy for photo-
synthesis is collected by an antenna system, made of many
homologous proteins belonging to the Lhc multigene fam-
ily (Green et al., 1991). These pigment proteins are orga-
nized around photosynthetic reaction centers to form su-
pramolecular complexes embedded into the thylakoid
membranes. The main light-harvesting chlorophyll a/b-pro-
tein complex, designated light-harvesting complex Ⅱ (LHC
Ⅱ), is most abundant and a highly complicated system
with a structural heterogeneity recognized at the levels of
DNA, apoprotein and pigment-protein holocomplex
(Kühlbrandt and Wang, 1991; Luca et al., 1999). The het-
erogeneity at the level of pigment-protein holocomplex from
the existence of non-identical LHCⅡ building blocs which,
based on structure in vitro studies performed on LHCⅡ
crystals and detergent solutions, are thought to be trimers
(Jonathan, 1987; Butler, 1988; Kühlbrandt, 1988). In higher
plants, LHCⅡ apoproteins of 25 – 28 kD (Lhcb1, Lhcb2
and Lhcb3) are encoded by nuclear multiple gene (lhcb1)
or smaller number nuclear genes (lhcb2 and lhcb3) (Jansson,
1992; Kühlbrandt, 1994). However, the extension of such
explanation is not true to green algae (Jackowski, 1998). It
is likely that some of the green algal LHCⅡ proteins will be
the functional homologues to some of the higher plant
proteins, but little or nothing is known about their roles in
construction of LHCⅡ and functions at present. Durnford
(1999) reported that green algae do not have the three types
of LHCⅡ polypeptides Lhcb1, Lhcb2 and Lhcb3 that
present in higher plants.
LHCⅡ is not only very important for the overall light-
harvesting process but it is also essential for the ultra-
structure of the thylakoid membranes and it plays impor-
tant roles in various regulatory mechanisms of the photo-
synthesis (Bassi, 1997). LHCⅡ is usually isolated from the
isolated thylakoid membrane stacks, so-called BBY particles
(Berthold et al., 1981), by using non-denaturing isoelectric
focusing (IEF) system followed by sucrose density cen-
trifugation (Bassi et al., 1988; Spangfort and Andersson
1989; Ruban et al., 1994; Jackowskia and Janson 1998).
Although IEF lets us obtain LHCⅡ subcomplexes, it takes
at least three days to isolate LHCⅡ with a limitation of the
product yield due to the limited loading capability of IEF
gel. In addition, the running costs of flat-bed IEF, involv-
ing the use of ampholites and a gel composed of ultradex
Acta Botanica Sinica 植物学报 Vol.46 No.8 2004916
(LKB), can be restrictive. Thus purification of LHCⅡ based
on more routine, less expensive chromatographic tech-
niques is of interest.
In this study, we describe a method to isolate LHCⅡ
directly from thylakoid membranes of marine alga (Bryopsis
corticulans) by single step detergent solubilization followed
by liquid chromatography, thereby avoiding the necessity
for the intermediate preparation of BBY (PS Ⅱ -enriched)
particles. It therefore offers an improved alternative and
more defined preparation of LHCⅡ, as compared with BBYs,
avoiding denaturing of the complex upon longer exposure
in vitro and impacts in protein purification procedure.
1 Materials and Methods
1.1 Membrane preparation
Green shiphonous alga, Bryopsis corticulans Setch. was
collected in the intertidal zone in Qingdao of China. The
green algal cells were homogenized in TSN buffer (10 mmol/
L Tris-HCl, pH 8.0, 200 mmol/L sucrose, 10 mmol/L NaCl)
and centrifuged at 10 000g for 10 min. The pellets were
osmotically broken in 10 mmol/L Tricine-NaOH, pH 8.0 and
the broken membrane fragments were collected by a cen-
trifugation at 12 000g for 10 min.
1.2 Membrane solubilization
The collected 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 concentration of 1 mg/mL.
After a centrifugation at 180 000g to remove unsolubilized
membrane fragments, the supernatants were subjected to
the chromatographic fractionations.
1.3 High performance liquid chromatography
The high performance liquid chromatography (HPLC)
was performed on SMART HPLC system (from Pharmacia).
Applied the supernatants of the membrane solubilization
to anion exchange chromatography on Mini Q PC 3.2/3
(3.2 mm ´ 30 mm, from Pharmacia) equilibrated with TMK
buffer containing 0.05% n-Dodecyl-b-D-Maltoside (DM).
Eluted the column with the same buffer at a linear gradient
of 0.1-1.0 mol/L NaCl. The LHCⅡ enriched fraction was
pooled up together and concentrated with AMICON
Centriprep-50 (50 kD cut off). The concentrated sample was
subjected to gel-filtration chromatography on Superose 12
PC 3.2/30 column (3.2 mm ´ 300 mm, from Pharmacia). The
column with TMK buffer containing 0.05% DM was eluted
isocratically.
1.4 Sucrose density gradient ultracentrifugation
Column-purified LHCⅡ preparations were loaded on
top of linear sucrose gradients prepared with 5%-25%
sucrose in TMK buffer containing 0.05% DM. The gradients,
onto which 1 mL of the LHCⅡ sample at a chlorophyll
concentration of 1 mg/mL were layered, were centrifuged at
270 000g for 12 h with SW 40 rotor (from Beckman).
1.5 Electrophoresis
Polypeptide composition was analyzed on 12% poly-
acrylamide gel containing 1% SDS and run in Laemmli sys-
tem (Laemmli, 1970). For partly denatured electrophoresis,
homogeneous 12% acrylamide gel was prepared without
SDS and run at 15 mA and 4 °C in the dark. Samples were
loaded without SDS. The SDS concentration in the running
buffer was 0.1%.
1.6 Analytical procedures
Chlorophyll concentration of the samples was deter-
mined according to Lichtenthaler’s method (Lichtenthaler,
1987).
1.7 Spectroscopy
Room-temperature absorption spectrum was recorded
on an UVKON-943 double beam UV/VIS spectrophotom-
eter at a spectral bandwidth of 2 nm.
2 Results
The aim of this study is to isolate LHCⅡ proteins di-
rectly from thylakoid membranes using mild chromato-
graphic techniques and therefore avoiding any intermedi-
ate step such as the separation of BBY-type membrane frag-
ments and isoelectric focusing (IEF) system. Chromato-
graphic fractionation of the solubilized membranes on Mini
Q PC 3.2/3 column produced single major peak (Fig.1). The
analysis of polypeptide composition shows that this peak
fraction contained predominantly LHCⅡ proteins (Fig.2).
It is obvious that LHCⅡ proteins remained in the peak
fraction, whereas most of the other membrane proteins went
through the column without binding. The anion exchange
chromatography with the Q type column performed a highly
selective separation of LHCⅡ from the thylakoid
membranes. Another gel-filtration chromatography (Fig.3)
was applied for further purification of the LHCⅡ sample
collected from Mini Q PC 3.2/3 column, since there were
still three thin bands present in the higher molecular weight
zone (Fig.2). SDS-PAGE analysis of polypeptide of the ma-
jor peak fraction eluted from Superose 12 PC 3.2/30 column
(Fig.3) indicates that the LHCⅡ sample was in relatively
high purity (Fig.2).
With the two consecutive runs of chromatographic
fractionation, LHCⅡ has been isolated directly from the
thylakoid membranes of B. corticulans. The absorption
properties of the purified LHCⅡ resemble to those of LHC
Ⅱ from higher plant, showing the main Chl a Qy absorption
CHEN Hui et al.: Isolation of the Main Light-harvesting Chlorophyll a/b-protein Complex from Thylakoid Membranes of
Marine Alga, Bryopsis corticulans by a Direct Method 917
band at 672 nm well separated from the Chl b peak at 654 nm
(Fig.4), which is characteristic of native LHCⅡ.
Sucrose density gradient is still up to date a useful tool
in separation of different conformations of protein complex.
The monomeric, trimeric and oligomeric subcomplexes of
LHCⅡ have been obtained in this work. Upon sucrose
gradient fractionation, the monomers, trimers and oligo-
mers were clearly separated in the sucrose density gradient
(Fig.5). These monomeric, trimeric and oligomeric
subcomplexes were solved in detergent containing buffer.
To assess the fractions harvested from the sucrose density
gradient, the materials of the fractions were subjected to
partially denaturing gel electrophoresis. The resolution of
the partially denaturing gel showed that oligomer stayed at
the upper place, trimer and monomer moved to lower places,
respectively (Fig.6).
Since the molecular weights of LHCⅡ proteins are in a
narrow region, these proteins are difficult to be identified
on normal SDS-PAGE gel. In this study, we tried to resolve
the LHCⅡ proteins on elongate SDS-PAGE gel. The SDS-
PAGE result shows that the purified LHCⅡ trimer con-
tains at least five proteins (Fig.7). No protein band beyond
the region of LHC proteins was found in the oligomeric
sample, indicating that this oligomeric complex is one of
the LHCⅡ subcomplexes.
3 Discussion
In this work, the choice of the detergent and its concen-
tration used in membrane solubilization and the column
used in chromatographic fractionation are critically impor-
tant to the success of the purification of LHCⅡ directly
from thylakoid membranes of B. corticulans. To examine
the experimental condition with which LHCⅡ proteins
obtained specific binding ability onto the Q type column,
we applied OG and DM detergents in the experiments of
membrane solubilization with different concentrations of
the detergents. The thylakoid membrane fragments were
treated with OG in a range of concentration of 0.8%-4.0%
and with DM in a range of concentration of 0.02%-2.00%.
Upon running Mini Q PC 3.2/3 column with the membrane
solubilizations, the LHCⅡ protein in the membrane
Fig.1. Chromatogram of anion exchanger of thylakoid membranes of Bryopsis corticulans solubilized with 3% n-Octyl-b-D-
glucopyranoside on Mini Q PC 3.2/3 column, detection at 430 nm ( ) and 280 nm ( ), respectively. The NaCl gradient was also
monitored (---------) with secondary Y-axis.
Fig.2. SDS-PAGE of thylakoid membranes of Bryopsis
corticulans (A), LHC II enriched fraction eluted from Mini Q PC
3.2/3 column (B) and major LHC II fraction eluted from Superose
12 column (C).
Acta Botanica Sinica 植物学报 Vol.46 No.8 2004918
Fig.3. Gel-filtration chromatography on Superose 12 PC 3.2/30 column of LHC II enriched fraction collected from Mini Q PC 3.2/3
column.
Fig.4. Absorption spectrum of the purified LHC II from Bryopsis
corticulans at room temperature.
Fig.5. Fractionation of LHC II mixture eluted from Superose 12
column on 5% – 25% sucrose linear gradient.
Fig.6. Monomeric (M), trimeric (T) and oligomeric (O)
subcomplexes of Bryopsis corticulans LHC II were resolved with
partly denatured electrophoresis.
solubilization treated with 3% OG obtained the highest spe-
cific binding ability onto Q type matrix, whereas other de-
tergent treatments resulted in nonspecific or low specific
binding ability of the LHCⅡ onto Q type matrix especially
at lower detergent concentrations. Meanwhile, for determi-
nation of the selective efficiency of anion exchange
column in use of separation of LHCⅡ from membrane
extracts, both Mini Q PC 3.2/3 column and DEAE
(Diethylaminoethyl, weak anion exchanger) Sepharose col-
umn were tested in anion exchange chromatography of the
solubilized membranes treated with the different concen-
tration of OG and DM detergents mentioned above. The
DEAE anion exchange column performed in less selective
binding of LHCⅡ proteins than Mini Q PC 3.2/3 column,
noting more polypeptide contaminants involving in LHC
Ⅱ fraction from DEAE Sepharose column (data not shown).
These phenomena implied that detergent used in the treat-
ments of membranes solubilization could impact surface
charges of the intrinsic membrane proteins with their struc-
tural specificity and binding quantity.
Purification of hydrophobic membrane proteins is still a
CHEN Hui et al.: Isolation of the Main Light-harvesting Chlorophyll a/b-protein Complex from Thylakoid Membranes of
Marine Alga, Bryopsis corticulans by a Direct Method 919
challenge for the biochemists. In order to solubilize mem-
brane proteins and keep them in a soluble state, detergents
must be present in every purification step. The protocols
of LHCⅡ purification exist that usually involved separa-
tion of BBY (PSⅡ -enriched) particles and IEF
electrophoresis, which often led to complicated and time-
consuming procedures. To ensure maintaining high activ-
ity of the purified proteins, the LHCⅡ purification method
described in this paper enables us to obtain LHCⅡ prepa-
ration in a workday and thus the native properties of the
LHCⅡ can be mostly preserved in the preparation. The
purification method with liquid chromatography enables
large-scale preparation of LHCⅡ proteins by increasing
column volume for enhance of column loading ability, which
we believe to satisfy the demand of large amounts of pro-
tein samples by researchers of protein crystals for further
investigation of its structure and function.
Our report is the first to show that it is possible to iso-
late LHCⅡ directly from thylakoid membranes by liquid
chromatography without the extensive biochemical purifi-
cation procedures involving separation of BBY particles,
stepwise centrifugation and IEF electrophoresis. It is thus
a fast and readily controlled procedure so far to isolate
LHCⅡ from the thylakoid membranes.
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