Indones. J. Nat. Pigm., Vol. 02, No.

1 (2020), 13–16

Intensity Ratio of LH2 Complexes from Rhodopseudomonas palustris and

Rhodobacter sphaeroides for Purity Determination

Monika N.U. Prihastyantia, Jovine M. Kurniawanb, Melisa M. Yusufb, Sherly S. Azmib,

Mustafavi C. Ilmib, Michelle L. Indartob, Stefan C. Hoetomob, Melina E. Wijayab, Greta Jatiyatib,
Tatas H.P. Brotosudarmoa,b, Heriyantoa,b,c,*
a
Ma Chung Research Center for Photosynthetic Pigments, Universitas Ma Chung, Malang 65151, East Java,
Indonesia
b
Department of Chemistry, Faculty of Science and Technology, Universitas Ma Chung, Malang 65151, East Java,
Indonesia
c
Department of Plant Physiology and Biochemistry, Jagiellonian University, ul. Gronostajowa 7, Krakow 30-397,
Poland
*Corresponding Authors: heri.yanto@machung.ac.id (Tlp. +62-341-550-171)

Article History: Received 02 March 2020, Revised 05 March 2020, Accepted 05 March 2020, Available Online 06 March 2020
Abstract
Light-harvesting complexes in photosynthetic bacteria has been studied as subject to understand the energy transfer processes
within the apparatus. The development of research on LH2 complexes opens an opportunity to develop biohybrid solar cell.
Purification procedures to obtain LH2 complexes were performed on two photosynthetic bacteria, i.e. Rhodospseudomonas (Rps.)
palustris and Rhodobacter (Rb.) sphaeroides. In this study, purification was initially carried out using series of centrifugation
towards the bacteria cell to acquire chromatophore of the bacteria. Application of detergent (lauryldimethylamine oxide, or LDAO)
to the chromatophore enabled the extraction of membrane containing photosynthetic apparatus. Further purification involved the
application of sucrose density gradient centrifugation and ion exchange chromatography. The purity of collected LH2 complexes
can be determined by calculating the ratio of AB850 : Aprotein. The resulting LH2 complexes from both bacteria showed relatively high
ratio (above 2) suggesting the purity of the complexes.

The Short Description: Purification steps involving sucrose density

gradient centrifugation (a) and application of anion exchange
chromatography (b) enables ones to determine the purity of LH2
complexes obtained from Rps. palustris and Rb. sphaeroides. The
application of sucrose density gradient centrifugation led to
separation of complexes according to the molecular weight, while
anion exchange chromatography would increase the purity of LH2
complexes.

(a) (b)
© 2020 MRCPP Publishing. All rights reserved.
http://doi.org/10.33479/ijnp.2020.02.1.13
Keywords: light-harvesting complexes, photosynthesis, photosynthetic bacteria, bacteriochlorophylls, polypeptides

INTRODUCTION (RC). The role LH1 complexes is to distribute solar energy to

Study of photosynthesis have been greatly assisted by
photosynthesis apparatus in purple photosynthetic bacteria,
such as Rhodopseudomonas (Rps.) palustris and Rhodobacter
(Rb.) sphaeroides, which are able to utilize solar energy to
produce their own food. Pigments like bacteriochlorophylls
and carotenoids in the photosynthetic bacteria have a great role
in absorbing solar energy. Antenna complexs are fabricated by
the interaction among α/β-polypeptide, bacteriochlorophyll a
(BChl a) and carotenoid (Car) molecules composing what so
called light-harvesting (LH) complexes which consists of LH1
and LH2 complexes. LH2 are peripheral antenna complexes
acting to collect light and transfer the solar energy to core
complex, which is constituted of LH1 and reaction center
Heriyanto et al. (2020)
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RC where photochemical reaction takes place. Each of such
complexes has different absorption properties. LH1 complexes
show absorption in near-infrared region at between 870–890
nm. On the other hand, two absorption peak are observed from
LH2 complexes at 800 nm and 850 nm which are made
possible due to different arrangment of BChl a molecules in
the complexes [1–2].
To investigate the properties and role of photosynthetic
apparatus, efforts have been delivered to obtain the
complexes. Series of experiments concerning on light-
harvesting complexes were succesfully performed by initially
purifying the complexes. Purification of LH2 was necessary in
the study of photophysical, electronic and vibrational
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 Indones. J. Nat. Pigm., Vol. 02, No. 1 (2020), 13–16
properties of dominant Cars in Rb. sphaeroides, i.e.
spiriloxanthin. Measurement of energy transfer in LH2
complexes of Rb. sphaeroides could take place through series
of LH2 purification which then followed by application of
polarization controlled two-dimensional electronic
spectroscopy allowed simultaneous measurement of energy
transfer within and between the two absorption bands at 800
nm and 850 nm [3–4]. LH2 purification steps should be
carefully executed especially when the complexes become the
subject of interest. For example, study on how light intensity
affected the creation of LH2 complexes in Rps. palustris which
resulted in different composition of apoproteins in the
complexes would required high purity of LH2 complexes. LH2
purification was also essential in research on the
characterisation of a pucBA mutant from Rps. palustris which
was combined with different intensity of light under which
they were grown to see the effect on LH2 complexes.
Interestingly, it was observed that under high light
illumination, the mutant could only assemble core complexes,
while LH2 complexes were created under low light intensity
[5–6].
Since obtaining LH2 complexes is a key success factor for
further analysis, herein we provide series of data that elucidates
the purification result of LH2 complexes from Rps. palustris
and Rb. sphaeroides.
EXPERIMENTAL
Chromatophore Extraction
Chromatophore was prepared from the cell of two
photosynthetic bacteria species, i.e. Rhodopseudomonas
palustris and Rhodobacter sphaeroides. The cell
(approximately 100 g wet cell) was suspended and
homogenized in Tris–HCl buffer (20 mM, pH 8) containing 5
mmol/L of Sodium-L-ascorbate. The mixture was sonicated for
10 minutes (amplitude 60 %) and centrifuged at 18,000 × g (30
minutes, 4 °C) to remove cell wall. Pellet was collected and
resuspended in 10 ml of Tris–HCl buffer followed by second
phase of homogenization and centrifugation. Chromatophore
was separated from cytochrome and other contaminants by
high speed centrifugation (70,000 × g, 90 min, 4 °C) then
washed three times with Tris–HCl buffer (20 mM, pH 7.8) and
high sped centrifugation. Collected chromatophore was stored
at freezer (–25 °C) prior to use.
Purification of LH2 Complexes
Chromatophore was solubilized in Tris–HCl buffer (20
mM, pH 8.0) and 1% of lauryldimethyl-N-oxide (LDAO) for 1
hour at 5 °C in dark condition. Sample was centrifuged (10,000
rpm, 10 min, 5 °C) to remove unsolubilized compounds.
Supernatant containing LH2 dan LH1-RC complexes was
collected and layered onto a sucrose step gradient consisted of
0.8 M, 0.6 M, 0.4 M and 0.2 M sucrose in 20 mM Tris–HCl
buffer (pH 8.0) containing 0.1 % LDAO followed by
centrifugation at 150,000 × g for 16 h at 4 °C).
LH2 complexes were carefully collected from the upper
pigmented band and purified by means of DE–52 cellulose
(Whatman, Maidstone, England) column. Prior to ellution, the
column was equilibrated using 20 mM Tris–HCl buffer (pH
8.0) containing 0.1 % LDAO. LH2 complexes were loaded
onto the column and washed through the column using
sequence of NaCl (50 mM, 100 mM, 150 mM) in Tris–HCl
buffer with LDAO. Desalting process was performed in the
collected complexes by washing the complexes with Tris–HCl
Heriyanto et al. (2020)
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buffer through a desalting column (PD-10). The purity of
collected samples from each purification process was
monitored using UV-Vis Spectrophotometer (UV 1700,
Shimadzu).
RESULTS AND DISCUSSION
Chromatophore from bacteria was collected with series of
centrifugation. Bacteria cell was centrifuged at least twice to
remove cell wall with low speed centrifugation and with high
speed centrifugation to remove contaminant in the collected
chromatophore. Figure 1 shows distinctive spectrum of each
condition from which we can observe that contaminant, such as
cell wall and cytochrome, have been removed resulting in
chromatophore with obvious Cars absorption in 497 nm. Such
absorption is hardly found in cell and unpurified
chromatophore due to its shoulderly shape.
5
cell
unpurified chromatophore
4
chromatophore
Absorbance (normalized)
3
2
1
0
300 400 500 600 700 800 900 1000
Wavelength (nm)
Figure 1. Absorption spectra (normalized at B850) of cell (—),
unpurified chromatophore (—), and chromatophore (—) following series
of centrifugation
Since bacteriochlorophylls are attached to the structure of
antenna complexes, the purity of them can be monitored
through the behaviour of the pigment by means of
spectrophotometer. Two types of BChl a and α/β-polypeptide
association in the structure of LH2 complexes lead to specific
absorption characteristic. Existed parallel to the membrane, 9
units of BChl a are bound to α/β-polypeptide pair separated 2.1
Å from center to center resulting in the absorption at 800 nm
region, which is assigned as B800. Other 18 BChl a units lie
perpendicular to the membrane, separated 9.5 Å from one to
another and are tagged as B850 due to the resulting absoprtion
at 850 nm [2].
Purification procedure continued with sucrose density
gradient which involved application of different concentration
of sucrose in Tris–HCl buffer. Having different molecular
density, LH2 and LH1 complexes will separated upon
ultrahigh speed centrifugation. Visually, ones can observe
distinctive color exhibited by bands in different layer of
sucrose. Pinkish red appeared at the bottom of the layers
followed by bold red on its top suggesting different molecular
weight of each band. LH2 complexes are formed by 9 α/β-
polypeptides association, each of which binds 3 molecules of
BChl a and two Car molecules giving a total 17 BChls and 18
Cars. As in LH1 complexes, 16 α/β-dimer units associated with
2 molecules of BChl a and a single Car in each units compile
the structure. Other experiments suggest there are 12 to 15 α/β-
subunits, alhtough 12 α/β-subunits might be to little to circle
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 Indones. J. Nat. Pigm., Vol. 02, No. 1 (2020), 13–16

1 1
Rps. palustris Rb. sphaeroides
0.8 0.8

Absorbance
0.6 0.6

Absorbance
0.4 0.4

0.2 0.2

0 0
300 400 500 600 700 800 900 1000 300 400 500 600 700 800 900 1000
Wavelength (nm) Wavelength (nm)

(a) (b) (c)

Figure 2. Collected bands upon ultra high speed centrifugation a); LH2 complexes from Rps. palustris b) and Rb. sphaeroides c).

the reaction center complexes [2,7–9]. Having more subunits Table 2. Ratio of AB850 : Aprotein of LH2 complexes collected from Rb.
sphaeroides
of polypeptides, RC-LH1 complexes were exhibited in lower
NaCl (mM) AB850 Aprotein Ratio (AB850 : Aprotein)
phase among the high concentration of sucrose solution. 50 0.004 0.048 0.08
Collected complexes revealed distinguish absoprtion spectra 100 0.004 0.205 0.02
characteristic. For example, the upper band of SDGC 0.908 0.388 2.34
complexes cleary shows absorption spectra at ~800 nm and 150 0.594 0.196 3.04
0.311 0.081 3.84
~850 nm suggesting the existence of LH2 complexes.
1.620 0.551 2.94
LH2 complexes from both Rps. palustris and Rb. 200 2.530 0.823 2.74
sphaeroides are displayed in Figure 2. Both species indicated 0.913 0.375 2.43
the existence of B800 and B850, with a slight shift in Rps.
palustris (801 nm and 854 nm). Through this step of 3
purification, more obvious spectra of carotenoid was obtained 50 mM NaCl; ratio 2.14
50 mM NaCl; ratio 1.45
due to the removal of free pigment that was extracted by 2.5
Absorbance (normalized)
50 mM NaCl; ratio 0.29
lowest concentration sucrose buffer, thus, appeared on the top
of the centrifuge tubes. Carotenoids in Rps. palustris consist of 2
rhodovibrin, rhodopin, spiriloxanthin, anhydrorhodovibrin and
lycopene as the dominant carotenoid. While in Rb. 1.5
sphaeroides, neurosporene, spheroidenone, spheroidene were
found alongside the before-mentioned carotenoids [10–12]. 1
Further purification applied the principal of ion exchange
chromatography that could separate molecules according to 0.5
their different charges in order to obtain higher purity of LH2
0
complexes. LH2 complexes from SDGC purification step was 300 400 500 600 700 800 900 1000
loaded onto a diethylaminoethyl (DEAE) which were Wavelength (nm)
previously suspended and equilibrated in Tris–HCl buffer (20 (a)
mM, pH 8) containing 0.1 % LDAO. LH2 complexes were 5
eluted through the resin by means of NaCl (50 to 150 mM) in 150 mM NaCl; ratio 2.34
150 mM NaCl; ratio 3.04
Tris–HCl buffer with various ionic strength. Protonated amine 150 mM NaCl; ratio 3.84
Absorbane (normalized)

4
group leads to positive charges in DEAE which enables them
to interact with negative charges molecule. Charged proteins in
the complexes were bound to the oppositely charged functional 3
groups in the DEAE resin [13]. When this procedure was
completely performed, lower intensity of protein and high 2
intensity of B800 and B850 was expected, suggesting the
removal of unwanted protein. Ratio of pigment-to-protein (at
1
850 nm and at 278 nm) was calculated from the corresponding
intensity to determine the purity. Higher ratio would mean
higher intensity of pigments, thus, resulting in higher purity of 0
300 400 500 600 700 800 900 1000
LH2 complexes.
Wavelength (nm)
(b)
Table 1. Ratio of AB850 : Aprotein of LH2 complexes collected from Rps.
palustris Figure 3. Absorption spectra (normalized at UV region) of purified LH2
NaCl (mM) AB850 Aprotein Ratio (AB850 : Aprotein) complexes using 50 mM NaCl for Rps. palustris a), and 150 mM NaCl
0.041 0.143 0.29 for Rb. sphaeroides b)
1.135 0.783 1.45
2.422 1.454 1.67 Table 1 and 2 show the ratio of AB850 : Aprotein of collected
50 3.408 2.454 1.34
3.612 2.458 1.47 LH2 complexes from Rps. palustris and Rb. sphaeroides,
2.498 1.170 2.14 respectively. Different concentration of NaCl to purify LH2
0.880 0.368 2.39 complexes may vary between species due to different amount
100 0.220 0.095 2.32 of unwanted proteins in the solubilized membrane. Highest
Heriyanto et al. (2020)
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 Indones. J. Nat. Pigm., Vol. 02, No. 1 (2020), 13–16

ratio of purified LH2 complexes from Rps. palustris (i.e. 2.39)
was obtained following the application of 50 mM NaCl. On the
other hand, higher concentration of NaCl (150 mM) was
needed to acquire higher ratio in LH2 complexes of Rb.
sphaeroides (i.e. 3.84). Moreover, Figure 3 reveals that the
absorption spectra of LH2 complexes from Rb. sphaeroides
suggested higher purity than that from Rps. palustris.
CONCLUSION
Absorption spectra of light-harvesting complexes from
photosynthetic bacteria reveal the purity on each step during
purification procedures. Sequence of LH2 complexes
purification involved detergent, series of centrifugation, the
application of sucrose density gradient centrifugation, and ion
exchange chromatography. The application of anion exchange
resin enabled ones to determine purity of collected LH2
complexes monitored from the intensity of B850 and protein in
UV region. Result suggested that highest AB850 : Aprotein ratio
from Rps. palustris was 2.39 obtained using 150 mM NaCl,
and from Rb. sphaeroides was 3.84 acquired from the
application of 200 mM NaCl.
Acknowledgement
The authors thank Universitas Ma Chung for the support.
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Abstrak

Prosedur pemurnian untuk mendapatkan kompleks antena penangkapan cahaya dilakukan terhadap dua jenis bakteri fotosintetik, yaitu
Rhodospseudomonas (Rps.) palustris dan Rhodobacter (Rb.) sphaeroides. Dalam penelitian ini, pemurnian diawali dengan beberapa kali
sentrifugasi terhadap sel bakteri supaya diperoleh kromatofornya. Penggunaan detergen (lauryldimethylamine oxide, atau LDAO) pada
kromatofor memungkinkan didapatkannya membran yang mengandung antena penangkap cahaya. Pemurnian selanjutnya meliputi
penggunaan sucrose density gradient centrifugation serta kromatografi pertukaran ion. Kemurnian dari kompleks antena penangkap cahaya
(LH2) dapat ditentukan melalui rasio AB850 : Aprotein. Hasil dari pemurnian kompleks LH2 menunjukkan rasio AB850 : Aprotein yang relatif tinggi (di
atas 2).

Kata kunci: kompleks antena penangkap cahaya, fotosintesis, bakteri fotosintesis, bakterioklorofil, polipeptida

Heriyanto et al. (2020)

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