Hemoglobin function in deep-sea and hydrothermal-vent endemic fish: Symenchelis parasitica (Anguillidae) and Thermarces cerberus (Zoarcidae)
1 Department of Zoophysiology, C.F. Møllers Alle, Building 131,
University of Aarhus, DK 8000 Aarhus C, Denmark
2 Department of Biology, Pennsylvania State University, University Park, PA
16802, USA
3 Department of Chemistry, University of California at San Diego, La Jolla, CA
92093, USA
4 Station Biologique de Roscoff, UPMC-CNRS-INSU, 29682 Roscoff Cedex,
France
* Author for correspondence (e-mail: roy.weber{at}biology.au.dk)
Accepted 6 May 2003
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Summary |
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Electrophoretically cathodic and anodic isoHbs from S. parasitica exhibit radical differences in O2 affinity and pH and organic phosphate (ATP) sensitivities, reflecting a division of labor as in other `class II' fish that express both Hb types. Remarkably, the cathodic Hb (I) lacks chloride sensitivity, and the anodic Hb (II) shows anticooperativity near half-saturation at low temperature. T. cerberus isoHbs exhibit similar affinities and pH sensitivities (`class I' pattern) but much higher O2 affinities than those observed in Hbs of the temperate, shallow-water zoarcid Zoarces viviparus, which, unless compensated, reveals markedly higher blood O2 affinities in the former species. The temperature sensitivity of O2 binding to T. cerberus Hbs and the anodic S. parasitica Hb, which have normal Bohr effects, is decreased by endothermic proton dissociation, which reduces the effects of ambient temperature variations on O2 affinity. In the cathodic S. parasitica Hb, similar reduction appears to be associated with endothermic conformational changes that accompany the oxygenation reaction.
Key words: hemoglobin, oxygen binding, enthalpy, deep-sea fish, hydrothermal vent, anguillid, zoarcid, Thermarces cerberus, Symenchelis parasitica
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Introduction |
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Fish show pronounced, well-documented adaptations in blood
O2-binding characteristics in response to environmental stresses
(Weber, 1996) that comprise
intraspecific adaptations (such as the decreases in the red cell levels of
organic phosphates, ATP and guanosine triphosphate (GTP), that increase
HbO2 affinity and occur in individual specimens) as well as
interspecific adaptations (between-species differences that are genetically
coded and commonly involve differences in Hb structure and heterogeneity).
Fishes show marked isoHb differentiation and may be categorized
accordingly. Whereas `class I' species, such as cyprinids and ciclids
(Gillen and Riggs, 1972),
possess only electrophoretically anodic Hb components with relatively low
O2 affinities and pronounced Bohr and Root effects (decreases in
O2 affinity and carrying capacity, respectively, at low pH that
promote O2 unloading in the respiring tissues and in the
swimbladder), `class II' fish (including eels, salmonids and some catfish)
additionally have cathodic Hbs that exhibit high intrinsic O2
affinities and low pH sensitivities
(Powers and Edmundson, 1972
;
Gillen and Riggs, 1973
; Weber
et al., 1976a
,
2000
;
Pellegrini et al., 1995
) and
may function as a reserve transport system when oxygenation of the anodic
components is compromised by acidification and hypoxia (Weber,
1990
,
2000a
).
With no information available on Hb function in hydrothermal-vent
vertebrates, we investigated isoHb differentiation, together with
HbO2 binding and its sensitivity to pH, temperature and red
cell effectors (organic phosphate and chloride ions), in the anguillid pugnose
eel Symenchelis parasitica and the zoarcid Thermaces
cerberus and carried out some comparative measurements on the Hb of the
temperate, shallow-water zoarcid Zoarces viviparous. Compared with
S. parasitica, which shows highly cosmopolitan distribution at depths
of up to 3000 m and lives at the borders of hydrothermal vents that it
casually visits for food
(Desbruyères and Segonzac,
1997), T. cerberus is endemic to hydrothermal-vent
environments, where it intertwines with, and feeds on, Riftia
(Dahlhoff et al., 1990
;
Geistdoerfer and Seuront,
1995
), which requires sufficient quantities of free sulfide to
support robust bacterial endosymbiosis
(Luther et al., 2001
).
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Materials and methods |
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Blood samples from the deep-sea species were drawn into EDTA-containing
syringes, frozen and stored at -80°C until use. Hb solutions were prepared
by addition of approximately two volumes of 0.02 mol l-1 Tris
buffer, pH 7.6 and stripped of organic phosphates on MB-1 mixed ion-exchanger
or by preparative isoelectric focusing. Other preparatory steps were carried
out at 0-5°C, as previously described (Weber et al.,
1987,
2000
). IsoHb composition was
investigated by electrophoresis on cellulose acetate strips (Gelman Science,
Ann Arbor, MI, USA) and isoelectric focusing in 110 ml(LKB) columns containing
Pharmacia (Biotech AB, Uppsala, Sweden) ampholines, pH 3.5-10 (0.55%) and 5-8
(0.18%). pH values of retrieved fractions were measured at 22°C using a
BMS Mk2 Blood Micro System (Radiometer, Copenhagen, Denmark). Z.
viviparus Hb was prepared from washed red cells and stripped by
filtration through a column of Sephadex G25 (fine) gel. All Hb solutions were
dialysed against three changes of 0.01 mol l-1 Hepes buffer, pH
7.67 containing 0.5 mmol l-1 EDTA and, where necessary,
concentrated by ultrafiltration (Millipore 10 000 NMWL Ultra-free-4 filters).
The Hb was frozen in 50 µl or 100 µl aliquots that were freshly thawed
for O2 equilibrium measurements.
Oxygenation equilibria of ultrathin (≤0.05 mm) layers of Hb solutions
were recorded using a modified O2-diffusion chamber
(Weber, 1981;
Weber et al., 1987
) in the
absence or presence of ATP (assayed using Sigma test chemicals) and 0.1 mol
l-1 KCl (assayed using a Radiometer CMT10 chloride titrator). The
pH was varied using Hepes buffers (final concentration 0.1 mol l-1;
Weber, 1992
). The overall heat
of oxygenation {
H' = R
loge[
logP50/(T1-1T2-1)],
where P50 is the half-saturation O2 tension, R
is the gas constant, and T1 and T2 are different
absolute temperatures; Wyman,
1964
} was investigated by measuring P50 values
at 5°C, 25°C and 35°C and at pH values near 6.8 and 7.5 and
interpolating the P50 at these two pH values from the
linear logP50 vs pH regressions.
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Results |
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O2-binding characteristics
S. parasitica Hbs I and II exhibit radically different
O2-binding properties. Stripped Hb I has a markedly higher affinity
than Hb II [P50 at pH 7.2 = 14 Torr (1.87 kPa)and 33 Torr
(4.40 kPa), respectively, at 25°C] and higher cooperativity at
half-saturation (n50=1.7 and 1.1, respectively; Figs
2,
3).
|
|
Hb I shows a slight, reverse Bohr effect
(=
logP50/
pH=0.08) that is unaffected
by chloride but changes to a slight, normal Bohr effect (
=-0.14) in the
presence of Cl- + ATP (Fig.
3A). By contrast, stripped Hb II has a pronounced Bohr effect
(
=-0.39) that is potentiated by both Cl- (
=-0.58) and
Cl- + ATP (
=-0.74) in accordance with increased anionic
binding at low pH. Hb II shows a reverse `acid' Bohr effect below pH 6.7
(Fig. 3A), which thus extends
to higher pH values than those occurring below pH 6.0 in anodic Hbs of trout,
carp and catfish Hbs (Binotti et al.,
1971
; Gillen and Riggs,
1977
; Weber et al.,
2000
). The P50, n50 and
values of the stripped hemolysate
(Fig. 3A) are intermediate
between those of Hb I and Hb II, indicating an absence of interaction between
the components.
In contrast to the moderate Cl- and ATP sensitivity of Hb II, Hb
I lacks Cl- sensitivity (Figs
2,
3) but exhibits strong
Cl- + ATP sensitivity. The persistence of a pronounced ATP effect
in Hb I at high pH (Fig. 2A) is
intuitively consistent with its high pI value, reflecting positively charged
sites. The induction of a normal Bohr effect of Hb I by ATP reflects
preferential phosphate binding at low pH and thus is analogous to the
increased normal Bohr effect in Hb II in the presence of ATP. The data at
5°C (Fig. 3B) confirm the
functional differentiation between Hb I and Hb II but show a larger
Cl- + ATP effect than at 25°C
(logP50 at pH 7.5 =
0.6 and
0.4,
respectively). Curiously, a distinct reverse Bohr effect (confirmed
repeatedly) is manifest in Hb II at 5°C in the presence of Cl-
+ ATP, although a large (normal) Bohr effect is seen in the absence of the
phosphate (Fig. 3B). With no
information on the structure of the Hbs, a molecular explanation cannot be
offered.
Given that cytoplasmic domains of the red cell membrane protein Band 3
(cd-B3) bind at the phosphate-binding site of human deoxyHb
(Walder et al., 1984), we
investigated the effect of trout cd-B3, a synthetic 10-mer peptide
corresponding to the amino terminus of trout Band 3 protein
(Jensen et al., 1998
), on
S. parasitica Hb but found no detectable effect over a wide range of
pH conditions (6.6-7.5; Fig.
3A).
Extended Hill plots (Fig. 4)
of the major S. parasitica isoHb (Hb II) indicate negative
cooperativity at extreme, low O2 tensions and positive
cooperativity only above 60% O2 saturation, indicating that
the molecules remain frozen in the deoxygenated `tense' conformation at low
O2 saturations. As is evident from
Fig. 4, increased temperature
as well as decreased pH lower O2 affinity of Hb II predominantly by
decreasing KT without significantly impacting
KR (the O2 association constants of the
low-affinity deoxy and the high-affinity oxy states of the molecules,
respectively), revealing greater Bohr effect and
H'
values in the deoxygenated compared with the oxygenated state. Curiously, at
low pH (7.0) and 5°C, Hb II shows distinct negative cooperativity
(n<1) at 30-50% O2 saturation in the absence of ATP
(Fig. 4), which correlates with
the high P50 value (and Bohr factor) found under these
conditions (Fig. 3B).
|
T. cerberus Hb exhibits a strikingly higher O2 affinity
than does Z. viviparous hemolysate [P50=9 Torr
(1.2 kPa) and 30 Torr (4.0 kPa), respectively, at pH 7.0] and a lower pH Bohr
factor (Fig. 5), whereby the
affinity difference between the two species increases with decreasing pH. The
Bohr factor decreased with increasing temperature ( at pH 7.0-7.5=-0.62,
-0.56 and -0.37 at 15°C, 25°C and 35°C, respectively), indicating
temperature dependence of ionization groups as reported in other vertebrate
Hbs (Antonini and Brunori,
1971
), and falls drastically at high pH (>8;
Fig. 5A). In contrast to the
marked functional differentiation between S. parasitica isoHbs,
T. cerberus isoHbs (I, II and III) show similar O2
affinities [P50=6-8 Torr (0.8-1.07 kPa) at pH 7.0 and
25°C], similar low chloride sensitivities, similar pronounced ATP effects
(Fig. 6) and similar
cooperativities (n50=
1.5).
|
|
Oxygenation enthalpies
The O2 affinities of S. parasitica and T.
cerberus Hbs at 5°C, 25°C and 33-35°C yield essentially
linear van't Hoff plots (Fig.
7), indicating temperature independence of the oxygenation
enthalpies (H'), and similar heat capacities in the
oxygenated and deoxygenated states of the Hb (cf.
Fago et al., 1997a
). The
oxygenation enthalpies comprise the exothermic intrinsic heat of heme
oxygenation (
H0), the heat of solution of oxygen
(
Hsol=
-13 kJ mol-1) and endothermic
contributions that include the oxygenation-linked dissociation of hydrogen
ions, organic phosphate or chloride ions (
HH,
HP and
HCl,
respectively). It follows that the Bohr effect and phosphate binding reduce
H' and that
H0 can
experimentally be assessed in the absence of oxygenation-linked ion binding
under pH conditions where there is no Bohr effect.
|
S. parasitica Hb II shows high temperature sensitivity
(H'=-52 kJ mol-1) at pH 8.0, where there is
almost no Bohr effect, and a lower sensitivity (-31 kJ mol-1) at pH
6.8, where the Bohr factor is pronounced, suggesting that the intrinsic value
(in the absence of proton and anion binding) exceeds -52 kJ mol-1.
As may be interpolated from Fig.
4, the heat of oxygenation is higher in the deoxygenated state
than at half saturation (
HT=
-70 kJ
mol-1 and
-40 kJ mol-1, respectively, at pH 7.0).
The cathodic Hb I, which has a slight, reverse Bohr effect (cooperativity
between proton and O2 binding), shows lower enthalpies
(
H'=-46 kJ mol-1 and -49 kJ mol-1,
respectively, at pH 6.8 and 7.5).
The temperature sensitivities of Hbs from the vent-endemic and temperate
zoarcids T. cerberus and Z. viviparus show corresponding
patterns (Fig. 7B) despite the
large difference in O2 affinities. At pH 8.4, where these Hbs only
show slight Bohr effects (normal and reverse, respectively;
Fig. 5A,B), the respective
enthalpies of -72 kJ mol-1 and -78 kJ mol-1 suggest that
the intrinsic heat for the reaction with O2 is intermediate
(75 kJ mol-1). The lesser reductions seen in T.
cerberus Hbs compared with Z. viviparus Hbs under physiological
conditions (
H'=-63 kJ mol-1 compared with -42
kJ mol-1, respectively, at pH 7.5, and -48 kJ mol-1 and
-31 kJ mol-1, respectively, at pH 6.8;
Fig. 7B) tally neatly with the
smaller Bohr factors in the former species.
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Discussion |
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A relevant consideration pertaining to deep-sea habitats is how high
hydrostatic pressure influences water O2 tension and the
O2 affinity of blood and Hb. Thermodynamic considerations indicate
that the solubility of O2 decreases whereas its partial pressure
increases with increasing pressure and depth
(Fenn, 1972). Increasing
hydrostatic pressure to 1000 atmos (101.3 MPa) raises the affinity of human
and menhaden (Brevoortia tyrannus) Hb approximately 2-fold without
disturbing the transition between the deoxygenated, low-affinity (T) state and
the oxygenated, high-affinity (R) states of the Hb
(Carey et al., 1977
).
Increasing pressure from 1 atmos (0.1 MPa) to 126 atmos (12.8 MPa) raises
O2 affinity of human whole blood, red cell suspensions and
hemolysate without affecting the sensitivity to 2,3-diphosphoglycerate (DPG;
Reeves and Morin, 1986
).
Similar affinity increases have been reported in most earlier studies,
although these often suffer from pitfalls associated with the use of buffers
with pressure-sensitive pK values and the pressure sensitivities of gas
solubility and spectral absorbances
(Reeves and Morin, 1986
).
O2 affinities: adaptive variation
Distinct adaptations to ambient conditions are seen. Thus
HbO2 affinities are much higher in vent-endemic T.
cerberus, which grazes amongst sulfide-metabolising Riftia, than
in S. parasitica, which frequents cold, O2-laden deep-sea
water and only casually visits the organic-rich vent areas. Also, whereas
HbO2 affinities in T. cerberus are higher than in
Z. viviparous, those in S. parasitica are lower than in the
eel Anguilla anguilla (Fig.
8). However, in contrast to the striking adaptations encountered
amongst Hbs of hydrothermal-vent invertebrates, the Thermarces and
Symenchelis Hb systems exhibit the same basic functional
differentiations as encountered in shallow-water class I and class II fish,
respectively. This aligns with the view that the regulatory burden for
environmental adaptations in vertebrates is shifted to higher (e.g. cellular
and organismic) levels of organisation than in invertebrates
(Weber and Vinogradov,
2001).
|
In humans, glycolytic enzymes compete with deoxyHb for binding cd-B3,
providing a mechanism whereby Hb oxygenation can govern red cell glycolytic
processes (Giardina et al.,
1995; Messana et al.,
1996
). The insensitivity of S. parasitica Hb to trout
cd-B3 (Fig. 3A) suggests that
S. parasitica Hb does not play a transducer role. This agrees with
data for salmonid (trout) isoHbs (Jensen
et al., 1998
; Weber,
2000a
) but contrasts with human and catfish (Hoplosternum
littorale) Hbs (Walder et al.,
1984
; Weber et al.,
2000
; Weber,
2000a
), whose O2 affinities are lowered by cd-B3,
possibly implicating these Hbs in regulating cellular metabolism in an
oxygenation-dependent manner (Weber,
2000b
).
IsoHb differentiation
The Hb systems of T. cerberus and S. parasitica appear to
be typical representatives of fish classes I and II, respectively
(Fig. 8).
IsoHbs from the vent-endemic eelpout T. cerberus have similar
O2 affinities and pronounced, normal Bohr effects that decrease
with increasing pH, where the proton-binding sites become neutralized. A
significant adaptation in T. cerberus Hbs appears to be the high
O2 affinity compared with that in the temperate eelpout Z.
viviparus [P50=8 Torr (1.07 kPa) and
24
Torr (3.2 kPa), respectively, at pH 7.0 and 25°C]. The affinity difference
will be further amplified in the presence of the natural compliment of red
cell effectors [P50 values become
20 Torr (2.67 kPa)
and
60 Torr (8 kPa) in the presence of ATP and 0.1 mol l-1
Cl-; Fig. 8].
Although the exact mechanism of high affinity in T. cerberus must
await the solution of their molecular structures, reduced sensitivities to
chloride and phosphate effectors appear not to be involved
(Fig. 8).
S. parasitica typifies class II fish, having cathodic Hb with
relatively high O2 affinity, a slight, reversed Bohr effect in the
absence of organic phosphate and a large effect of ATP (that normalizes the
Bohr effect) as well as an anodic Hb with relatively low affinity and marked
Bohr and ATP effects as found in other anguillids
(Weber et al., 1976a;
Fago et al., 1995
;
Tamburrini et al., 2001
) and
catfish (Garlick et al., 1979
;
Powers and Edmundson, 1972
;
Weber et al., 2000
;
Fig. 8). Unexpectedly, the
affinities of stripped S. parasitica Hbs [P50=14
Torr (1.89 kPa) and 33 Torr (4.4 kPa) for Hb I and II, respectively, at pH 7.2
and 25°C] are low compared with those obtained by the same technique in
eel and catfish cathodic and anodic Hbs [P50=
2 Torr
(0.23 kPa) and
8.5 Torr (1.13 kPa), respectively;
Weber et al., 1976a
; Fago et
al., 1995
,
1997b
;
Weber, 2000a
] but are similar
to those in trout Hbs [P50=
17-20 Torr (2.27-2.67 kPa)
at 20°C; Weber et al.,
1976b
] all of these species are classified as class
II.
The pH insensitivity of the cathodic Hbs favors O2 binding under
acidotic conditions (burst activity, acid influx or lactate secretion in the
swimbladder; Powers, 1972;
Weber, 1990
). In S.
parasitica, however, the division of labor between the cathodic Hb I and
anodic Hb II may be of limited physiological significance due to the low
abundance of Hb I (cf. Fig. 1).
The higher sensitivity to ATP in cathodic Hb I compared with Hb II aligns with
Hbs of the eel Anguilla (Weber et
al., 1976a
; Fago et al.,
1995
) and the Amazon fishes Myllossoma, Pterygoplicthys
and Hoplosternum (Martin et al.,
1979
; Weber and Wood,
1979
; Weber et al.,
2000
) but contrasts with rainbow trout (Oncorhynchus
mykiss), whose cathodic Hb I is insensitive to phosphates. This confirms
that the `model' trout Hb system is exceptional rather than prototypical. An
intermediate situation appears in the South African mudfish Labeo,
where the phosphate sensitivity of cathodic Hb I is markedly lower than those
of the anodic isoHbs (Frey et al.,
1998
). In cathodic eel and Hoplosternum Hbs, the
ß-chain amino acid residues implicated in phosphate binding are
Val1(NA1), Glu/His2(NA2), Lys82(EF6) and Lys/Ser143(H21), and the marked
reverse Bohr effects (
=0.2 and 0.38, respectively) in the absence of
phosphates are attributed to the close proximity of these positively charged
residues in the T-state, which reduces their affinity for protons. The smaller
reversed Bohr effect (
=0.08) in cathodic Hb I of S. parasitica
may thus indicate a lower density of positive charges at the phosphate-binding
site.
A significant finding is the low Cl- sensitivity of S.
parasitica Hb I (Fig. 3).
Based on studies of mammalian/human Hb, two schools of thought exist as
regards the molecular mechanism of the Cl- effect: (1)
oxygenation-linked Cl- binding at specific sites
(Fronticelli et al., 1994)
[one between Val1(NA1) and Ser131(H14) on the
chain and another
between Lys82(EF6) and Val1(NA1) on the ß chain;
Fantl et al., 1987
;
Riggs, 1988
] and (2) a general
neutralization of excess positive charges that destabilize the T-state in the
central cavity (Perutz et al.,
1994
). Although the Cl- sensitivities of abnormal human
Hbs support the latter view, the Hb of the high-altitude Andean frog
Telmatobius peruvianus [where loss of Cl- sensitivity
correlates with acetylation of NA1 of the
chains (as in fish) and
replacement of polar
chain Ser131(H14) by nonpolar Ala] provides
evidence for the implication of specific
chain sites (Weber et al.,
2002). Elucidation of the primary structures of S. parasitica Hb I
and Hb II promises valuable insight into the molecular basis for
differentiated Cl- sensitivities
(Fig. 3).
Cooperativity
Another striking observation is the anticooperativity
(n50=0.6) observed in the major S.
parasitica isoHb at low temperature (5°C) and neutral pH (7.0)
(Fig. 3B), which correlates
with anticooperativity (n=
0.64) at 30-50% O2
saturation (Fig. 4).
Correspondingly low values (n50=0.6) observed for CO and
O2 binding in Hbs of the deep-sea Antimora rostrata
(Noble et al., 1975
) and
Coryphaenoides acrolepsis (R. E. Weber and F. C. Knowles,
unpublished), respectively, raise the possibility that allosteric interactions
may be differently expressed at atmospheric pressures compared with high
hydrostatic pressures. However, no shape changes are seen in
O2-binding curves of human blood and Hb with hydrostatic pressures
of 1-126 atmos (Reeves and Morin,
1986
). n50 values below unity at low pH are
diagnostic of Root effect Hbs that secrete O2 into the swimbladders
and retinae of fish (Pelster and Weber,
1991
). Values of <1 can result from two populations of
dissimilar heme groups, which could represent different isoHbs or
and
ß chains of the sets of Hbs (Noble et
al., 1986
). Obviously, the former possibility cannot explain the
n<1 regions in the oxygenation curves of purified Hb II
(Fig. 4).
Heats of oxygenation
The linear van't Hoff plots (Fig.
6) indicate that the enthalpy of oxygenation is temperature
independent, unlike in the Antarctic fish Dissostichus mawsoni, where
convex van't Hoff plots reflect increased temperature sensitivities at
decreasing temperatures and marked changes in the heat capacity difference
upon oxygenation (Fago et al.,
1997a).
Given that S. parasitica Hb I lacks significant oxygenation-linked
Cl- binding and only shows a slight Bohr effect, its
H' in the absence of anions may be expected to
approximate
H0. However, the values found (-46 kJ
mol-1 to -49 kJ mol-1) are low compared with those for
zoarcid Hbs (
-75 kJ mol-1) and human Hbs (-78 kJ
mol-1) (Weber,
1992
) at high pH, where the Bohr effect (oxygenation-linked proton
dissociation) is almost zero. Curiously also, the overall heat of oxygenation
of Hb I was not reduced by ATP addition (at pH 7.0,
H'=-44 kJ
mol-1 to -45 kJ mol-1 in the absence and presence of
ATP, respectively; not shown). Given the small variability of the intrinsic
heats of oxygenation of metal-containing gas-binding proteins
(Klotz and Klotz, 1955
), these
observations indicate the presence of other endothermic reactions that reduce
H'. A possible candidate is endothermic allosteric
transitions, as in cathodic trout Hb I, where the low temperature sensitivity
for CO binding is attributed to conformational changes conditioned to the
molecules in the T-state (Wyman et al.,
1977
). In S. parasitica Hb II, however, the decreased
temperature sensitivity is seen at high saturation
(Fig. 4). In effect, this is
analogous to bluefin tuna (Thunnus thynnus) Hb, where temperature
insensitivity (which now reduces outward transport of heat and helps to
maintain warm bodies) is attributable to the dissociation of a large number of
Bohr protons late in the oxygenation process
(Ikeda-Saito et al., 1983
;
Weber and Wells, 1989
), and
with tench (Tinca tinca red blood cells, where endothermic proton
dissociation occurs at high saturation
(Jensen, 1986
). In each case,
the resultant reduction temperature sensitivity will limit O2
affinity variations in the Hb in the face of variable environmental
temperatures.
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Acknowledgments |
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References |
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