Temperature Tolerant Hemoglobin Variant of Barbus sharpeyi
Moahmmad Reza Dayer,
Mohammad Saaid Dayer
Hemoglobin is a tetrameric protein of α2β2
subunits. The main function of hemoglobin is to pick up oxygen from surrounding
environment. Since oxygen solubility in water is very low the availability of
dissolved oxygen is affect by different factors e.g., temperature, salinity
and pH. Temperature increment accounted as a more threatening factor for fish
in regions with hot climate. In the present work, through conducting different
experiment and using different methods such as circular dichroism, differential
scanning calorimetry we decided to study hemoglobin stability against temperatures.
Barbus sharpeyi was used as a model habitat in hot climate. Our results
show that Barbus sharpeyi hemoglobin shows high melting high transition
temperature of 68°C which tolerate against thermal denaturation for long
period. The wide transition range of 56-68°C indicates that B.S. can adapt
and survive in hot climate unexpectedly.
Received: March 30, 2013;
Accepted: April 24, 2013;
Published: June 14, 2013
Hemoglobin is a respiratory protein found in vertebrate erythrocytes that takes
part in biological activities such as oxygen and CO2 transport, H2O2
dispersion, electron transfer (Mojtahedi et al.,
2008; Brittain, 2002; Clementi
et al., 2007), oxygen storage and buffering (Baumann
and Dragon, 2005) with good performance. However, the most important function
of hemoglobin remains to be oxygen transportation. Hemoglobins obtained from
different kinds of animals show structure-function relationships which enable
their adaptation to unfavorable environmental conditions such as low oxygen
pressure (Bonini-Domingos et al., 2007). This
relationship provides an interesting example of molecular mechanism acquired
during animal evolution that helps animal to meet its need for oxygen (Clementi
et al., 1994). Unlike Antarctic ice fish, Channichthyidae which lack
hemoglobin as an oxygen carrier, various species of fish kingdom obtain oxygen
with aid of hemoglobin as an oxygen carrier (Sidell and
O'Brien, 2006). Therefore, hemoglobin in these animals is expected to binds
enough oxygen even under harsh and threatening conditions. This implys that
fish hemoglobin may have experienced a high degree of evolutionary structural
changes conferring sophisticated regulatory mechanisms as per fish circumstances.
Oxygen solubility in water is only about 3.5 mg 100 mL-1 and yet
influenced by different environmental variables e.g., temperature, pH, salinity,
ionic composition and environmental pollutants. Henceforth aquatic habitats
differ from oxygen availability point of view. Oxygen deficiency poses a serious
problem on fish survival and cause fish to exert different mechanism for adaptation
such as synthesis of a wide spectrum of hemoglobins (hemoglobin isoforms) using
different genes and evolving their gas exchanging organs (Di
Prisco et al., 2000). The first mechanism is adapted by some temperate
fish species such as Barbus grypus (Dayer et
al., 2011). This species possesses different hemoglobin isoforms with
different oxygen affinity that enable oxygen capture at various oxygen levels.
Another possible mechanism that may function especially in warm climate is the
presence of thermally stable variant of hemoglobin that functions at high temperature
(Clementi et al., 1994; Val
and de Almeida-Val, 1988; Val, 1986). Barbus sharpeyi
(Gunther, 1874), the most resistant fish to hot climate in Kuzestan Province
of Iran, having a tetrameric hemoglobin and a single electrophretic band is
believed to use this kind of mechanism to resist against the hot climate (Mukhaysin
and Jawad, 2012; Mohammadi and Marammazei, 2000).
The present work aimed to answer two important questions in relation to temperature
resistance of B.S. hemoglobin. 1) What are structural characteristics of B.S.
hemoglobins and how these characteristics confer tolerance against the hot climate
in Khuzestan? 2) Are there structural bases for interdependence of the body
temperature (Tb) and transition temperature (Tc) for hemoglobin?
MATERIALS AND METHODS
Materials: All materials used were of analytical grade and were purchased
from sigma chemical Co.
Hb purification: Barbus sharpies were collected from Khuzestan
fish reproduction center. Blood samples were drawn from the caudal vein by heparinized
syringes. Immediately after sampling, erythrocytes were centrifuged at 1000
x g for 5 min at 4°C and washed three times with isotonic 1.7% NaCl solution.
The crude lysate was achieved by adding 1 vol. of ice-cold. Stroma was removed
by centrifugation at 31000 x g for 30 min at 4°C. We used a Sephadex G-25
column (1.5 x 45 cm) that was equilibrated with a 100 mM phosphate buffer pH
7.0. Elution was carried out with a 0.1 M phosphate buffer pH 7.0 at a flow
rate of 25 mL per h.
Circular dichroism experiments: Circular Dichroism (CD) spectroscopy
is appropriate technique to study secondary and tertiary structures of proteins
(Quddus and Ma, 2003). We used of circular dichroism
to monitor structural changes of the proteins.
Ultraviolet (UV) CD spectra were measured with a Aviv 215 CD spectropolarimeter
equipped with a temperature Peltier controller. Thermal unfolding of hemoglobin
was studied between 25 and 67°C in a 0.1 cm-thick quartz cuvette with a
0.1 cm optical path length. The starting temperature of the hemoglobin solution
was adjusted to 25°C and then (stepwise or gradually) increased. The sample
was allowed to equilibrate for 1 min at each temperature point. Then, a full
wavelength scan was performed in the UV region between 190 and 260 nm. For measurement
of the α-helical content of the proteins 222±2 nm wavelength scans
were Selected Because CD method is sensitive at 222 nm to α-chain content
in globins (Greenfield, 1996; Ranjbar
et al., 2006). The fractional change in the ellipticity at 222±2
nm was calculated according to under Equation:
where, Eabs (T) is the ellipticity at 222 nm at temperature T, Emax
is the ellipticity at the maximum temperature (°C) used and E25
is the ellipticity at 25°C (Jiang et al., 2001).
Spectroscopic experiments: Absorption spectra were recorded with a Shimadzu
model UV-3100 (Japan) spectrophotometer and a thermostatically controlled cell
compartment with a Haak D8 water bath. Spectrophotometric measurements were
studied between 30 and 80°C. The spectra were recorded after 3 at every
temperature. The absorbance were recorded at 280 nm. The concentration of hemoglobin
solutions in the experiments was 2.5 mg mL-1.
DSC experiments: Differential scanning calorimetric measurement of B.S
hemoglobins were carried out in an ultra-sensitive Scal-1 microcalorimeter (Moscow,
Russia) with a 0.3 mL cell at a heating rate of 2 K min-1. Temperature
ranges of 20 to 80°C were selected for heat transfer record. At this stage
of the experiment, the buffer baseline was subtracted and the data were normalized
with respect to protein concentration. To prevent a possible degassing of the
solution during the heating process, the pressure was maintained at 2 atmospheres
during all DSC runs. This software enabled us to deconvolute Cpex
(excess Cp) profile to a number of sub peaks the energetic domains
that contributed to observed DSC profile. Tm is one of application
DSC that study in different experiments (Chottanom and Srisa-Ard,
2011; Baimark, 2009) Tm is the transition temperature
at Cp,max and it is a criterion of protein stability.
Figure 1 represents heat capacity changes in constant pressure
for B.S. hemoglobin obtained from DSC experiment. In this graph the highest
value of Cp belongs to the temperature midpoint or melting temperature of hemoglobin
(Tm) which is calculated to be 68°C. Turbidity measurement at
630 nm (data not shown) shows that B.S. hemoglobin unlike other hemoglobin used
remain stable for long time of up to 1 h which in turn indicate more s conformation
for B.S hemoglobins. There are miscellaneous works used Tm as an
index for hemoglobin stability (Baimark and Srisuwan, 2012;
Srisa-Ard et al., 2008; Kumaresan
et al., 2011; NDri et al., 2007).
Figure 2 shows changes in absorbance of hemoglobins at the
wavelengths of 280 nm versus temperature as other measure of structural alteration
in B.S. hemoglobin. The maximum point of this thermal profile is defined as
the critical point equals to protein melting point (Tm). Figure
3 is pulled out from the data of Fig. 2. Indeed this graph
depicts the derivation of Fig. 2 and shown as dY/dX against
temperature. This derivation was made to sharps the graph point showing Tm.
The maximum point in Fig. 3 belongs to B.S. Tm.
This finding is completely agreeing with DSC findings shown in Fig.
1 form B.S. melting temperature of 68°C.
||Plot of Cp (heat capacity at constant pressure)
against temperature. The peak maximum indicates the hemoglobin melting temperature,
Tm. The data used here is obtained from DSC experiments. Hemoglobin
concentration of 2 mg mL-1 and 50 mM phosphate buffer, pH 7 was
||Changes in hemoglobins optical density at 280 nm against temperature.
Hemoglobin concentration of 4 mg mL-1 and 50 mM phosphate buffer,
pH 7 was used
||Differential plot of the data presented in Fig.
2 illustrated as dY/dX against temperature to magnify B.S. hemoglobin
melting temperature, Tm
||Change in hemoglobin secondary structure with temperature
(in %). The data were obtained using circular dichroism method. Hemoglobin
concentration of 2 mg mL-1 and 50 mM phosphate buffer with pH
7 was used
||Fractional change at in circular dichroism ellipticity at
222 nm with temperature increment for B.S. hemoglobin. Hemoglobin concentration
of 2 mg mL-1 and 50 mM phosphate buffer with pH 7 was used
Figure 4 shows the effect increasing temperature on hemoglobin
secondary structures as percent in contrast to the total secondary structure
content. The secondary structures of α-helix, anti parallel, parallel,
beta-tern and random coil were calculated using Aviv software (www.avivbiomedical.com/circular.php).
As it obvious the main secondary structure of hemoglobin in its native state
is α-helix structure. Upon heat treatment this structure is decreased and
the random coil instead increased to the same degree. However other secondary
structures of anti parallel, parallel and beta-tern structures not significantly
changed. Figure 5 indicate the fractional change in B.S. hemoglobin
CD ellipticity at 222 nm. This curve give us the maximum transition temperature
(Tc) of 68°C.
||Plot of fractional ellipticity at 222 nm showing critical
temperature, Tc for B.S. hemoglobin. Hemoglobin concentration of 2 mg mL-1
and 50 mM phosphate buffer, pH 7 was used
Figure 6 was plotted so as to determine transition temperature
ranges that appear to be ranged from 56-68°C. The cross part of the best-fitting
lines were regarded as the beginning and end of the accelerated transition temperature
range (Tc) as previously shown by Digel et
Protein stability at high temperature (60-70°C) is primarily due to higher
interaction between hydrophobic groups of protein (Bull
and Breese, 1973; Irback and Sanderlin, 2000; Wagschal
et al., 1999; Kumar et al., 2000;
Chothia et al., 1976). The hydrophobic properties
of protein are considered as a factor stabilized the tertiary structure of protein
(Bigelow and Channon, 1976; Chothia
et al., 1976). Perutz (1983) had shown
that protected hydrophobic domain insure protein thermal stability during evolution
and maintain their performance in thermophilic conditions. The melting temperature
of 68°C that reported here for B.S. (Fig. 1-6)
indicates high thermal stability for B.S. hemoglobins tertiary structure. This
thermal stability ultimately enabled Barbus sharpeyi to habitat the extreme
conditions of higher temperature in Khuzestan Province. As Turi
et al. (1981) proposed that proteins as active and functional units
of living organisms answered to the environmental stimuli via changing their
three dimensional conformation, we postulate this logic may be responsible for
B.S. resistance for such a harsh conditions. Digel et
al. (2006) calculated Tc for Ornithorhynchus anatinus
(with body temperature 31-33) and Tachyglossus aculeatus (with body temperature
31-33) to be 34°C in contrast to our finding for B.S. indicates the more
stability of B.S. hemoglobin. Based on these findings Digel
et al. (2006) expressed that Nature knows by protein structure
where body temperature must be set. Transition temperature (Tc)
for B.S. as depicted in Fig. 6 is calculated in the range
56-68°C (Kinderlerer et al., 1973). Considering
these temperature range we can conclude that B.S. with its stable hemoglobin
can adapt hot climate and pick up dissolved oxygen and survive easily.
Present findings confirm that B.S. erythrocytes contain more compact and more
stable conformation of tetrameric hemoglobin with higher transition temperatures
when contrasted to human hemoglobin. Unlike Barbus grypus which uses
the synthesis of different hemoglobin isoforms as a mechanism of adaptation.
Barbus sharpeyi employs a temperature resistant form of hemoglobin of
highly enough oxygen affinity to survive the hot climate of Khuzestan Province
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