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Biohelica : Vol.1(1):1- 4, 2010

A Scientific Journal of Biological Sciences
Comparative study of protein profile of eight benthic marine
macroalgae by SDS PAGE
S. Chakraborty
1
, S.C. Santra
2
and T. Bhattacharya
3
1
Dept. of Biological and Environmental Science, N.V.Patel College of Pure and Applied Sciences,
V.V. Nagar, Gujarat-388120, INDIA
2
Department of Environmental Science, University of Kalyani, Nadia, West Bengal - 741235
3
P.G. Dept. of Environmental Science and Technology,Institute of Science and Technology for
Advanced Studies & Research, (ISTAR),V. V. Nagar 388120, Gujarat, INDIA
(Received 25 March 2010; accepted 28 April 2010)
SDS PAGE was tested as an analytical tool for the isolation and identification of proteins from eight
marine benthic macroalgae (Catenella, Polysiphonia and Gelediella of Rhodophyceae, Rhizoclonium,
Enteromorpha, Lola and Ulva of Chlorophyceae and Dictyota belonging to Phaeophyceae,) thriving in the mangrove
vegetation of Sunderban, India. Chracteristic protein banding pattern was observed for each genus. The
pattern for Dictyota consists of 7 bands located between 29 and 205 kDa with the presence of triplicate bands
at 43 kDa. The pattern for Gelediella consists of four bands between 43 and 205 kDa. Rhizoclonium consists
of six bands lying between 3 and 205 kDa. Four bands between 3 to 43 kDa were characteristic of Lola.
Catenella exhibited six bands within a wide range of 6.5 to 205 kDa. Polysiphonia consists of three bands
within a wide range of 3kDa to 205kDa. Enteromorpha showed very interesting pattern of 4 clear bands, while Ulva
showed nearly 7 bands. SDS PAGE appears to be suitable for the identification of benthic marine macro algae.
Key Words: Bands, Gel electrophoresis, macroalgae, protein
INTRODUCTION
Innumerable works done on whole cell
protein analysis of algae have revealed that they
are rich in protein content and also potentially
important for containing many bio-active
substances which could be skillfully isolated and
employed for various food, feed, pharmaceutical
and cosmetic industry. Therefore protein
characterization of marine algae is a major factor
deciding the success of experiments in algal
biochemistry. In some species like Palmaria
palmate and Porphyra tenera proteins can
represent 35 to 47% of the dry weight. Spirulina,
a fresh water micro-alga is well known for its
high protein content (70% of the dry matter).
Among the algal proteins, it is worth noting the
occurrence of phycobiliproteins in red and blue
algae (blue phycocyanin in Spi rul i na,
phycoerythrin in red algae) (Fan-jie et al., 1984).
But the experiment of SDS PAGE (Sodium
Dodecyl Sulphate Polyacrylamide Gel) for algae
is a formidable task because extraction of
proteins from brown algae is difficult due to the
occurrence of phenolic compounds (Ragan and
Glombitza, 1986), Pigments and large amounts
of polyanionic cell-wall mucilages mainly
consisting of alginates (Mabeau and Kloareg,
1987). Phenolic compounds can destroy native
protein structure as they attach and upon
oxidizing conditions, couple covalently to them
by tanning effect (Loomis and Battaile, 1966).
Alginates from the cell wall form highly viscous
solutions disturbing extraction and purification
procedures for proteins. Furthermore they
revealed, together with other components of
brown algal cell walls, ion-exchange properties
(Kloareg et al., 1987). Apart from nutritional study,
Gel Electrophoresis of algal protein is also
performed for purification and characterization
of novel antibacterial protein from marine algae.
Quite a distinct number of works was done on
SDS PAGE of algae up to date. Two dimensional
1
Corresponding author: su_kalyanc@yahoo.co.uk
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gels were run for whole cell extract of model
alga Synechocystis sp. (Simon et al., 2002).
Membrane protein separation of
Chlamydomonas reinhardtii was done by Hippler
et al., (2001). Red carotenoid astaxanthin was
characterized in unicellular green algae
Haematococcus pulvialis under environmental
stress condition (Lorenz and Cysewski, 2000).
In this study, we analyzed the protein content and
protein profile of 8 species of marine macroalgae
abundant in Sunderban mangrove estuary of
India.
MATERIAL AND METHODS
Sample collection and preservation
Eight species of benthic algae, which are found
in large quantities throughout the ecosystem,
were collected, in food grade plastic bags from
Marichjhapi in the east, Canning, Gosaba and
J harkhali in the central part, and Patharpratima
and Dhanchi island in the western part of
Sunderban (between latitude 2131-2240 North
and longitude 8805-8906 East). The algal
forms were hand picked from substratum like
mud, concrete surface and bark and
pneumatophores of trees. The samples were
washed twice with seawater followed by fresh
water and single distilled water to remove the
adhering impurities, sand debris and epiphytes.
Among these algal taxa four species
(Rhi zocl oni um ri pari um, Enteromorpha
intestinalis, Lola capillaris and Ulva lactuca)
belonged to green algae, Dictyota ceylinica to
brown algae and three species (Catenella
repens, Polysiphonia mollis and Gelidiella
acerosa) belonged to red algae.
Extraction and estimation of protein
The Lowry method (Lowry et al., 1951)
was used for protein determination. The amount
of protein present was determined comparing to
authentic BSA standard (Sigma, USA) and
expressed in terms of percentage of dry weight
basis.
Fig 1. Protein bands by SDS PAGE of the eight benthic macroalgae
(M - Protein Molecular Marker, D - Dictyota ceylinica, G - Gelidiella acerosa,
R - Rhi zocl oni um ri pari um, L - Lol a capi l l ari s, C - Catenel l a repens,
P - Polysiphonia mollis, E - Enteromorpha intestinalis, U - Ulva lactuca)
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Gel Electrophoresis of protein
Electrophoresis of proteins in Poly
acrylamide gel was carried out in buffer gels (non-
denaturing) as well as in Sodium Dodecyl sulfate
(SDS) containing (denaturing) gels. Seperation
in buffer gels relied on both the charge and size
of the protein whereas it depended only on the
size in SDS gels. Poly acrylamide gels were
formed by polymerizing acrylamide with a cross
linking agent (bisacrylamide) in the presence of
a catalyst (persulphate ion) and chain initiator
(TEMED). Solutions were degased by evacuation
prior to polymerization. The samples after mixing
with equal volume of sample buffer were loaded
in the wells and the gel was run starting with 60
V and then to 120 V. The plates were then
disassembled and the gel was stained with silver
stain. With respect to the marker, the molecular
weights of the protein bands in the samples were
estimated (Sambrook & Russell2001).
RESULTS AND DISCUSSION
The protein content showed marked
individual variation (Table 1) with a highest
average value of 40.87% of the dry matter in Lola
and lowest, 3.33% of the dry matter in Dictyota.
These values were consistent with earlier studies
on other marine algae (Norziah and Ching, 2000).
SDS PAGE revealed characteristic pattern of
protein bands of all the eight benthic algae, with
selected over expressed bands (Fig 1). Their
results showed a high degree of homogeneity
along with characteristic pattern of protein bands
among all strains. This suggests that protein
expression is a phenotypic character, which is
regulated by both genotype and environmental
factors and differences in protein profiles must
be a reflection either of underlying differences in
the regulation of gene expression or in post-
translational modification of common proteins.
Similar work was carried out by Rouxel et al.,
(2001) on four red algae, where he found six
protein characteristic protein bands.
Brown alga Dictyota showed the presence
of total seven subunits, one between 97.4 kDa
and 205 kDa, two between 43 kDa and 66 kDa,
three prominent bands adjacent to band of
ovalbumin at 43 kDa. Previously this protein was
found by Rouxel et al., (2001) in C. crispus.
Another band corresponding to carbonic
anhydrase of 29 kDa was also observed. 29 kDa
protein was earlier reported in Alexandrium
catenella by Bustamante et al. (2003).
In Gelediella, 4 bands were observed
corresponding to ovalbumin at 43 kDa with
trailings of protein bands at positions 205 kDa
and between 97.4 kDa and 66 kDa. A glycoprotein
noncovalently associated with cell-wall
polysaccharide of the red microalga
Porphyridium sp.of 66 kDa was reported by
Prakash et al.(2004). Rhizoclonium, belonging to
Chlorophyceae exhibited 6 bands with two bands
corresponding to unknown molecular weight
between 97.4 kDa and 205 kDa, one band
between 97.4 kDa and 66 kDa two bands
adjacent to protein carbonic anhydrase of 29 kDa.
Another band of molecular weight below 3kDa
was also observed in this alga. Lola again
belonging to Chlorophyceae showed only 4
overexpressed protein bands out of which one
corresponded to 43 kDa, two near 29 kDa and
one below 3 kDa. Catenella, however showed a
different pattern of protein bands. This might hint
at its phylogenetic position also in
Rhodophyceae. It showed a total of 6 bands, out
of which, one was found higher than 205 kDa,
one adjacent to 66kDa, one at 43 kDa, one
between 20.1kDa and 29 kDa, one at 14.3 kDa
and another at 6.5 kDa. Mainly 4 bands were found
in Polysiphonia, a red alga. Two prominent bands
were found with one between 205 kDa and 97.4
kDa and the other between 97.4 kDa and 66 kDa.
Another band was seen between 20.1 kDa and
29 kDa. The most distinct band was found
between 6.5 kDa and 3 kDa. Enteromorpha
showed very interesting pattern of 4 clear bands.
Three bands were between 43 and 66 kDa and
one band of 14.3 kDa. Ulva however belonging
to Chlorophyceae again showed 5 individual
bands, one corresponding to Myosin band of the
marker, one between 97.4 and 205 kDa, one
bovine serum albumin band of 66 kDa, one of
29 kDa and one slightly more than 20.1 kDa.
Previously SDS PAGE was tested as an
analytical tool for Ulva and Enteromorpha
showed a reference pattern composed of 7
bands located between 69.9 and 15.5 kDa with
the presence of triplicate bands at 29.5, 26.3 and
22.9 kDa. (Rouxel et al., 2001).
ACKNOWLEDGEMENT
Authors are thankful to UGC, New Delhi, India
for funding the present study.
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