Short Communication

Langmuir-Blodgett Film of
Phycobilisomes from Blue-Green Alga Spirulina platensis

CHEN Chao¹, ZHANG Yu-Zhong¹*, CHEN
Xiu-Lan¹, ZHOU Bai-Cheng^1,2, GAO Hong-Jun³

( 1State Key Laboratory of Microbial Technology,
Shandong University, Ji ‘nan 250100, China; 2Institute of Oceanology, the
Chinese Academy of Sciences, Qingdao 266071, China; Nano-Physics and Its Device
Laboratory, Institute of Physics, the Chinese Academy of Sciences, Beijing
100080, China )

Abstract        The
phycobilisomes were isolated from blue-green alga Spirulina platensis, and
could form monolayer film at air/water interface. The monolayer film of
phycobilisomes was transferred to newly cleaved mica, and coated with gold.
Scanning tunneling microscope was used to investigate the structure of the
Langmuir-Blodgett film of phycobilisomes. It was shown that phycobilisomes in
the monolayer arrayed in rows with core attaching on the substrate surface and
rods radiating towards the air phase, this phenomenon was similar to the
arrangement of phycobilisomes on cytoplasmic surface of thylakoid membrane in
vivo. The possible applications of the Langmuir-Blodgett film of phycobilisomes
were also discussed.

Key
words     pirulina
platensis; phycobilisome; Langmuir-Blodgett film; scanning tunneling microscope
(STM)

The
phycobilisomes (PBS) are the photosynthetic light-harvesting complexes in
blue-green algae and red algae[1－5]. They have been exhaustively studied with biochemical,
biophysical, and electron microscope methods, especially those from blue-green
algae[6]. Phycobilisomes are composed of several kinds of phycobiliproteins and
colorless polypeptides which assembled in specific configuration for optimized
energy transfer to downstream photosynthetic complexes[7,8]. According to their
spectroscopic properties, phycobiliproteins are divided into phycoerythrin(PE),
phycoerythrocyanin(PEC), phycocyanin(PC) and allophycocyanin(APC). Because of
their physical and spectroscopic properties, phycobiliproteins are widely used
as labeling reagents for a variety of fluorescence detection applications,
including flow cytometry, and are attractive as one of the most promising
molecules for bioelectronics [9,10]. The photosynthetic light harvesting
pigment complexes in green algae and higher plants were located in thylakoid
membrane, otherwise, the PBS is arranged in rows on thylakoid surface with the
core attached to the membrane surface[4].

In our previous
work, it was shown that the phycobilisome in Spirulina platensis is composed of
APC and C-phycocyanin(C-PC). C-PC is assembled into the rod of the
phycobilisome, and APC is stacked in the core of the phycobilisome. The rods
radiated from the core to different directions, and the APC-rich phycobilisome
core is then attached to the photosynthetic membrane, allowing the efficient
light energy transfer to photosynthetic system II (PSII)[11]. The three
dimensional structure of the C-PC was observed with STM[12]. We has also found
that the water-soluble R-PE could self-assembly into rod-like structures when
absorbed on the surface of HOPG, and could form two-dimensional
Langmuir-Blodgett film at the air/water interface, its structure was observed
by STM[13]. Facci et al.[14] studied the structure of monolayer of reaction
center of photosynthetic bacterium and its property of light-electricity
conversion with STM. But to date, there was no report on the ability of
phycobilisomes to form two-dimensional film by LB technique.

In this paper,
it was found that phycobilisomes could form two-dimension Langmuir-Blodgett
film at the air-water interface.

1    Materials and Methods

1.1   Isolation of phycobilisomes

The
phycobilisomes were isolated from blue-green alga Spirulina platensis according
to the procedure of Gantt and Lipschultz[1] with some modifications. The
fluorescence property at 77 K showed that the isolated PBS was intact.

1.2   reparation of LB film

The monolayer of
PBS was prepared on Sixing film deposition system (Jilin
University, China)
with a surface area of approximate 648 cm2. Deionized ddH2O (pH 5.6) was used
as the subphase. π-A curve
measurement was carried out by spreading a 2% ethanol/water solution containing
about 0.4 g/L PBS onto the subphase surface, and ethanol solvent was allowed to
evaporate for 15 min before compressing the monolayer at a rate of 0.5 cm2/s.
Surface pressure was measured with Wilhelmy plate. Monolayer was compressed to
a surface pressure of 15 mN/m, and was allowed to stabilize for at least 40 min
before dipping down the mica matrix. The mica was a suitable substrate for
transferring PBS monolayer because it has a negatively charged surface, which
was similar to the thylakoid membrane surface. The lifting speeds were 5 mm/min
upward and 15 mm/min downward. The transfer ratio for PBS in the upward
collection was approximately 0.85, and no deposition took place during downward
motion. In order to enhance the electroconductivity of the film, the mica
matrix onto which PBS monolayer was deposited was coated with gold and the
thickness of gold film should be controlled as thinner as possible so as to
minimize its interference. However, a continuous gold layer should meanwhile be
formed to have good electroconductivity. In order to obtain good STM images, only
one layer of PBS monolayer was transferred onto the mica matrix.

1.3   TM experiments

STM experiments
were carried out in ambient environment with a CSTM-9100 STM machine
(manufactured by Institute of Chemistry,
the Chinese Academy of Sciences). STM measurement was performed with normal constant current mode,
using tungsten tips made by electrochemical etching. All STM images were
presented from raw data without any smoothing and filtering.

2    Results

Fig.1 is the 77 K fluorescence spectrum of PBS from Spirulina platensis
excited at 580 nm. The major emission peak was at 685 nm, typical of intact
PBS, and another minor maximum at 623 nm. PBS showed F685 excitation maxima at
617 nm and 650 nm, which were the absorption maxima of C-PC and APC,
respectively. From Fig.1, it could also be deduced that C-PC was the major
source of F685. These results indicated that the isolated PBS from Spirulina
platensis was intact.

Fig.1       Fluorescence
spectra of phycobilisomes isolated from Spirulina platensis at 77 K

PBS is a water-soluble
phycobiliprotein complex. But usually its pure aqueous solution was not satisfactory
for spreading in the preparation of LB film. Thus, it was crucial to find a
suitable spreading solvent, in which this water-soluble compound could spread
to form monolayer, but not denature in the meantime. Results showed when PBS
was dissolved in 2% (V/V) ethanol/water solution, it can keep intact, and the
proteins will not denature (data not shown).

Fig.2 was the surface pressure-area isotherm at room temperature of PBS
monolayer at the air/water interface. From Fig.2, it could be resulted that PBS
could form monolayer when they were dissolved in 2% ethanol/water spreading
solution. Usually, water-soluble molecule are apt to be desorbed from air/water
interface to subphase (water), so the monolayer of these compounds was not
stabilized. Since the interfacial concentration of PBS used in our experiment
was far less than the limiting interfacial concentration of proteins, the
desorption can be neglected. The

Fig.2       Surface
pressure-area isotherm of phycobilisome monolayer at the air/water interface

The quantities of phycobilisomes
deposited were 32 μg. The subphase was composed of deionized ddH2O (pH 5.6).
All measurements were carried out at room temperature. change in the area of
PBS monolayer at the air/water interface with time at constant surface pressure
of 15  mN/m was presented in Fig.3.
From the illustration, it can be found that the area of PBS monolayer at the
air/water interface hardly changed within 3 h after 15 min post-preparation,
indicating that the monolayer of PBSwas very stable once the monolayer was
formed at the air/water interface.

 

Fig.3       Changes
in areas of phycobilisome monolayer with time

One PBS
monolayer was transferred onto the newly cleaved mica, coated with gold and its
structure was observed by STM. The STM images of PBS LB film were shown in Fig.4.
It was shown that the phycobilisomes arranged orderly in the LB film, most PBS
arrayed in rows [Fig.4(A), shown along the line]. Single PBS could be
distinguished, with the core of PBS attached to mica surface, and rods of PBS
radiated towards the air phase [Fig.4(B), shown by the arrows], which was in
agreement with its arrangement on thylakoid membrane surface in vivo. No
obvious defect was observed in the scanning area of 830 nm×830 nm. These results showed that
intact PBS had good ability to form two-dimensional film, and the film could
easily be transferred onto mica matrix.

Fig.4 ATM image of phycobilisome monolayer

(A) Scan area: 830 nm × 830 nm. (B) Scan
area: 460 nm × 260 nm. It=0.60 nA; Vbias=510 mV.

3    Discussion

Due to their
stability, high fluorescence yield, large stokes’ shifts between absorption and
emission[9], the phycobiliproteins are widely used as labeling reagents for a variety
of fluorescence detection applications, including flow cytometry[10]. Recently,
the stabilized phycobilisomes were used as fluorochromes for the detection,
which had far greater fluorescence intensity per binding event than a single
phycobiliprotein molecule[15,16]. Chronakis et al.[17] reported that the
protein-pigment complexes isolated from the phycobilisome of Spirulina
platensis are quite capable of forming Langmuir-Blodgett films. The protein
layer spreading at the air/aqueous interface also has a higher collapse
pressure than the common food proteins. We also found that the R-phycoerythrin
from marine red alga Polysiphonia urceolata can be easily prepared for
two-dimensional film with Langmuir-Blodgett technique[13]. In this paper, it
was found that PBS could also form two-dimensional Langmuir-Blodgett film at
the air/water interface. With special property of photophysics and
photochemistry, phycobiliproteins and phycobilisomes might be acted as a kind
of useful materials for crystallization electronics and bioelectronics
research. Moreover, STM could be used to study the property of
light-electricity conversion and to manipulate the molecules or complexes in
the Langmuir-Blodgett film with STM tip[14,18]. On the base of this research,
the Langmuir-Blodgett films of phycobilisomes might finally be used as
functional organic biological materials for data storage and molecular switch.

References

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3Glazer AN. Phycobilisomes: Structure and
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4     MacColl
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5     Yamanaka
G, Glazer AN, Williams RC. Molecular architecture of a light-harvesting
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8     Bryant
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9     Kronick
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10    Oi
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11    Zhang
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12    Zhang
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Tseng CK, Pang SJ. Study on the structure of C-phycocyanin in Spirulina
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13    Zhang
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Biophys Sin, 2002, 34: 99－103

14    Facci
P, Erokin V, Nicolini C. Scanning tunneling microscopy of a monolayer of
reaction centers. Thin Solid Film, 1994, 243: 403－406

15    Morseman
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detection of proteins on membranes. Biotechniques, 1999, 26: 559－563

16    Telford WG, Moss MW, Morseman JP, Allnutt FC. Cyanobacterial stabilized phycobilisomes
as fluorochromes for extracellular antigen detection by flow cytometry. J
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17    Chronakis
IS, Galatanu AN, Nylander T, Lindman B. The behavior of protein preparations
from blue green algae (Spirulina platensis strain Pacifica)
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18    Wiesendanger
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______________________________________________

Received: May
26, 2003        Accepted:
July 9, 2003

This work was supported by the grants from
the National High Technology R&D Program (863 Program) of China
(No. 2002AA302213) and the Natural Science Foundation of Shandong Province,
China

*Corresponding author: Tel, 86-531-8364326;
Fax, 86-531-8565610; e-mail, [email protected]

Updated at: 2003-10-09

钝顶螺旋藻(Spirulina platensis)藻胆体Langmuir-Blodgett膜

陈超¹         张玉忠¹*        陈秀兰¹         周百成^1,2         高鸿钧³

( 1山东大学微生物技术国家重点实验室， 济南 250100； 2中国科学院海洋研究所， 青岛 266071； 3中国科学院物理研究所纳米物理与器件实验室，
北京 10008 )

摘要       从钝顶螺旋藻中分离制备完整藻胆体，
然后滴加于空气/水界面上， 应用LB膜技术制备藻胆体LB膜。 结果表明， 藻胆体在空气/水界面上具有很好的成膜性能。 将藻胆体LB单层膜转移到刚揭开的云母表面， 喷一层金， 然后用扫描隧道显微镜观察。
结果表明， 藻胆体在Langmuir-Blodgett膜中的排列方式与其在体内类囊体膜表面的排列方式类似，
一排排聚集在一起， 然后排列成膜。 藻胆体的“核”吸附在云母表面， 而藻胆体的“杆”伸向外面。
由于钝顶螺旋藻易于规模化培养， 藻胆体容易批量制备，
加之藻胆体具有的独特的光物理、 光化学特性和良好的成膜性能，
以及本身就是纳米量级的颗粒（50~70 nm）， 预示着藻胆体在纳米光电子器件中具有很好的应用前景。

关键词   钝顶螺旋藻； 藻胆体； Langmuir-Blodgett膜； 扫描隧道显微镜(STM)