R-00292-2002-R1-Weber et al. 1
Novel Mechanism for High-Altitude Adaptation in
Hemoglobin of the Andean Frog Telmatobius peruvianus
ROY E. WEBER1, HRVOJ OSTOJIC2, ANGELA FAGO1, SYLVIA DEWILDE3, MARIELOUISE VAN HAUWAERT3, LUC MOENS3 and CARLOS MONGE4
1
Department of Zoophysiology, University of Aarhus, 131 C.F. Møllers Alle, DK 8000 Aarhus
C, Denmark, 2Clinicum, Laboratorio Automatizado, Iquique, Chile, 3Biochemistry Department,
University of Antwerp, Universiteitsplein 1, B-2610 Antwerpen, Belgium, 4Laboratorio de
Transporte de Oxígeno, Universidad Cayetano Heredia, Lima 31, Peru.
Running title: Altitude adaptation in frog hemoglobin
Address for Correspondence and proofs:
Roy E. Weber, Department of Zoophysiology, University of Aarhus, 131 C.F. Møllers Alle, DK 8000
Aarhus C, Denmark. Telephone: +45 8942 2599; Fax: +45 8619 4186;
E-mail address: [email protected]
Abbreviations used (defined when first-used): DPG, 2,3-diphosphoglycerate; NTP, nucleoside triphosphate;
P50, half-saturation O2 tension; Pm , median O2 tension; n50 , Hill cooperativity coefficient at P50; nmax , the
maximum cooperativity, MWC, Monod, Wyman and Changeux; KT and KR, O2 association constants of
low-affinity (tense) and high-affinity (relaxed) states, respectively, of Hb; qH, the number of interacting O2
binding sites; M, Bohr factor ('log P50/'pH); 'G, free energy of cooperativity; 'Happ, apparent heat of
oxygenation.
R-00292-2002-R1-Weber et al. 2
(Abstract)
In contrast to birds and mammals, no information appears to be available on the molecular adaptations for
O2 transport in high-altitude ectothermic vertebrates. We investigated hemoglobin (Hb) of the aquatic
Andean frog Telmatobius peruvianus from 3800 m altitude as regards isoform differentiation, sensitivity
to allosteric cofactors and primary structures of the D and E chains, and carried out comparative O2binding measurements on Hb of lowland Xenopus laevis. The three T. peruvianus isoHbs show similar
functional properties. The high O2 affinity of the major component results from an almost complete
obliteration of chloride sensitivity, which correlates with two D-chain modifications: blockage of the Nterminal residues and replacement by nonpolar Ala of polar residues Ser and Thr, at position D131(H14)
in human and X. leavis hemoglobins, respectively. The data indicate adaptive significance of D-chain
chloride-binding sites in amphibians, in contrast to human hemoglobin where chloride appears mainly to
bind in the cavity between the E chains. The findings are discussed in relation to other strategies for highaltitude adaptations in amphibians.
Keywords: amphibians, chloride binding, hypoxia, organic phosphates, oxygen transport
R-00292-2002-R1-Weber et al. 3
HOW IS OXYGEN TRANSPORT to metabolising tissues secured at high-altitude? In contrast to
intensive investigations in birds and mammals (6,39,59), the molecular strategies for O2 transport in highaltitude ectothermic vertebrates remain unexplored, despite greater variations in environmental conditions
(temperature, pH, O2 tension, etc.) and lesser capacities for homeostatic regulation of internal physical
and chemical conditions compared to homeothermic vertebrates, and a long-standing interest in highaltitude, aquatic amphibians (1,24).
The anuran genus Telmatobius (that variously is referred to as frogs or toads) occurs in the
Andes mountains at altitudes from 2000 m to over 4000 m (14) where aerial O2 tensions fall from
approximately 159 mm Hg at sea level to 92 mm Hg. The hypoxic stress is compounded in aquatic
species, particularly at night when photosynthetic activity in the ponds ceases (21). T. culeus found in lake
Titicaca at 3812 m, has reduced, poorly-developed lungs but exhibits compensatory physiological and
behavioural adaptations (24) that include an “over-sized”, folded skin, which is penetrated by cutaneous
capillaries and ventilated by ‘bobbing’ behaviour under hypoxia, and small erythrocytes and higher
erythrocyte counts, blood O2 affinities and O2 carrying capacities than anurans living at sea level (24).
Subspecies of Bufo spinulosus living at sea-level and at 3100 to 4100 m in the Andes analogously exhibit
increasing blood-O2 affinities with altitude (43).
The O2 affinity of blood is a product of the intrinsic O2 affinity of the Hb molecules and the
erythrocytic effectors that modulate Hb-O2 affinity. Compared to mammals that use 2,3
diphosphoglycerate (DPG) and fish that use ATP [often in conjunction with the more potent effector
guanosine triphosphate) (60) as organic O2-affinity modulators, amphibian red cells carry both ATP and
DPG in widely varying relative concentrations (22). Moreover, as seen in Rana temporaria (13), and R.
catesbeiana (49-51) individual amphibian isoHb components may exhibit functionally-significant
interactions.
Aiming to identify the molecular adaptations for O2 transport in high-altitude amphibians we
investigated isoHb differentiation and the interactive effects of pH, temperature, chloride ions, ATP and
DPG on O2 binding in T. peruvianus Hb, carried out some comparative measurements on Hb from the
R-00292-2002-R1-Weber et al. 4
lowland, aquatic toad Xenopus laevis and determined the primary structures of the D and E chains of the
major T. peruvianus isoHb.
METHODS
Animals,Hb preparation and isolation. Telmatobius peruvianus of either sex was collected at
3800 meter’s altitude from small streams near the Andean village Cancosa at the Bolivian boarder in
North Chile. Frogs used (n=5) weighed 17.2 (2.5) g, and measured 4.9 (0.2) cm (snout-vent). Blood
samples were taken within 12 hours of descent to the sea-level laboratory at Iquique. Electrophoresis on
cellulose acetate strips at pH 8.6 indicated Hbs with the same anodic migration rates in all specimens.
Specimens of the lowland African clawed toad Xenopus laevis were purchased from Blades Biological,
Cowden UK. Animal handling followed the “Guiding principles for research involving animals and
human beings” (2).
Hb purification was carried out at 0-5°C as previously described (61). Hb heterogeneity was
investigated by isoelectric focussing in 110 ml LKB columns (Bromma, Sweden) in 0.87% ampholines
(pH 6.7 - 7.7) (46). Separated isoHb fractions were dialysed against 0.01 M HEPES buffer containing
5·10-4 M EDTA, pH 7.7 (at 5°C) and stored at –80°C in 0.1 ml aliquots that were freshly thawed for
molecular and functional characterization. Hemolysates were stripped of organic phosphates using MB1
mixed ion-exchange resin (BDH Chemicals, Poole, England).
O2-equilibrium measurements. O2 equilibria of thin (~0.01 mm) layers of Hb dissolved in 0.1 M
HEPES buffers were measured using a modified diffusion chamber as previously described (57,58). The
P50 [half-saturation O2 tensions (1 mm Hg = 0.133 kPa)] and n50 (cooperativity coefficient at 50% O2
saturation) values recorded represent individual data point interpolated from Hill plots (Log
([OxyHb]/[Hb]) vs. Log PO2; correlation coefficient r > 0.995) that were generated on the basis of at least
4 equilibration steps between 30 and 70% O2-saturation. The pH values were measured in oxygenated
subsamples equilibrated to the same temperatures (61). O2-equilibrium curves focusing on extreme O2
saturations (<2% and >98%) were analyzed by end-weighted fitting (61) of the two-state Monod-WymanChangeux (MWC) equation (40) to the data. The overall heat of oxygenation ('Happ that includes
R-00292-2002-R1-Weber et al. 5
contributions from oxygenation-linked reactions) was evaluated from the van’t Hoff equation (4,63).
Primary structure determinations. Separation of the D and E chains, enzymatic digestions, and
isolation and amino acid sequence analyses of the peptides were carried out as previously described (61).
RESULTS
Hb heterogeneity. Isoelectric focussing revealed one major component, Hb II, and two minor
ones, Hb I and Hb III, with isoelectric points of 7.34, 7.45 and 7.30, and relative abundances of 81:12:7
respectively (Fig. 1). At pH 7.55 T. peruvianus isoHbs I, II and III show practically identical P50 values
(Table 1) that correspond with that of the composite hemolysate (P50 = 7.3 at 20°C, pH 7.5) (Fig. 1, inset).
In conjunction with corresponding results at pH 7.1 (not shown) this indicates the absence of functionallysignificant interactions between the individual isoHbs under the experimental conditions. Cooperativity in
O2 binding at half-saturation (n50) was pronounced (2.8) and pH independent in Hbs I and II (Fig. 3) but
lower (2.2) in Hb III.
Effector sensitivities and allosteric interactions. Strikingly, the O2 affinity of T. peruvianus Hb
II is almost insensitive to chloride ions, despite pronounced effects of [ATP+Cl-] and [DPG+Cl-] (Figs. 2
& 3, Table 1). The potentiation of the Bohr effect by chloride (associated with increased ionization of the
positively-charged sites with falling pH) was correspondingly small (M = -0.16, compared to -0.43 and 0.52, in the presence of Cl-, [ATP+Cl-] and [DPG+Cl-]). The higher O2 affinities and increased anion
sensitivies observed at 10° than at 20°C (Fig. 3A) reflect exothermic oxygenation and linked endothermic
dissociation of allosteric effactors that reduce 'Happ (Table 1). Hb I showed the same O2 affinity trends as
the major component (Hb II) but slightly lower anion sensitivities (Table 1, Fig. 3C).
In the absence of anions X. leavis and T. peruvianus Hbs show almost identical O2 affinities (P50
= 7.3 mm Hg at 20°C) (Fig. 2). In the presence of 0.1 M chloride, however, X. laevis Hb exhibits a much
lower affinity than T. peruvianus Hb, revealing preservation of a pronounced chloride effect, and a larger
Bohr effect (M = -0.40 compared to –0.16 in T. peruvianus) (Fig. 3; Table 1).
Dose-response curves (Fig. 4) show that ATP and DPG exert the same effects on the O2 affinity
of T. peruvianus Hb . The maximum slope of the log P50 vs log [phosphate] plots approximates 0.25,
R-00292-2002-R1-Weber et al. 6
tallying with O2-linked binding of one phosphate molecule per deoxyHb tetramer. The maximum
ATP/DPG-induced logP50 shift is smaller than that for DPG and human Hb (Fig. 4) and the phosphate
concentrations required for half the maximum change in log P50 indicates an apparent dissociation
equilibrium constant in T. peruvianus Hb that is an order of magnitude higher than for human Hb and
DPG (Kd = 7.9 $10-4 compared to 0.71$10-4 M; Fig. 4).
Extended Hill plots for T. peruvianus Hb II at 10 and 20 °C and in the absence and presence of
ATP and the derived MWC parameters are shown (Fig. 5 and Table 2). The number of interacting O2
binding sites (qH) estimated by fitting this allosteric parameter along with the others was 3.760.10 (n=4)
indicating a stable tetrameric Hb structure, which also is reflected by the exact superimposition of
extended Hill plots obtained at different Hb concentrations (Fig. 5). In contrast to most vertebrate Hbs
where organic phosphates primarily reduce the O2 association constant of the low-affinity, tense state of the
Hb molecules (KT) (55,62) thus increasing the free energy of cooperativity ('G), ATP also decreases the
association constant of the high-affinity relaxed state (KR), and reduces 'G (Table 2). However, the KR
values need to be viewed with caution due to difficulties in measuring the last few per cent saturation of
the oxygenation curve (37).
Primary structure. The primary structures of the D and E chains of Hb II are shown (Fig. 6).
Whereas the E chain was directly accessible for Edman degradation, N-terminal sequencing of the intact
globin chains showed that the D chain was blocked. Attempts to deblock the chain failed to give clear-cut
results, so that the four/five N-terminal amino acid residues of this chain are not known. The primary
structures of both globin chains were reconstructed from relevant peptides. Each sequence was obtained at
least twice. The obtained sequences were aligned unambiguously with known amphibian sequences (Fig.
6).
In contrast to Rana esculenta and R. catesbeiana E chains that lack the first six N-terminal
residues compared to most other vertebrate Hbs (5,16), T. peruvianus E chains consist of 145 amino acid
residues. Of these 93 (64.13 %) are identical with X. laevis E-1 chains and 87 (56.55 %) with human Hb.
The differences in T. peruvianus compared to X. laevis are concentrated in the N-terminal region where
only 8 of the 24 N-terminal E chain residues are identical. Although common in fish Hbs, acetylation of
R-00292-2002-R1-Weber et al. 7
the -amino group of the chains as found in T. peruvianus HbII is rare in amphibians and has only been
reported in E - III larval (56), D-III larval (38) and D-C chains (49) of R. catesbeiana.
DISCUSSION
The hypoxic challenge at altitude where O2 loading may be critical is compounded in aquatic
habitats – as indicated by increased blood O2 affinities encountered in lowland amphibians with increasing
reliance on water as the respiratory medium (32) and the higher blood-O2 affinities in predominantly
aquatic T. culeus and X. laevis than in predominantly terrestrial Rana and Chiromantis species (Fig. 7A).
These inter-species correlations accord with observations that hypoxic exposure increases blood-O2
affinity by decreasing red cell DPG levels in the salamander Ambystoma tigrinum (66) and raises plasma
catecholamine levels (that may increase O2 affinity through red cell swelling (29,42,42)) in the toad Bufo
marinus (3). However, 10-11 days’ hypoxic acclimation did not change blood-O2 affinity in the
salamander Desmognathus quadramaculatus (36).
The ATP-induced Hb-O2 affinity shifts (Fig 2) and the difference in affinities between stripped Hb
and whole blood in T. peruvianus and X. laevis (Fig. 7B) reveal pronounced capacities for effector
modulation in both species. The similar effects of ATP and DPG on the O2 affinity of T. peruvianus Hb
(Fig. 4) tally with similar magnitudes of ATP and DPG binding constants in human Hb (25) and suggest
that the differences in erythrocytic NTP/DPG ratios (~3.0 and ~0.8, respectively, in Lake Titicaca T.
culeus and X. laevis (22)) do not contribute to the species differences in blood O2 affinity. The
observation that ATP alone decreases the O2 affinity of T. peruvianus Hb slightly more than ATP+Cl(Fig. 3) may be attributed to competition of the two anions for the same sites in the central cavity (19,44)
and neutralization of the positively-charged phosphate binding sites by chloride.
The anuran Hbs show distinctive structure-function relationships. In comparison to human Hb, T.
peruvianus HbII shows a less tight binding of ATP and DPG (Fig.4), despite conservation of the
positively-charged organic phosphate binding sites in the cavity between the E chains, viz., N-terminal
Val, E2(NA2)His, E82(EF6)Lys, and E143(H21)Lys that replaces histidine in human Hb. In X. laevis the
deletion of the first E-chain residue (Fig. 6) could bring the N-terminus closer to the bound cofactor and
R-00292-2002-R1-Weber et al. 8
preserve phosphate sensitivity despite the loss of His(NA2). The Bohr effect of the stripped T. peruvianus
HbII is small despite the presence of E146His(HC3) and a negatively-charged residue (Glu) in position
E94(FG1) that contribute about half of the anion-independent Bohr effect in human Hb (30,47). In
contrast to the majority of vertebrate Hbs, where allosteric effectors decrease the affinity of the T state of
the deoxygenated molecule (53,54,62), frog Hbs may also be modulated in the R state, as evident from
the ATP sensitivity of T. peruvianus HbII (Fig. 4 and Table 2) and the pH effect in Rana temporaria Hb
(13). The molecular mechanism underlying these effects must await the elucidation of the crystal
structures of deoxy and oxy forms of amphibian Hbs.
The similar O2 affinities in Hbs I, II and III and in the stripped hemolysate (Table 1, Fig. 1)
indicate the absence of functionally-significant interactions between the individual isoHbs under the
tested conditions. This contrasts with R. catesbiana where aggregation of the major tetrameric
components B and C, to form a low-affinity BC2 trimer-of-tetramers is manifested at corresponding pH
and Hb concentrations as tested here (50,51).
What, if any, are the distinguishing molecular adaptations to altitude in T. peruvianus Hb?
Comparison with lowland X. laevis Hb shows that the major difference resides with the effects of anions.
Although stripped Hbs from the two species show almost identical O2 affinities and pronounced
[ATP+Cl-] effects, T. peruvianus Hb shows a drastically suppressed chloride sensitivity ('log P50 = 0.10
compared to 0.32 in X. laevis and >0.4 in human Hb, Figs. 2 & 7B). In the absence of other changes this
will enhance O2 loading under hypoxia without the need for reducing erythrocytic organic phosphate
levels and thus allosteric regulatory capacity. In contrast to short-term hypoxic challenges that evoke
adaptive changes in erythrocytic phosphate levels (41), obligate residence at high-altitude appears to be
associated with the presence of high-affinity (iso)Hbs - as previously illustrated in homeothermic
vertebrates. However, in contrast to the bar-headed goose and Rüppell’s griffon (7,12,45) that may fly at
9000 and 11300 m above sea level, where the high intrinsic O2 affinity is attributed to amino acid
substitutions located at the D1E1 or D1E2 interface (26,33,63) and llama Hbs, where high blood affinity is
achieved through loss of E-chain phosphate-binding residues, the high affinity in T. peruvianus HbII
results from a loss of anion sensitivity that correlates with D subunit amino acid substitutions.
R-00292-2002-R1-Weber et al. 9
Two schools of thought exist as regards O2-linked chloride binding to human Hb, which has been
proposed to occur either at ‘localized’ (19) or at ‘delocalized’ (44) sites. The ‘localized’ binding sites are
an D-chain site [lying between the D1Val-NH+3 group and E-OH of D131(H14)Ser and the side chain of
D131Ser(H14)] and a E-chain site [between E1Val and the H-NH3+ group of E82Lys] (47). Evidence for
their involvement comes from X-ray diffraction studies of crystallized human Hb specifically
carboxymethylated at D1Val (18) and the crystal structure of the human Hb mutant, E (V1M+H2) [where
E1Val(NA1) is exchanged for Met and E2(NA2)His is deleted] that document the implication of the Nterminal residues in O2-linked chloride binding and the chloride-dependent Bohr effect (19). The
‘delocalized’ mechanism proposed by Perutz et al. (44) builds on the view (9) that excess positive charges
in the water-filled cavity between the E chains destabilize the T-state, and that chloride ions diffusing into
the cavity of deoxygenated human Hb reduce O2 affinity by partially neutralizing the repulsion between
these charges, thus reducing the free energy of the T-structure. This mechanism is supported by
observations that amino acid substitutions that increase central cavity electropositivity cause a
proportional increase O2 affinity and vice versa (44).
Whereas the mechanism of chloride binding in human Hb remains unresolved, our data indicate
predominant importance of specific (‘localized’) D-chain sites in amphibian Hbs. Thus the D-chain
residues (1Val and 131Ser in humans) are conserved in X. laevis Hb (where polar Ser at 131 is substituted
by polar Thr and the N-terminal residues are free) that shows pronounced chloride sensitivity, but
eliminated in T. peruvianus Hb (where 131 is occupied by nonpolar Ala and the D chain N termini are
acetylated) that shows strongly reduced chloride sensitivity. That chloride additionally may bind in the
central cavity between the E chains (in competition with organic phosphates) is indicated by the
observation that ATP alone has a greater effect on O2 affinity of T. peruvianus Hb than ATP in the
presence of 0.1 M chloride (Fig 3).
In contrast to evidence for ‘localized’ chloride-binding (above), there is no evidence from the
central cavity amino acid exchanges for greater ‘delocalized’, oxygenation-linked chloride binding in X.
laevis than in T. peruvianus Hb. Perutz et al. (44) list 5 D-chain and 14 E-chain polar residues in the
central cavity of human Hb that may affect O2 affinity by increasing or reducing the excess positive
R-00292-2002-R1-Weber et al. 10
charge. Compared to X. laevis Hb, T. peruvianus Hb II shows one -chain and six E-chain exchanges at
these positions. These are (the helix notation refers to human Hb): D133(H16)SerGly (that represents
loss of a polar site), E1(NA1)<no residue>Val (that does not affect charge), E2(NA2)GlyHis (that
increases positive charges), E104(G6)LysVal (that reduces positive charge), E135(H13)AspGly (that
reduces negative charge and thus increases net positive charge) and E101(G3)LeuAla,
E132(H10)HisLys and E136(H14)Ala Gly (that are electroneutral). Assuming equivalence of
structural factors, these exchanges may thus be expected to increase the number of positive charges in the
central cavity and consequently the intrinsic O2 affinity and the chloride effect in T. peruvianus compared
to X. laevis Hb. Such effects are not evident from our data.
Other evidence indicates that ‘localized’, D-chain chloride binding also may be implicated in
adaptations encountered in some mammalian Hbs. The high O2 affinities of Hb from Andean camellid
vicuna (31) and of embryonic pig Hbs Gower I and Heide I that have ]-(-type) chains (64) are associated
with a D130AlaoThr replacement, which introduces a hydroxyl group that may interfere with chloride
binding at neighboring D131Ser. Also, the almost complete lack of chloride effects in human embryonic
Hbs Gower I and Portland (]2H2 and ]2Q2) (68) correlates with an analogous D131Sero]131Val
substitution to that here reported for T. peruvianus Hb II.
In conclusion this study shows a novel molecular mechanism for high altitude adaptation in
ectotherm vertebrates that involves a reduction in chloride modulation of Hb-O2 affinity via loss of
specific chloride binding sites on the D-chains and still allows for phosphate modulatory capacity. It
should, however, be borne in mind that the molecular adaptations supporting tissue O2 supply are but part
of a symphony of organismic, cellular and molecular adjustments expressed in high altitude animals
(39,63). As has become well-established, hypoxia elicits a fall in (preferred) body temperature, which in
anurans, appears to be adenosine- and lactate-mediated (10,11,67). The low body temperatures – that are
naturally experienced by Telmatobius living in cold streams of melted snow – impart a range of possible
advantages. Apart from raising the O2 content of the water, it increases blood O2 affinity, as dictated by
the exothermic nature of the Hb-oxygenation reaction. Also, it decreases metabolic rate and lowers tissue
R-00292-2002-R1-Weber et al. 11
O2 demands, which in cold-submerged Rana temporaria is associated with increased reliance on
carbohydrate metabolism and maintenance of homeostatic ATP levels (17). Hypoxia may, however, also
have beneficial effects under certain conditions (48). Several studies show that O2 deprivation may
protect tissues of homeo- as well as ectothermic vertebrates against subsequent hypoxic/ischemic
episodes (15,20,34). In Rana pipiens and goldfish anoxic exposure moreover induces changes in the
antioxidant system that minimize subsequent effects of oxidative stress (23,34). Telmatobius may be an
excellent model for studying adaptations to chronic hypoxemia.
Text footnotes
(Acknowledgements)
Supported by the Danish Natural Science Research Council (SNF) and the Fund for Scientific Research
Projects, Flanders, Belgium. We thank Anny Bang (Aarhus) for valuable technical assistance.
R-00292-2002-R1-Weber et al. 12
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Figure Legends
Fig. 1. Preparative isoelectric focussing of T. peruvianus hemolysate, showing one major (II) and two
minor (I and III) isoHb components. o, absorption values at 540 nm; ', pH at 25°C. Horizontal bars,
fractions pooled for analyses. Right inset, diagram of column at the end of focussing. Left inset, O2
equilibrium curves of o, stripped hemolysate; c, Hb I; V, Hb II; U, Hb III; Buffer, 0.1 M HEPES, [KCl],
0.10 M; [heme], 0.15 mM; 20 °C.
Fig. 2. O2 equilibrium curves of stripped (A) T. peruvianus Hb II and (B) X. laevis Hb at pH 7.0 and 20°C
in the absence of added anions (U) and in the presence of Cl- ({) and Cl-+ATP (z). Buffer, 0.1 HEPES;
[Heme], 0.15 mM (Hb II); ATP/Hb ratio: >100.
Fig. 3. Effects of pH on P50 and n50 values of stripped T. peruvianus and X. laevis Hbs in the absence and
presence of 0.1 M chloride and saturating phosphate concentrations. A, T. peruvianus Hb II at 10°C; B, T.
peruvianus Hb II at 20°C; C, T. peruvianus Hb I at 20°C, and D, X. laevis Hb at 10 and 20°C; U, no
effectors; S, Cl-, , ATP; ‹, ATP+Cl-; ’, DPG+Cl-; dotted lines, 10°C; continuous lines, 20°C; [Heme],
0.15 mM (Hb II) and 0.09 mM (Hb I); Other details as in Fig. 2.
Fig. 4. Dose-response curves showing effects of ATP (U) and DPG (‘) on P50 of T. peruvianus Hb II.
pH, 7.0; [Cl-], 0.10M; [Heme], 0.15 mM. , effect of DPG on human Hb (pH, 7.3; [Cl-], 0.05M, after ref.
(8)); Temperature, 20 °C. Arrows and dotted lines show maximum phosphate-induced changes in P50.
Dashed straight lines indicate slopes of 0.25.
Fig. 5. Extended Hill plots of T. peruvianus Hb II at 10°C (‘, …,¡) and 20°C (U,SS) in the absence
(open symbols) and presence (closed symbols) of saturating ATP concentrations (ATP/Hb 134). Y,
fractional O2 saturation; [Heme], 0.26 mM (, , U, S), and 0.16 mM (…).
R-00292-2002-R1-Weber et al. 20
Fig 6. Amino acid sequences of the D and E chains of Telmatobus Hb II compared with those of human, X.
laevis and Rana Hbs. X. laevis laevis E, major adult Hb E1 chain (Ac.no.P02132); Rana catesbeiana E,
adult Hb E-chain (Ac.no. P02135); X. laevis D, major adult Hb D1 chain (Ac.no. P02012); Rana
catesbeiana BD, adult Hb DB chain (Ac. P51465); Rana catesbeiana CD, adult Hb DC chain (Ac.no.
P55267). The protein sequence data for the D and E chains of Telmatobius peruvianus Hb II here reported
are registered in the Swiss-Prot Protein Data Bank under accession numbers P83113 and P83114,
respectively.
Fig. 7. O2 affinities of anuran blood and Hbs. A, P50 values for whole blood (cross-hatched columns),
stripped hemolysates (hatched columns) and isoHbs (open and speckled columns) in the presence of Cl-,
showing the log P50 shifts induced by saturating ATP concentrations (stacked solid columns).
Experimental conditions for T. peruvianus and X. laevis Hbs, pH 7.5 and 20°C. Conditions for blood
measurements: Rana temporaria, pH 7.65, 20°C (35,65); R. catesbeiana, pH 7.55, 24°C (35); R.
brevipoda, pH 7.72, 25°C (52); Chiromantis petersi, pH 7.65, 25°C (27); X. laevis laevis, 7.6, 25°C (28);
[Cl-] in Hb solutions, 100 mM (0.05 M for C. petersi). For Telmatobius, blood values (*) pertain to T.
culeus at pH 7.54 and 18°C (24) and Hb values to T. peruvianus, 20 °C. B, Comparison of the P50 values
of stripped Hbs in the absence of effectors (open columns), in the presence of 100 mM Cl- (speckled
columns), Cl-+ATP for T. peruvianus and X. laevis Hbs and DPG+Cl- for human Hb (solid columns) at
the indicated temperature and pH values. The data for human Hb are from Table 6.2 in ref. (25). [Heme],
0.14 mM (T. peruvianus), 0.50 mM (X. laevis) and 0.60 mM (human Hb).
Table 1. Oxygen equilibrium characteristics of isolated Telmatobius HbI and HbII and stripped Xenopus Hb.
Telmatobius Hb II
°C
20
P50*, M
pH
Stripped
3
Cl-
P50
P50
7.5
7.0
7.33
7.83
8.22
9.90
ATP
Telmatobius Hb I
10
P50
P50
7.5
7.0
-0.06
3.76
3.93
-0.16
4.34
M
26.67
10-20
2
20
'H
P50
P50
7.5
7.0
7.5
7.0
-0.04
-46.1
-47.8
7.52
8.82
5.20
-0.16
-44.1
-39.2
7.42
11.8
16.5
-0.29
M
'H
20
P50
P50
7.5
7.0
-0.14
7.31
9.21
9.14
-0.18
12.3
21.0
M
14.0
22.9
-0.43
9.52
15.1
-0.40
-26.7
-26.0
12.3
20.4
-0.44
Cl-+DPG
14.4
26.1
-0.52
8.97
15.6
-0.48
-32.6
-35.5
13.0
23.9
-0.53
&
'H values in kJ$mole-1
10-20
'H
'H
7.5
7.0
-0.20
-44.7
-51.1
19.5
-0.40
-33.6
-30.0
37.6
-0.50
-30.3
-34.2
M
-32.3
Cl-+ATP
*P50 values in mm Hg;
Xenopus hemolysate
Table 2. MWC and derived parameters for Telmatobius HbII at 10 and 20 °C in the absence and presence of
saturating ATP concentrations. The MWC model was fitted with the number of interacting binding sites (qH)
fixed at 4. (Other details as in Legend of Fig. 4).
°C
pH
Effector
10
10
20
20
7.082
7.070
7.185
7.082
ATP
ATP
P50
Pm
n50
nmax
Kt
5.64 5.36 3.03 3.07 0.028
14.45 13.44 2.73 2.79 0.016
9.59 9.09 2.87 2.91 0.019
22.73 21.37 2.76 2.81 0.0094
SE
0.0018
0.0014
0.0016
0.00077
Kr
4.93
1.78
2.13
0.88
SE
1.46
0.917
0.703
0.295
L
'G
rms
4.9x105
3.3x105
1.4x105
1.3x105
11.97
10.58
11.23
10.73
0.045
0.060
0.068
0.061
P50 and Pm (half-saturation and median O2 tension values) in mm Hg; KT , KR [O2 association constants of low-affinity
(tense) and high-affinity (relaxed) states respectively] and SE (standard error) in (mm Hg)-1; n50 and nmax,
cooperativity coefficients at half-saturation and maximal values, respectively; L, allosteric constant [T]/[R]
in the absence of O2; 'G (free energy of heme-heme interaction) in kJ$mole-1; rms, root mean square error.
Fig. 6; Weber et al.
Globin fold
Human E
Telmatobius E
Xenopus laevis E
Rana catesbeiana E
N 1
5
10
15 1
5
10
161
5 7C
1
5 71
5
10
15
20
A A
B
C
D
D
E
123
12345678
VHLTPEEKSAVTALWGKV--NVDEVGGEALGRLLVVYPWTQRFFESFGDLSTPDAVMGNPKVKAHGKKVLGAFSDGLA
VHLTDQEIKYINAIWSKV--DYKQVGGEALARLLIVYPWTQRYFSTFGNLASADAISHNSKVIAHGEKVLRSIGEALK
-GLTAHDRQLINSTWGKL--CAKTIGQEALGRLLWTYPWTQRYFSSFGNLNSADAVFHNEAVAAHGEKVVTSIGEAIK
------GGSDVSAFLAKV--DKRAVGGEALARLLIVYPWTQRYFSTFGNLGSADAISHNSKVLAHGQRVLDSIEEGLK
Human D
V-LSPADKTNVKAAWGKVGAHAGEYGAEALERMFLSFPTTKTYFPHF-DLSH-----GSAQVKGHGKKVADALTNAVA
?????DDRSHILAIWPSVASHGADYGGEALYRLFLSNPQTKTYFPNF-DFHK-----DSPQIKAHGKKVVDALTEASK
Telmatobius D
-LLSADDKKHIKAIMPAIAAHGDKFGGEALYRMFIVNPKTKTYFPSF-DFHH-----NSKQISAHGKKVVDALNEASN
Xenopus laevis D
Rana catesbeiana BD -PFSASDRHDITHLWEKMAPNVEFLGEEAMERLFKSHPKTKTYFSHL-NVEH-----GSAAVRAQGAKVLNAIGHASK
Rana catesbeiana CD -ALNCDDKAHIRAIWPCLASHAEQYGAEALHRMFLCHPQTKTYFPNF-DFHA-----NSAHLKNHGKKVMNALTDAVK
Human E
Telmatobius E
Xenopus laevis E
Rana catesbeiana E
E
1
5
9F
1
5
10
15 19G
1
5
10
15
21H
F
F
G
G
H
H
C
12345678
12345
12345
123
HLDNLKGTFATLSELHCDKLHVDPENFRLLGNVLVCVLAHHFGKEFTPPVQAAYQKVVAGVANALAHKYH
HLDNLKGY-AKLSQYHSEKLHVDPANFVRFGGVVVIVLAHHFHEEFTPEVQAAFEKAFGGVADAVGKGYH
HMDDIKGYYAQLSKYHSETLHVDPLNFKRFGGCLSIALARHFHEEYTPELHAAYEHLFDAIADALGKGYH
HPZBLKAYYAKLSERHSGELHVDPANFYRLGNVLITVMARHFHEEFTPELQCALHSSFCAVGEALAKGYH
Human D
Telmatobius D
Xenopus laevis D
Rana catesbeiana BD
Rana catesbeiana CD
HVDDMPNALSALSDLHAHKLRVDPVNFKLLSHCLLVTLAAHLPAEFTPAVHASLDKFLASVSTVLTSKYR
HLDNINGALSKLFDLHAFELRVDPGNFPLLAHHINVTIAVMFPDDKFDIAHHQLDKFLAAVGGSLTSKYR
HLDNIAGSMSKLSDLHAYDLRVDPGNFPLLAHNILVVVAMNFPKQFDPATHKALDKFLATVSTVLTSKYR
NLDHLDEALSNLSDKHAHDLRVDPGNFHLLCHNILVVLAIHFPEDFTPRAHAAFDKFLAAVSETLYSKYR
HLDHPEASLSSLSDLHAFTLRVDPGNFALLSNNILVVVAVHHSDKLSYETHQALDKFLNVVSGLLTSKYR