Oxygen binding and its allosteric control in hemoglobin of
the primitive branchiopod crustacean Triops cancriformis
Ralph Pirow
1
, Nadja Hellmann
2
and Roy E. Weber
3
1 Institute of Zoophysiology, University of Mu
¨
nster, Germany
2 Institute of Molecular Biophysics, Johannes Gutenberg University of Mainz, Germany
3 Zoophysiology, Institute of Biological Sciences, University of Aarhus, Denmark
The Branchiopoda are an ancient, primitive and,
except for the Cladocera, conservative group of crusta-
ceans [1]. The earliest known representatives were mar-
ine and occurred % 500 million years ago in the Upper
Cambrian [2]. Present-day branchiopods are predomin-
antly freshwater animals, and the fossil records indi-
cate that marine branchiopods invaded freshwater
habitats early in evolution. Two of the four extant
Keywords
allosteric control; Crustacea; hemoglobin;
oxygen binding
Correspondence
R. Pirow, Institute of Zoophysiology,
Hindenburgplatz 55, University of Mu
¨
nster,
D-48143 Mu
¨

Hbs. Remarkably, Mg
2+
ions increased oxygen affinity solely by displacing
the equilibrium between the tense and relaxed conformations towards the
relaxed states, which accords with the original MWC concept, but appears
to be unique among Hbs. This effect is distinctly different from those of
ionic effectors (bivalent cations, protons and organic phosphates) on anne-
lid, pulmonate and vertebrate Hbs, which involve changes in the oxygen
affinity of the tense and ⁄ or relaxed conformations.
Abbreviations
Hb, hemoglobin; i
r
, i
t
, interaction parameters of the cooperon model; K
i
, Adair constants of the i th oxygenation step; K
r
, K
s
, K
t
, oxygen-
binding constant for a particular conformation; K
ab
, oxygen-binding constant for a particular conformation; m, number of Mg
2+
-binding sites
per oxygen-binding site; MWC, Monod–Wyman–Changeux; n
50

Devonian and the Late Carboniferous [3], respectively.
The transition from the marine to the physicochemically
more extreme inland water environments represented a
great challenge for the physiological systems involved
in regulating the internal milieu. Given the primitive
and conservative morphological characteristics of
many extant branchiopods, which seem to have chan-
ged little over long periods of time, it may be pre-
sumed that prehistoric adaptations to a highly variable
environment remained preserved and essentially unsup-
plemented by physiological ‘innovations’, allowing us
to gain insight into homeostatic mechanisms operative
in early crustaceans [4,5].
The tadpole shrimp Triops cancriformis (Notostraca)
is one of the ‘oldest’ extant branchiopods; it was found
to be inseparable from Triassic (250–205 million years
ago) fossils on the basis of morphological criteria [6].
Notostracans comprising the two genera Triops and
Lepidurus inhabit temporary water bodies that com-
monly exhibit extreme physicochemical conditions.
For desert ephemeral pools in south-western North
America, a typical branchiopod habitat, Scholnick [7]
reported large diurnal variations in oxygen tension
(40–200 mmHg), carbon dioxide tension (0.07–
3 mmHg), pH (7.5–9.0), and temperature (17–35 °C)
during summer months. Horne [8] similarly observed
large diurnal fluctuations in oxygen concentration
(1–6.5 mgÆL
)1
) and temperature (17–30 °C) in North

static regulatory burden from the organ to the mole-
cular level compared with vertebrates. This view is
corroborated by the fact that exposure to hypoxic
conditions increases hemolymph hemoglobin (Hb)
concentrations in Triops spp. [16–18]. This homeostatic
response may be complemented by changes in the
functional properties of the protein.
The extracellular Hbs of invertebrates are commonly
high-molecular-mass complexes which exhibit high
variability in oxygen-binding properties and their sensi-
tivities to pH and ionic effectors [15]. So far, nothing
appears to be known about the allosteric control of
Hb–oxygen binding and its significance for the regula-
tion of internal oxygen conditions in branchiopod
crustaceans. This lack of knowledge contrasts with the
detailed information available on the structure of sev-
eral branchiopod Hbs [19,20]. To probe Hb function,
its molecular correlates and organismic regulation in
the phylogenetically ancient crustaceans, we investi-
gated the oxygen-binding characteristics, their sensitivi-
ties to pH, temperature and bivalent cations, and the
allosteric mechanisms controlling oxygen binding in
Hb from T. cancriformis.
Results and Discussion
Physicochemical characteristics of
Triops hemolymph
The in vivo pH of the hemolymph in the dorsal sinus
of T. cancriformis was 7.52 ± 0.02 (n ¼ 3 animals) at
20 °C. A markedly lower pH value of 7.1 (presumably
measured at 23 °C) has been given for T. longicaudatus

increased the n
50
from 1.9 to 2.9 and decreased the P
50
from 16.0 to 9.4 mmHg, respectively.
The oxygen-binding characteristics of purified Hb
(Fig. 1B,D) were comparable to those of dialyzed
hemolymph. The P
50
of purified Hb, for example, was
only 2–3% lower than that of dialyzed hemolymph
under the same buffer and temperature conditions
(Tris ⁄ Bis-Tris, pH 6.7–8.1, 20 °C). Experiments using
Hepes as an alternative buffer revealed a somewhat
higher P
50
than with Tris ⁄ Bis-Tris (Fig. 1D). At
pH 7.5, for example, which represents the in vivo pH
condition, the Hepes-buffered Hb showed a P
50
of
14.0 mmHg.
The oxygen-binding properties of whole hemolymph
were examined at three CO
2
levels at 20 °C (Fig. 2).
Strong alkalinization of hemolymph induced by CO
2
-
free conditions yielded an extreme affinity (P

in the largest species, i.e. the notostracans (body length
10–100 mm [24]). This negative correlation extends to
the smallest branchiopods such as the cladocerans
(0.2–6 mm), which at high ambient oxygen tension rely
predominantly on simple diffusion. Several lines of
evidence [25–27], including the reduction in oxygen
uptake when Hb–oxygen binding is blocked by carbon
monoxide [28] and the striking induction of Hb under
hypoxia in euryoxic species such as Daphnia magna
(1–16 g HbÆL
)1
) [29], indicate that the high-affinity
Hbs of cladocerans (P
50
¼ 1.2–8.3 mmHg) function as
oxygen carriers mainly at low ambient oxygen tension.
Large branchiopods, in contrast, invariably require
convective transport of oxygen. The moderate oxygen
affinity (P
50
¼ 6.8–14 mmHg) and the high concentra-
tion of Hb (8–25 g HbÆL
)1
) [17,21] (this study) in Tri-
ops spp. suggest that the respiratory protein mediates
circulatory oxygen transport over a wide range of ambi-
ent oxygen tensions i n Notostraca. The remarkably
C
pH
6.5 7.0 7.5 8.0

50
) and (C) half-saturation oxygen tensions (P
50
)at20°C
(circles) and 10 °C (squares) in Tris ⁄ Bis-Tris-buffered (dialyzed)
hemolymph. (B, D) Effects of pH on n
50
and P
50
of purified Hb in
Hepes buffer (diamonds) and Tris ⁄ Bis-Tris buffer (triangles) at
20 °C.
Oxygen partial pressure P
O2
(mmHg)
0 1020304 0
Fractional oxygen saturation Y
0.0
0.5
1.0
0 % CO
2
1 % CO
2
2 % CO
2
whole hemolymph
Fig. 2. Oxygen-binding curves of T. cancriformis Hb in whole hemo-
lymph at three different CO
2

50
from 6.5 to
13.3 mmHg at pH 7.44 (Fig. 1C). The pH-dependence
of n
50
was virtually unaffected by temperature
(Fig. 1A). The temperature-dependence of the P
50
val-
ues at pH 7.0 and pH 8.0 corresponded to the overall
heats of oxygenation of )51.3 and )45.6 kJÆmol
)1
,
respectively, which include the heat of oxygen dissolu-
tion and the heat of proton dissociation from oxygen-
ation-linked acid groups. The reduction in the overall
heat of oxygenation with increasing pH correlates with
an intensification of the Bohr effect at higher pH and
the endothermic nature of Bohr proton release [31]. In
the physiological context, the reduction in oxygen
affinity with increasing temperature may favor oxygen
delivery to the tissues in synchrony with temperature-
induced increases in oxygen consumption rates, but
Table 1. Oxygenation characteristics of branchiopod Hbs. Data from whole hemolymph (WH) were obtained under normocapnic conditions
(0.03% CO
2
). In cases where information on the experimental buffer conditions was lacking, the extraction buffer type is given together with
a question mark.
Group ⁄ species
P

Daphnia magna
Pale 3.8 1.3 20 7.2 Phosphate [71]
Pale 8.3 20 WH [72]
Pale 7.7 1.6
a
25 7.2 Bis-Tris ⁄ propane [57]
Red 1.6 1.5 20 7.2 Phosphate [71]
Red 2.5 20 WH [72]
Red 2.6 1.8
a
25 7.2 Bis-Tris ⁄ propane [57]
Daphnia pulex
Hb-1 2.6 2.2 17 7.45 Tris (?) [73]
Hb-3 1.2 1.4 17 7.45 Tris (?) [73]
Moina macrocopa 2.1 20 7.2 Phosphate (?) $0 [70]
a
Calculated from the Adair constants;
b
dLogP
50
⁄ dpH.
R. Pirow et al. Allosteric control of O
2
binding in crustacean Hb
FEBS Journal 274 (2007) 3374–3391 ª 2007 The Authors Journal compilation ª 2007 FEBS 3377
may also compromise oxygen loading in warm hypoxic
water.
Effect of bivalent cations on oxygen binding
The addition of Mg
2+

effect and the greater sensitivity of oxygen affinity to
Mg
2+
than Ca
2+
are moreover consistent with a previ-
ous study [21], which showed that univalent cations
such as Na
+
and K
+
(added as chloride salts) had no
significant effect on T. longicaudatus Hb. The differen-
tial effects of Mg
2+
and Ca
2+
show that specific prop-
erties of cationic effectors other than net ionic charge,
such as size and the stereochemical orientation of their
charges, influence oxygen affinity of Triops Hb.
Allosteric control mechanisms and their
physiological significance
To reveal the allosteric control mechanisms, high-reso-
lution oxygen-equilibrium curves of dialyzed hemo-
lymph (in Tris ⁄ Bis-Tris buffer) and purified Hb
(in Hepes buffer) were measured at different pH values
(pH 6.7–8.3) and Mg
2+
concentrations (0–100 mm).

1
and K
N
reveals an apparently unique
heterotropic control mechanism for bivalent cations.
In contrast with the heterotropic interactions so far
described for annelid (Arenicola marina [32]), pulmo-
nate molluscan (Biomphalaria glabrata [33]) and ver-
tebrate [34] Hbs, where modulation of oxygen affinity
by ionic effectors invariably involves changes in K
1
and K
N
, increasing Mg
2+
concentrations raised the
oxygen affinity of T. cancriformis Hb without affecting
K
1
and K
N
.
The physiological significance, if material, of the
effects of bivalent c ations on Triops Hb is not clear. T he
cation concentrations in T. cancriformis hemolymph
(Ca
2+
, 1.6–3.1 mmolÆkg
)1
;Mg

2+
concentra-
tions continue to be regulated [35].
C
[M
g
2+
], [Ca
2+
] (m
M
)(m
M
)
12 5102050100
log P
50
0.7
0.8
0.9
1.0
1.1
1.2
D
[M
g
2+
]
12 5102050100
A

on
Mg
2+
(circles) and Ca
2+
(squares) in Tris ⁄ Bis-Tris-buffered (dialyzed)
hemolymph at pH 7.1 (grey, filled symbols) and pH 7.8 (open sym-
bols), where the dotted lines extrapolate to the values in the
absence of bivalent cations at the same pH. (B, D) Effects of Mg
2+
on n
50
and P
50
of purified Hb in Hepes buffer (diamonds) at pH 7.6.
Allosteric control of O
2
binding in crustacean Hb R. Pirow et al.
3378 FEBS Journal 274 (2007) 3374–3391 ª 2007 The Authors Journal compilation ª 2007 FEBS
The proton and cation insensitivities at low satura-
tions contrast with the pH-dependent divergence of
oxygen-binding curves at high saturation (> 95%)
(Fig. 4A,B). Increasing pH enhanced the affinity for
binding the last oxygen molecule. Accordingly,
the Adair constant for the last oxygenation step
(K
N
) increased from 0.49 mmHg
)1
at pH 6.71 to

monoxide poisoning of Hb resulted in no depression
of the rate of oxygen consumption [21]. However,
these observations may merely indicate that the oxygen
requirements of the tissues may be satisfied by the oxy-
gen carried in physical solution in the hemolymph, at
least at rest and under normoxic conditions. Against
the background of the limited systemic (circulatory)
regulatory capacities in Triops, Hb becomes the key
control component of the oxygen-transport cascade
from environment to cell. Hb enables the animal
to maintain aerobic respiration under environmental
hypoxia by increasing the convective conductance for
oxygen in the circulatory system [39]. When oxygen
loading and unloading spans the steep part of the
oxygen-equilibrium curve, Hb also exerts a stabilizing
effect on the hemolymph oxygen tension (‘oxygen buf-
fering’) [40], thereby reducing the risk of oxidative
stress to the tissues. The oxyregulatory function of Hb
may be enhanced by the Bohr effect, which enables the
animal to optimize oxygen loading to the hemolymph
at the respiratory surfaces via hyperventilation and res-
piratory alkalosis under conditions of environmental
oxygen deficiency.
B
% Ox
y
Hb
99
98
95

1
2
C
log P
O2
0.0 0.5 1.0 1.5 2.0
log [Y/(1–Y)]
-1
0
1
2
dialyzed hemolymph in Tris buffer purified Hb in Hepes buffer
–log K
1
pH 8.30
pH 7.50
pH 7.12
pH 6.69
pH 7.77
pH 7.44
pH 6.71
[Mg
2+
] pH
100 7.78
15 7.80
0 7.77
[Mg
2+
] pH

The intercepts of these dashed lines
with the horizontal (dotted) line at log
[Y ⁄ (1–Y)] ¼ 0 correspond to the negative
logarithms of the Adair constants of the first
and last oxygenation step (K
1
and K
N
).
R. Pirow et al. Allosteric control of O
2
binding in crustacean Hb
FEBS Journal 274 (2007) 3374–3391 ª 2007 The Authors Journal compilation ª 2007 FEBS 3379
Structure–function relationships
The multimeric Hb of T. cancriformis is composed of
two subunit types, TcHbA (60–70%) and TcHbB
(30–40%), which have polypeptide masses of 35775
and 36055 Da, respectively; each carry two heme
groups and assemble into disulfide-bridged dimers that
comprise a homodimer of TcHbA and a heterodimer
[20]. These dimers assemble into three native isoforms,
one 16-mer and two 18-mer species. Only the larger
18-meric species seems to possess the heterodimer con-
taining subunit type TcHbB. Thus, several structural
levels are present: the first level is the di-domain sub-
unit, which carries two oxygen-binding sites (see
Fig. 6A). The next level is the disulfide-bridged dimer
D. As it seems unlikely that eight or nine copies of
these dimers oligomerize into a big lump, additional
substructures such as D

as well as the uncertainty in the remaining parameters.
In the Adair formalism none of the parameters are
shared between the curves, thus, the total set contains
24 free parameters. Despite this very large number of
parameters, the root mean squared error (rmse) is sig-
nificantly larger than that of most other models tested
(Table 2, model b). Furthermore, the residuals are not
randomly distributed (Fig. 5B), indicating systematic
deviations between the data and the fit. The obvious
next level to take into account is a functional coupling
of two dimers (Fig. 6A, substructure D
2
), involving an
interaction between eight oxygen-binding sites. This
Table 2. Comparison of the goodness of fit of different oxygen-binding models. Each model was applied to the set of six oxygen-equilibrium
curves of purified Hb shown in Fig. 4B,D. Shown are the total number of (shared and curve-specific) parameters, SSR, the degrees of free-
dom (DF), the root mean squared error (rmse ¼
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
SSR=DF
p
), and the best-fit parameter values of each model. Shared parameters and curve-
specific parameters are given as single values and range of values, respectively. K values are in mmHg
)1
.
Model Number of parameters SSR DF rmse Best-fit values of model parameters
(a) Two-state MWC 9 (¼ 6 · 1 +3) 300 105 1.69 K
T
¼ 0.034 log L ¼ 5.2–6.6
with shared q K
R

¼ 0.074
K
R
¼ 1.546
log M ¼ 5.9–10.2
q ¼ 5.3–8.0
(f) 4 · 2 Cooperon model 14 (¼ 6 · 2 +2) 32 100 0.56 K
T
¼ 0.025 log L ¼ 8.3–10.8
z ¼ 4 dimeric cooperons K
R
¼ 1.448 i
T
¼ 4.6–11.3
with i
R
fixed to unity i
R
¼ 1 (fix)
(g) 4 · 2 Cooperon model 20 (¼ 6 · 3 +2) 20 94 0.46 K
T
¼ 0.026 log L ¼ 5.6–6.7
z ¼ 4 dimeric cooperons K
R
¼ 0.274 i
T
¼ 3.6–9.6
with curve-specific i
R
i

r
are shared among the six binding curves,
and the allosteric equilibrium constant (L) is specific
for each curve. If one allows curve-specific values for
the size of the allosteric unit (q), a reasonably good fit
is obtained, but the values for q vary between
4.1 ± 0.2 and 5.4 ± 0.3 (means ± 95% confidence
interval) (Table 2, model c). If one fixes q to the same
value for all curves, a poor value for the rmse is
obtained (Table 2, model a) and the residuals are non-
randomly distributed (Fig. 5A), indicating that the
MWC model does not reflect the complexity of the
oxygen-binding process for Triops Hb.
At the next level of complexity, we allowed one
more conformation for the allosteric unit within the
framework of the MWC model, and employed a three-
state model (Eqn 8). In this case, the value for the size
of the allosteric unit (q) is still highly variable, ranging
from 5.3 to 8.0 (Table 2, model e), disfavoring this
model too. Alternatively, we extended the simple
MWC model to take a possible heterogeneity in the
cooperative interactions because of the different sizes
of the oligomers (16-mers and 18-mers; Fig. 6A) into
account. This extension is based on superimposition of
two binding curves corresponding to two types of mole-
cules, each having an oxygen-binding characteristic
that obeys the MWC formalism (Eqn 9). The size of
the allosteric unit (q) is allowed to differ between these
two types, but for each molecule species, q is shared
among the binding curves obtained at different effector

0.00
0.02
G
H
Fractional ox
yg
en saturation Y
0.0 0.2 0.4 0.6 0.8 1.0
Residuals
-0.02
0.00
0.02
0.0
pH 8.30 (0)
pH 7.12 (0)
pH 6.69 (0)
pH 7.60 (100)
pH 7.58 (50)
pH 7.50 (0)
tetramerous Adair
2-state MWC (curve-specific q
-state MWC (2 species)
3-state MWC (curve-specific q)
4×2 cooperon (i
R
= 1) 4×2 cooperon (curve-specific i
R
)
3×4 nested MW
ctomerous Adair

approach are present in a ratio of 15 : 85, indicating
that the main part of the oxygen-binding curve is dom-
inated by one species. Altogether, these results indicate
that models allowing hierarchical functional properties
need to be applied.
The cooperon model includes both KNF-type and
MWC-type interactions [41,42]. It describes a basic
dimeric cooperon (the ab dimer in the case of verteb-
rate Hb), in which cooperative interactions are allowed
according to the induced-fit mechanism. The change in
the binding affinity for the second oxygenation step
compared with the first is quantified via a parameter i.
A value for i larger than unity indicates positive coop-
erativity, and a value smaller than unity indicates neg-
ative cooperativity. The dimeric cooperon is nested
into a higher-level oligomer formed by a number of z
cooperons, which are regulated according to the MWC
mechanism. Thus, for each conformation r and t, a
specific interaction parameter (i
r
and i
t
) is considered.
We applied this model by equating the dimeric cooper-
on with the di-domain subunit of the T. cancriformis
Hb (Fig. 6A). The resulting fit describes the data
somewhat better than the three-state model, with the
additional plus that the values for the parameters do
not violate model-inherent assumptions. The best fit
for this model was achieved for a variant where four

two levels of allosteric units, which both function
according to the MWC model, are embedded into each
other. This model has been successfully applied to
describe the allosteric interactions in hierarchically
structured, multimeric proteins such as arthropod
hemocyanins [45,46], annelid Hbs [47], and chaperonin
GroEL [48]. The nested MWC model was fitted to the
data using different combinations of the size (w) of the
basic allosteric unit and the number (s)ofw-sized
basic allosteric units. In order to keep the number of
free parameters as small as possible, the influence of
effectors such as protons and Mg
2+
was directly inclu-
ded in the model (Eqns 14–16, 18–20). The results
obtained for different combinations of s and w are
shown as contour maps (Fig. 7), which also visualize
the influence of variations in the numbers of Mg
2+
-
binding sites (m) and proton-binding sites (h) per
oxygen-binding site.
di-domain subunit
disulfide-bridged dimer D
possible sub-structures
possible stoichiometries of native Hb isoforms
D
2
D
3

scheme of the nested MWC model. ( A) T. cancriformis Hb consists
of di-domain subunits, which carry two heme groups and form
disulfide-bridged dimers. Three possible assemblies of dimers (D
2
,
D
3
and D
4
) have been suggested as building blocks of the native
16-meric and 18-meric Hb isoforms [20]. (B) Nested 2 · 4 MWC
model showing the conformational states (tR, rR, rT and tT) and
transitions for a nested, basic allosteric unit containing w ¼ 4 oxy-
gen-binding sites. A number of s ¼ 2 copies of the basic allosteric
unit assemble into a larger structure. This s · w assemblage can
adopt two overall conformations, R and T, which impose con-
straints on the conformations of the constituent basic allosteric
units. The conformational equilibria are described by the allosteric
constants l
R
, l
T
, and L.
Allosteric control of O
2
binding in crustacean Hb R. Pirow et al.
3382 FEBS Journal 274 (2007) 3374–3391 ª 2007 The Authors Journal compilation ª 2007 FEBS
These contour maps suggest that the size of the basis
allosteric unit is a functional tetramer (i.e. w ¼ 4),
which is accommodated by the disulfide-bridged,

rR
<K
rT
and L<<l
R
<l
T
) found in anne-
lid Hbs (Macrobdella decora [47]) and arthropod
hemocyanins [45,46].
On the basis of the parameters of the 2 · 4 nested
MWC model, conformational distributions were calcu-
lated for three different situations. Under the condi-
tions used for the measurement of oxygen-binding
curves, the conformation tT is not strongly populated
(Fig. 8A–C). Thus, neither protons nor Mg
2+
displace
the conformational distribution sufficiently towards the
tT state to visibly shift the lower asymptote of the Hill
plot. This also explains the relatively large errors in
the parameter describing the effector binding in the tT
Size of the basic allosteric unit w
3456
Number of coupled allosteric units s
4
3
2
1
1/4 1/2 1

region excludes the 4 · 4 combination (SSR ¼ 34.6) but includes
the 2 · 4 combination (SSR ¼ 22.1). The confidence region also
includes all combinations of m and n (SSR < 21.9), for which the
3 · 4 nested model was tested. Note that the error contour maps
are truncated at SSR levels higher than 40.
Table 3. Best-fit parameter combinations of the nested MWC
model. The best fit of oxygen-binding data from T. cancriformis Hb
gave a model that assumed a basic allosteric unit with w ¼ four
oxygen-binding sites and a functional coupling of s ¼ two to three
allosteric units. The parameters refer to the presence of one oxy-
genation-linked acid group and half a Mg
2+
-binding site per oxygen-
binding site. Given are the oxygen-binding constants (K
ab
), the pK
ab
values, and the Mg
2+
-binding constants (z
ab
) for the four conforma-
tions (ab ¼ tT, rT, tR, rR). The allosteric equilibrium constants (l
T
°,
l
R
°, L°) refer to the reference condition at pH 6.5 and zero Mg
2+
concentration. The SSR was taken as a measure of the goodness

pK
rT
7.65 ± 1% 7.63 ± 1%
pK
tR
8.16 ± 1% 8.15 ± 1%
pK
rR
7.95 ± 1% 7.84 ± 1%
z
tT
(mM
)1
) 0.016 ± 50% 0 ± 0.147
z
rT
(mM
)1
) 0.023 ± 20% 0.024 ± 18%
z
tR
(mM
)1
) 0.009 ± 33% 0.011 ± 25%
z
rR
(mM
)1
) 0.025 ± 21% 0.028 ± 18%
log l

the effector-induced phenomena visualized by the Hill
plot are reflected in the conformational distribution
and can be rationalized by the values for the effector
binding constants.
From the detailed analysis of oxygen-binding curves
measured at different effector concentrations, some
information about the functional properties can be
gained. Firstly, the functional coupling extends beyond
the disulfide-bridged dimer D. Secondly, the structural
hierarchy is mirrored in the functional properties, and
a certain complexity in the binding process involving
several conformational substates is present. No indica-
tion of negative cooperativity (for example in the
values for i
r
and i
t
) could be found. Thus, if any
heterogeneity is present at the subunit level, it is not
sufficient to influence the results of the analysis. A lack
of significant functional heterogeneity despite the exist-
ence of different subunit types is known for other
proteins such as the hemocyanins [49–51].
Both the cooperon model and the nested MWC
model identify substructure D
2
(Fig. 6A) as a possible
higher-level allosteric unit. However, D
3
may also be

D.
Actually, a hybrid model consisting of a 2 · 4 nested
MWC model and the Hill equation, the latter descri-
bing the oxygen binding of the attached 9th dimer in
an effector-independent manner, gave an SSR similar
to that of the 3 · 4 nested MWC model. The parame-
ters of this hybrid model (data not shown) typically
differed by less than 10% from those obtained for the
pure 2 · 4 nested MWC model.
Although the physiological significance of the pres-
ence of 16-mers and 18-mers remains unresolved, the
Hb of the branchiopod crustacean T. cancriformis
at constant pH 7.6
pH
6.5 7.0 7.5 8.0
log [ , L]
-8
-6
-4
-2
0
Mg
2+
-free conditions
log [l
R
, l
T
]
1

0.0
0.5
1.0
D
tT
tR
rR
rT
Y
Fig. 8. Conformational distributions (A–C) and effector sensitivities of the allosteric equilibrium constants of the 2 · 4 nested MWC model
(D). The conformational distributions as a function of oxygen partial pressure (PO
2
) were calculated for three different conditions (Hepes buf-
fer and 20 °C): at pH 7.60 and 100 m
M Mg
2+
(A), and in the absence of Mg
2+
at pH 8.30 (B) and pH 6.69 (C). The conformational states are:
tT (dotted lines), tR (dashed lines), rR (solid lines), and rT (dotted ⁄ dashed lines). Oxygen saturation (Y) is given by circles. (D) Dependence of
the allosteric equilibrium constants (l
T
, l
R
, L, L) on pH under Mg
2+
-free conditions (left) and on Mg
2+
concentration at constant pH 7.6 (right).
Error bars represent the standard errors of the allosteric equilibrium constants for the reference state at pH 6.5 and zero Mg

without contact with air [33]. Hemolymph osmolality was
measured in three individual organisms using a cryoscopic
osmometer (Osmomat 030; Gonotec, Berlin, Germany).
The chloride concentration was measured in a pooled sam-
ple of hemolymph using a CMT 10 chloride titrator (Radio-
meter). The heme concentration was determined from the
a-absorption peak of oxygenated Hb at 576 nm using a
millimolar absorption coefficient of 10.4 LÆmmol
)1
Æcm
)1
determined for T. cancriformis Hb (R. Pirow, unpublished
data). Hb concentration (mg proteinÆmL
)1
) was deduced
from heme concentration by assuming that the T. cancrifor-
mis Hb is composed of 37-kDa subunits each carrying two
heme groups [20].
Preparation of dialyzed hemolymph
Dialyzed hemolymph solutions were prepared from two
pooled samples (500 lL each) of pure hemolymph drawn
from 60 animals. Hemolymph cells were removed by cen-
trifugation (7000 g, 10 min) at 4 °C. To remove possible
cofactors to Hb–oxygen binding, the Hb-containing super-
natant was repeatedly dialyzed against 0.01 m Tris ⁄ HCl
buffer (pH 7.5 at 5 °C) using 4 mL centrifugal filter devices
with a molecular mas cut-off of 30 kDa (Amicon Ultra-4;
Millipore, Schwalbach, Germany). Any cofactor that may
have been present was thereby diluted to less than 1.2% of
its native concentration. The retentate was finally concen-

)1
. The
samples were kept on ice until oxygen-binding measure-
ments.
Oxygen-binding curves
The various types of Hb-containing samples (whole hemo-
lymph, dialyzed hemolymph, purified Hb) were investigated
in two campaigns using different experimental set-ups.
Oxygen-binding curves of dialyzed hemolymph were
recorded at different pH values obtained by adding (to a
30-lL aliquot of Hb stock solution) BisTris buffers
(pH 6.7–7.3) and Tris buffers (pH 7.4–8.1) to a final buffer
concentration of 0.1 m and distilled water to a final sample
volume of 100 lL. pH measurements were carried out in
duplicate, at the same temperature as the oxygen-equilib-
rium measurements, using % 50-lL subsamples and the
above-described pH meter. The effects of inorganic bivalent
ions on oxygen binding were examined by adding accurate
volumes of standard (40 mm, 100 mm,1m) solutions of
MgCl
2
and CaCl
2
. In addition to the experiments with dia-
lyzed Hb, three oxygen-binding curves of a pooled sample
of whole hemolymph were recorded at 0%, 1%, and 2%
CO
2
. Oxygen equilibria were determined on 3–6-lL sub-
samples using a modified gas diffusion chamber [32,52]

sthoff gas mixing pump which mixed air with pure
nitrogen. Oxygen saturation was evaluated from absorbance
relative to the values obtained for the samples equilibrated
with air and nitrogen, respectively. As deduced from earlier
experiments, the Hb is practically saturated with oxygen
at normoxic conditions. Possible errors introduced by a
slightly incomplete saturation when equilibrating with air
were taken into account by an adjusting parameter for nor-
malization to 100% saturation. This parameter remained
above 0.97 in all cases.
The possibility of met-Hb formation during the oxygen-
equilibrium measurements was checked at the end of the
experiment by recording absorbance spectra of the recov-
ered Hb sample. The met-Hb content was derived from
the peak wavelength of the Soret peak using a calibration
curve, which was generated from different linear combina-
tions of the absorption spectra of fully oxygenated Hb
(peak wavelength 415.7 nm) and fully oxidized Hb
(404.3 nm). Gaussian curve-fitting was used to determine
the exact position of the Soret peak. The recovered Hb
samples had a met-Hb content of 4.1 ± 2.4% (mean ±
SD, n ¼ 19).
Determination of oxygen-equilibrium parameters
Oxygenation data based on at least five equilibrium steps
between 0.05 and 0.95 fractional saturation (Y) were con-
verted into Hill plots {log [Y ⁄ (1–Y)] against log PO
2
, where
PO
2

and T
2
are the absolute
temperatures (283 and 293 K, respectively).
Analysis of oxygen-binding curves
Precise equilibrium measurements for extended Hill plots
that emphasize extreme (low and high) oxygen saturation
values were carried out as previously described [53]. Six
equilibrium curves (Fig. 4B,D), which were measured under
four pH conditions and at three Mg
2+
concentrations in
Hepes buffer, were analyzed using different models for
cooperativity. The binding polynomial (B
mod
) for each
model (Adair, MWC, three-state, cooperon, nested MWC)
is given below. From B
mod
, the degree of saturation (
^
Y)is
obtained by differentiation according to:
^
Y ¼
1
N
xoB
mod
=ox

À
^
Y
i
Þ
2
s
2
Y
i
ð4Þ
where Y
i
and
^
Y
i
are the measured and predicated satura-
tions of the ith point, and k is the number of data points.
Adair equation
To determine the minimum number of interacting binding
sites necessary to describe the data, the binding curves were
analyzed by a tetramerous and octomerous Adair equation
[54,56]. The native Hb isoforms of T. cancriformis contain
32 and 36 oxygen-binding sites [20], respectively, but often
it is not mandatory to assume that all binding sites of large
respiratory proteins are coupled in a cooperative manner.
Allosteric control of O
2
binding in crustacean Hb R. Pirow et al.

effects of temperature and pH on the overall oxygen affin-
ity was calculated from the geometric mean of the Adair
constants [54]:
P
m
¼
Y
N
i¼1
K
i
!
À
1
N
ð6Þ
MWC model and three-state model
The MWC model [60] is based on two conformations
(r and t), which are in equilibrium (L) and have different
binding affinities for the ligand (K
r
, K
t
). The number of
molecules that are coupled so that they always adopt the
same conformation is defined by the size of the allosteric
unit (q):
B
MWC
¼

q
1 þ L
s
þ L
t
ð8Þ
For a situation where two molecule species with oxygen-
binding characteristics obeying the MWC formalism are
present as fractions q and 1–q, the saturation function
(Y
2-MWC
) is given by:
Y
2-MWC
¼ qY
1
þð1 À qÞY
2
ð9Þ
The individual saturation functions Y
1
and Y
2
are derived
from B
MWC
with species-specific values for K
r
, K
t

x
2
Þ
z
1 þ L
ð10Þ
Nested MWC model
Oxygen-binding curves were also analyzed in terms of the
nested MWC model [43,44], which takes hierarchies within
the quaternary structure of multimeric proteins into
account. In this model it is assumed that a number of w
binding sites are functionally coupled to form an allosteric
unit which, in accordance with the standard MWC model
[60], can adopt two basic conformations (r and t). A num-
ber of s copies of these basic allosteric units assemble into
a larger allosteric unit containing q ¼ s · w oxygen-
binding sites. This s · w structure can adopt two overall
conformations (R and T) which impose constraints on the
conformations of the constituent (nested) w-sized allosteric
units. When the s · w structure is in the R-state, the nes-
ted w-sized allosteric units can adopt two conformations,
B
Fractional ox
yg
en saturation (Y)
0.0 0.2 0.4 0.6 0. .0
Standard deviation (s
Y
) of Y
0.00

2
binding in crustacean Hb
FEBS Journal 274 (2007) 3374–3391 ª 2007 The Authors Journal compilation ª 2007 FEBS 3387
rR and tR. Two alternative conformations, rT and tT, are
available when the overall conformation is T (Fig. 6). The
equilibrium between the unliganded R
0
and T
0
states of
the overall conformation is described by the equilibrium
constant L ¼ [T
0
] ⁄ [R
0
]. The allosteric equilibrium con-
stants l
R
and l
T
correspond to the concentration ratio of
the unliganded states of the basic allosteric unit, l
R
¼
[tR
0
] ⁄ [rR
0
] and l
T

s
þ K B
w
rT
þ l
T
B
w
tT
ÀÁ
s
ð12Þ
with
K ¼ L
ð1 þ l
R
Þ
s
ð1 þ l
T
Þ
s
¼
½rT
0

s
½rR
0


l
R
¼ l
Ã
R
ðQ
tR
=Q
rR
Þ
wm
ð14Þ
l
T
¼ l
Ã
T
ðQ
tT
=Q
rT
Þ
wm
ð15Þ
K ¼ K
Ã
ðQ
rT
=Q
rR

is given by:
l
Ã
R
¼ l

R
ðq
tR
=q
rR
Þðq

rR
=q

tR
Þ
ÂÃ
wh
ð18Þ
l
Ã
T
¼ l

T
ðq
tT
=q

q
ab
¼ 1 þ 10
pK
ab
ÀpH
ð21Þ
where l
R
°, l
T
°, and L° represent the allosteric equilibrium
constants in the absence of Mg
2+
at pH 6.5 (reference con-
dition), whereas q
ab
and pK
ab
refer to the proton-binding
polynomial and the pK value of the oxygenation-linked
acid group for the particular conformation. q
ab
° refers to
the reference state at pH 6.5 and zero Mg
2+
concentration.
Parameter constraints included the following lower and
upper boundary values: K
ab

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