The detection of Hg2+ by cyanobacteria in aqueous media

Article abstract of DOI:10.1039/b821687h

A tetrapyrrole-based chromophore was obtained through the methanolysis of C-phycocyanin extracted from Spirulina platensis, and was found to act as a selective receptor for Hg²⁺ at physiological pH conditions. The Royal Society of Chemistry 200

Full text of DOI:10.1039/b821687h

View Article Online / Journal Homepage / Table of Contents for this issue
COMMUNICATION
www.rsc.org/chemcomm | ChemComm
The detection of Hg2+ by cyanobacteria in aqueous mediaw
Moorthy Suresh, Sanjiv K. Mishra, Sandhya Mishra* and Amitava Das*
Received (in Cambridge, UK) 3rd December 2008, Accepted 3rd March 2009
First published as an Advance Article on the web 2nd April 2009
DOI: 10.1039/b821687h
A tetrapyrrole-based chromophore was obtained through the
methanolysis of C-phycocyanin extracted from Spirulina
platensis, and was found to act as a selective receptor for
Hg2+ at physiological pH conditions.
excitation with a 543 nm laser source. C-PC was extracted
from a Spirulina platensis culture by following a reported
procedure.7 Phycocyanobilin in C-PC was obtained from 1.0 g
of previously extracted, purified and freeze-dried C-PC
through soxhlet extraction (16 h), using 150 ml of methanol
as the solvent. The dark blue extracted solution was filtered to
remove protein residue and concentrated to 5 ml under
reduced pressure. To this, 10 ml of distilled water was added
and the resultant solution was dialysed against double-distilled
water for 24 h in the dark at 10 1C in order to remove any
unwanted lower molecular weight water soluble impurities,
using a HiMedia DM 004 Dialysis membrane-110. Finally, the
solution inside the dialysis sack was collected and lyophilized
to yield the desired compound as a dark blue solid mass, and
the spectrum recorded for this was found to be identical to the
one reported for phycocyanobilin.8 Methanolysis is known to
cleave the thioether linkage that links cysteine moiety of the
protein unit, and in C-PC, an ethylidine group is formed on
the free bilin at the site of the cleavage of the thioether
linkage.9 The purity of the phycocyanobilin thus obtained
was confirmed by exact mass measurement, elemental analysis,
Spirulina platensis is a highly efficient light harvesting cyano-
bacteria that contains two phycobiliproteins, namely allophyco-
cyanin and C-phycocyanin (C-PC).1 C-PC is the major light
harvesting pigment protein present in the antenna rods of
Spirulina platensis.2 Tetrapyrrole-based chromophoric units,
which are present in C-PC, are the basic components of
phycobiliproteins and are covalently attached to a protein
unit through a cysteine residue with a thioether linkage. This
linear tetrapyrrole chromophore, called phycocyanobilin, acts
as the light absorbing centre and plays a vital role in photo-
synthesis.1d,e,3 Spirulina platensis is a blue-green, but red
fluorescent algae and is known to be an efficient adsorbent
for Hg2+. This cyanobacterial species is generally grown in
aqueous environments and is increasingly being used as a
protein supplement.4 Thus, there is huge risk of Hg2+ entering
the food chain through the use of Spirulina platensis grown
in contaminated water.5 This warrants a serious need for
Hg2+ detection in Spirulina platensis and this has prompted
us to explore the possibility of detecting adsorbed Hg2+ in
cyanobacteria.
1
HPLC and H NMR studies (see the ESIw).
The red fluorescence nature of Spirulina platensis arises due
to the presence of fluorescent phycocyanobilin in the cyano-
bacteria. Earlier spectral studies with Spirulina platensis
revealed that it has a strong absorption band in the wavelength
range of 500–620 nm, while a sharp emission maximum is
centered at around 650 nm.7 We studied the fluorescence
behaviour of Spirulina platensis using a laser confocal fluor-
escence microscope, before and after exposing it to the Hg2+
solution, and the images recorded are shown in Fig. 1. The
confocal laser images revealed that the strong red fluorescence
of Spirulina platensis was completely quenched when exposed
to a 10 mM solution of Hg2+ for 10 min at 25 1C. To check the
reversible binding of Hg2+ to the receptor functionality in
Spirulina platensis, the treated culture was further exposed to a
The strong red fluorescence of Spirulina platensis is known
to be quenched upon Hg2+ adsorption. Thus, the study of the
fluorescence as the output signal could be used as a tool for
monitoring the amount of Hg2+ in the body of water where it
exists. Furthermore, this opens up the possibility of developing
a naturally occurring fluorescent marker for Hg2+, present in
aqueous environments and living cells. However, the exact
nature of the receptor functionality for Hg2+ in this cyano-
bacteria is not known. This prompted us to investigate the
probable binding site for Hg2+ in Spirulina platensis. To address
this issue we have probed Hg2+ binding to (i) Spirulina
platensis, (ii) C-PC extracted from Spirulina platensis and
(iii) the tetrapyrrole-based chromophore unit that was isolated
from C-PC following methanolysis (Scheme 1),6 through
binding-induced changes in electronic and fluorescence spectral
patterns. The ON-OFF fluorescence signalling behaviour of
the Spirulina platensis when exposed to Hg2+-solution was
also studied through confocal imaging experiments after
Central Salt and Marine Chemicals Research Institute (CSIR),
G.B. Marg, Bhavnagar, 364002, Gujarat, India.
E-mail: amitava@csmcri.org. E-mail: smishra@csmcri.org;
Fax: 91 278 2567562; Tel: 91 278 2567760 (672)
w Electronic supplementary information (ESI) available: Spectral
data, HPLC profile, HR ESIMS and confocal images. See DOI:
10.1039/b821687h
Scheme 1 A 3.0 A resolution single crystal structure of C-PC
extracted from Spirulina platensis (showing the tetrapyrrole-based
chromophoric unit in red)14 and the methanolysis process.
ꢀc
This journal is The Royal Society of Chemistry 2009
2496 | Chem. Commun., 2009, 2496–2498

View Article Online
t1 = 1.50 Æ 0.001 ns (w2 = 1.17) (FWHM for excitation
source is 0.2 ns). Time resolved emission studies, carried out in
the presence of added Hg2+ (lmon = 642 nm; lexc = 340 nm),
revealed a much faster fluorescence decay, with t1 = 0.44 Æ
0.008 ns (w2 = 1.418). A shorter lifetime presumably reflects
the efficient fluorescence quenching of the Hg2+-bound chromo-
phore owing to an efficient spin–orbit coupling. Fluorescence
studies with varying [Hg2+], revealed that the emission band
at ca. 660 nm became little more prominent with a gradual
decrease in intensity. The association constant (Kf) evaluated
for the formation of the Hg2+–C-PC complex was found to be
(2.7 Æ 0.2) Â 104 MÀ1. A residual emission was presumably
due to the presence of trimeric C-PC in phosphate buffer.11
The C-PC trimer has less conformational freedom than its
monomer and thus, has a higher emission quantum yield
(Z3) as compared to the corresponding monomeric form.
Presumably, this trimeric form with lower conformation flexi-
bility is less available for coordination to Hg2+ and accounts
for the residual emission in phosphate buffer. To check the
possibility of any conformational change that may be asso-
ciated with binding to Hg2+ ions, we recorded the CD spectra
of C-PC in the absence and presence of added Hg2+. C-PC
showed a positive CD band at B631 nm, with an associated
shoulder at 590 nm along with a negative band at 343 nm
corresponding to a weak absorption at 350 nm. A strong
negative CD band at 229 nm was also observed. This band is
actually crucial for estimating the secondary structure compo-
nents of C-PC and calculations indicated that the secondary
structure has a 65.5% a-helix, 8.5% b-helix and 29% random
coiled structure. C-PC is known to be denatured in the
presence of added acid and this is known to cause a large
decrease of the red absorption, with an associated reduction in
the CD band. Upon denaturation, the chromophore that is
present in C-PC gets relaxed from a linear extended form to a
cyclic-helical conformation.11 A similar reduction in the
absorption band at 610 nm was observed upon addition of
increasing [Hg2+], while the CD bands at 344 and 624 nm
disappeared (Fig. 3). Thus, to develop a better insight, we
studied the binding phenomena of phycocyanobilin with
Hg2+. Phycocyanobilin was extracted from C-PC through
methanolysis, and it does not have a S-atom to compete with
the tetrapyrrole unit for coordination to Hg2+. The absorp-
tion maximum for phycocyanobilin in the visible region is
centered at 590 nm, and upon excitation at 590 nm, an
emission maximum at 640 nm was observed in phosphate
buffer at pH 7.2. A systematic decrease in the emission
intensity of phycocyanobilin was observed (Fig. 4) with
Fig. 1 Confocal fluorescence and bright field images of (a) Spirulina
platensis, (b) cells stained with 10 mM Hg2+ in 1 : 1 water : ethanol, v/v
for 10 min at 25 1C (lexc = 543 nm) and (c) Hg2+ supplemented cells
loaded with 20 mM L1 for 10 min at 25 1C. Magnification = 10Â.
20 mM solution (1 : 1 water : ethanol, v/v) of L1 (a rhodamine
based Hg2+ sensor) (see ESIw) and images were recorded at
different time delays. L1 is known to bind to Hg2+ and
associated binding constant is (8.0 Æ 0.1) Â 105 MÀ2 L2 at
25 1C.10 Preferential binding of Hg2+ to L1 caused the
recovery of the bleached red fluorescence of the chromophore
present in Spirulina platensis; the change in fluorescence was
monitored through confocal laser microscopy and the images
recorded are shown in Fig. 1.
To probe the probable binding site for Hg2+ in Spirulina
platensis, we studied the binding properties of C-PC and
tetrapyrrole-based chromophores. Absorption and emission
spectra of C-PC in the absence and presence of varying [Hg2+
]
were recorded at pH 7.2 in a phosphate buffer. The absorption
spectrum of C-PC showed an absorption maximum at 617 nm,
which is characteristic for phycocyanobilin pigments (Fig. 2).
Spectrophotometric titration of C-PC with varying [Hg2+] in a
phosphate buffer (pH 7.2) showed a systematic decrease in
absorbance at 617 nm, with an associated isosbestic point at
555 nm, which signifies the presence of two species in equilibrium.
A fluorescence experiment was carried out in pH 7.2 phos-
phate buffer solution and C-PC showed an emission maximum
at 638 nm upon excitation at 555 nm. However, this emission
was found to be almost quenched upon addition of Hg2+
.
Similar experiments were repeated with various alkali, alkaline
earth and transition metal ions; while changes were either
insignificant or small when compared to that for Hg2+ (see the
ESIw). Time-correlated single photon counting (TCSPC)
studies revealed that upon excitation with 340 nm laser light,
C-PC exhibited a single exponential emission decay, with
Fig. 2 (a) Absorption and (b) emission spectra of C-PC (2.57 Â 10À8 M)
in phosphate buffer (pH 7.2) upon titration with varying [Hg2+
]
Fig. 3 CD spectra of C-PC (1.0 Â 10À7 M) (a) at different [Hg2+] =
(0–7.8 Â 10À4M); lexc = 555 nm.
(0–7.35 x 10À4 M) and (b) at different pH.
ꢀc
This journal is The Royal Society of Chemistry 2009
Chem. Commun., 2009, 2496–2498 | 2497

View Article Online
is more favorable for coordination to Hg2+. Thus, spectral
and photophysical data tend to suggest the tetrapyrrole core as
the probable binding site for Hg2+. Furthermore, the possi-
bility of binding of Hg2+ to the pendant –COOH fragment of
C-PC could be nullified based on the FTIR studies. As
mentioned earlier, phycocyanobilin produced through the
methanolysis of C-PC does not have an S-atom to compete
or complicate the binding phenomena; though it has a pendant
–COOH functionality. FTIR spectra clearly revealed that the
sharp stretching frequency at 1654 cmÀ1 remains unaffected
before and after addition of Hg2+ to the phycocyanobilin
(see the ESIw). Thus, our experimental results tend to support
the binding of Hg2+ to C-PC and its spectral response is
primarily due to the coordination of the Hg2+ to the
denaturated cyclic-helical conformer of the tetrapyrrole unit
in C-PC, and thereby, in Spirulina platensis also.
Fig. 4 Fluorescence titration of the phycocyanobilin chromophore
(1.9 Â 10À5 M); (a) [Hg2+] = 0–4.6 Â 10À4 M in phosphate buffer at
pH 7.2 (lexc = 590 nm) and in (b) [Hg2+] = 0–1. 2 Â 10À4 M in water
(lexc = 601 nm).
increasing [Hg2+]. A systematic titration profile revealed that
emission was completely quenched when an aqueous solution
was used for the studies. However, in phosphate buffer
solution (pH = 7.2), complete quenching was not observed
as it was in the case of titration with C-PC. The TCSPC studies
with phycocyanobilin in phosphate buffer (pH = 7.2) revealed
In conclusion, our studies tend to suggest that the
tetrapyrrole-based chromophore unit of C-PC is an efficient
receptor for Hg2+ and could account for the Hg2+ uptake by
Spirulina platensis.
that upon excitation with
a 340 nm laser source, a
bi-exponential emission decay with t1 = 7.95 Æ 0.024 ns
(55.18%) and t2 = 4.24 Æ 0.16 ns (44.82%) (w2 = 1.13) was
observed (Fig. 5)—where two different decay constants are
presumably associated with different conformers of phyco-
cyanobilin they may exist in equilibrium. The association
constant for Hg2+–phycocyanobilin complex formation was
CSIR and DST (India) have supported this work. M. S
acknowledges CSIR for a research fellowship. S. M and A. D
thank Dr P. K. Ghosh (CSMCRI, Bhavnagar) for his support.
The authors also thank Dr M. V. Krishnasastry of NCCS,
Pune for helping us in recording confocal images.
evaluated12 and was found to be (2.5 Æ 0.2) Â 104 MÀ1
.
Time resolved emission studies in the presence of added
Hg2+ ions (monitored at 642 nm with lexc = 340 nm),
revealed a bi-exponential decay of the excited state, with
t1 = 3.22 Æ 0.11 ns (10.67%) and t2 = 0.532 Æ 0.0074 ns
(89.33%) (w2 = 1.39). The longer component presumably
reflects the fluorescence decay associated with free phyco-
cyanobilin, while the shorter and major component reflects
the emission decay associated with the phycocyanobilin bound
to Hg2+. For efficient coordination to Hg2+, the conforma-
tional requirement for the tetrapyrrole unit is to adopt the
cyclic-helical form. Such a structure is predicted by van Thor
et al. through detailed NMR and molecular modelling
studies.13 The cyclic-helical form for an analogous tetrapyrrole
compound was found to be the energy minimized one.
Notes and references
1 (a) S. Boussiba and A. E. Richmond, Arch. Microbiol., 1979, 120,
155–159; (b) A. K. Padyana, V. B. Bhat, K. M. Madyastha,
K. R. Rajashankar and S. Ramakumar, Biochem. Biophys.
Res. Commun., 2001, 282, 893–898; (c) A. K. Padyana and
S. Ramakumar, Biochim. Biophys. Acta., 2006, 1757, 161–165;
(d) A. N. Glazer, in Advances in Molecular and Cell Biology, ed.
E. E. Bittar and J. Barber, JAI Press, Greenwich, CT, 1994,
pp. 119–149; (e) W. A. Sidler, in The Molecular Biology of
Cyanobacteria, ed. D. A. Bryant, Kluwer, Dordrecht, The
Netherlands, 1994, pp. 139–216; (f) A. N. Glazer, Methods
Enzymol., 1988, 167, 291–303; (g) A. N. Glazer and L. Stryker,
Trends Biochem. Sci., 1984, 423–427; (h) K. E. Apt, J. L. Collier
and A. R. Grossman, J. Mol. Biol., 1995, 248, 79–96.
2 X. Wen, H. Gong and C. Lu, Plant Physiol. Biochem., 2005, 43,
389–395.
A closer look at similarities in the spectral response and
respective decay constants for C-PC and phycocyanobilin in
the presence of added Hg2+ tends to suggest the linear
3 (a) R. J. Beuhler, R. C. Pierce, L. Friedman and H. W. Siegelman,
J. Biol. Chem., 1976, 251, 2405–2411; (b) E. Fu, L. Friedman and
H. W. Siegelman, Biochem. J., 1979, 179, 1–6.
4 K. Chojnacka, A. Chojnacki and H. Gorecka, Chemosphere, 2005,
59, 75–84.
tetrapyrrole unit as a probable binding site for Hg2+
.
5 A. Cain, R. Vennela and L. K. Wu, Biosource Technology, 2008,
99, 6578–6586.
6 D. Isailovic, H.-W. Li and E. S. Yeung, J. Chromatogr., A., 2004,
1051, 119–130.
7 (a) S. K. Mishra, A. Shrivastav and S. Mishra, Process Biochem.,
2008, 43, 339–345; (b) A. Patel, S. Mishra, R. Pawar and
P. K. Ghosh, Protein Expression Purif., 2005, 40, 248–255.
8 B. L. Scheram and H. H. Kroes, Eur. J. Biochem., 1971, 19, 581–594.
9 B. Samuel and C. Juan, Plant Physiol., 1984, 76, 7–15.
10 M. Suresh, A. Shrivastav, S. Mishra, E. Suresh and A. Das, Org.
Lett., 2008, 10, 3013–3016.
Furthermore, CD spectral studies revealed that denaturation
took place in the presence of Hg2+ and upon denaturation, the
tetrapyrrole-based chromophore relaxed from linear extended
form to a cyclic-helical conformation—a conformation which
11 M. Kupka and H. Scheer, Biochim. Biophys. Acta, 2008, 1777,
94–103.
12 K. Hirose, J. Inclusion Phenom. Macrocyclic Chem., 2001, 39,
193–209.
13 J. Jasper van Thor, M. Mackeeny, I. Kuprov, R. A. Dwek and
M. R. Wormaldy, Biophys. J., 2006, 91, 1811–1822.
14 L. Satayanarayana, C. G. Suresh, A. Patel, S. Mishra and
P. K. Ghosh, Acta Crystallogr., Sect. F, 2005, 61, 844–847.
Fig. 5 Fluorescence lifetime decay spectra for C-PC and phyco-
cyanobilin in the absence and presence of Hg2+. lexc = 340 nm LED.
ꢀc
This journal is The Royal Society of Chemistry 2009
2498 | Chem. Commun., 2009, 2496–2498