Am J Physiol Regul Integr Comp Physiol 305: R300–R312, 2013. First published May 29, 2013; doi:10.1152/ajpregu.00014.2013.
Lack of conventional oxygen-linked proton and anion binding sites does not impair allosteric regulation of oxygen binding in dwarf caiman hemoglobin Roy E. Weber,1 Angela Fago,1 Hans Malte,1 Jay F. Storz,2* and Thomas A. Gorr3,4* 1
Zoophysiology, Department of Bioscience, Aarhus University, Aarhus, Denmark; 2School of Biological Sciences, University of Nebraska, Lincoln, Nebraska; 3Institute of Veterinary Physiology, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland; and 4Center for Pediatrics and Adolescent Medicine, University Medical Center Freiburg, Freiburg, Germany Submitted 24 January 2013; accepted in final form 22 May 2013
Weber RE, Fago A, Malte H, Storz JF, Gorr TA. Lack of conventional oxygen-linked proton and anion binding sites does not impair allosteric regulation of oxygen binding in dwarf caiman hemoglobin. Am J Physiol Regul Integr Comp Physiol 305: R300 –R312, 2013. First published May 29, 2013; doi:10.1152/ajpregu.00014.2013.—In contrast to other vertebrate hemoglobins (Hbs) whose high intrinsic O2 affinities are reduced by red cell allosteric effectors (mainly protons, CO2, organic phosphates, and chloride ions), crocodilian Hbs exhibit low sensitivity to organic phosphates and high sensitivity to bicarbonate (HCO⫺ 3 ), which is believed to augment Hb-O2 unloading during diving and postprandial alkaline tides when blood HCO⫺ 3 levels and metabolic rates increase. Examination of ␣- and -globin amino acid sequences of dwarf caiman (Paleosuchus palpebrosus) revealed a unique combination of substitutions at key effector binding sites compared with other vertebrate and crocodilian Hbs: 82Lys¡Gln, 143His¡Val, and 146His¡Tyr. These substitutions delete positive charges and, along with other distinctive changes in residue charge and polarity, may be expected to disrupt allosteric regulation of Hb-O2 affinity. Strikingly, however, P. palpebrosus Hb shows a strong Bohr effect, and marked deoxygenation-linked binding of organic phosphates (ATP and DPG) and CO2 as carbamate (contrasting with HCO⫺ 3 binding in other crocodilians). Unlike other Hbs, it polymerizes to large complexes in the oxygenated state. The highly unusual properties of P. palpebrosus Hb align with a high content of His residues (potential sites for oxygenation-linked proton binding) and distinctive surface Cys residues that may form intermolecular disulfide bridges upon polymerization. On the basis of its singular properties, P. palpebrosus Hb provides a unique opportunity for studies on structure-function coupling and the evolution of compensatory mechanisms for maintaining tissue O2 delivery in Hbs that lack conventional effector-binding residues. allosteric interaction; Bohr effect; carbon dioxide; crocodilians; oxygen-binding THE ROLE OF HEMOGLOBIN (Hb) in transporting O2 to respiring tissues is governed by its intrinsic O2 affinity, its sensitivity to the effectors that modulate Hb-O2 affinity, levels of those effectors in the red blood cells, and the O2 tensions for loading and unloading O2 at the respiratory surfaces and in tissues, respectively (91). Each of these factors may vary among different species and may also undergo reversible changes within the same individual animals in response to changes in environmental conditions, metabolic requirements, and mode of life. In vertebrate Hbs that comprise two ␣ chains and two  chains, O2 binding at the heme groups triggers a transition of
* T. A. Gorr and J. F. Storz contributed equally to this work. Address for reprint requests and other correspondence: R. E. Weber, Zoophysiology, Dept. of Bioscience, Bldg. 1131, Aarhus Univ., DK1000 Aarhus, Denmark (e-mail:
[email protected]). R300
the protein molecules from the low-affinity, tense (T) state to the high-affinity, relaxed (R) state, which is basic to cooperativity in O2 binding. Hb-O2 affinity is modulated by allosteric effectors, chiefly protons (low pH) and CO2 that decrease O2 affinity (increase O2 unloading in the tissues via the Bohr effect), and organic phosphate and chloride ions that commonly reduce Hb-O2 affinity by preferential binding at specific sites of the molecules in the T state (62, 91). Thus, whereas protons mainly bind at 146His (the COOH-terminal histidines of the  chains), the organic phosphates [typically 2,3-diphosphoglycerate (DPG) in mammals, and ATP in ectothermic vertebrates] interact with seven amino acid residues in the cavity between the  chains (1Val of one chain; and 2His, 82Lys, and 143His of both  chains), CO2 binds at the unprotonated NH2-terminal residues of both chains, and Cl⫺ ions at one ␣ chain site (between ␣1Val and ␣131Ser) and one  chain site (between 1Val and 82Lys) (42, 53, 62, 70, 78). Crocodilian Hbs exhibit striking, distinguishing characteristics. In contrast to the vast majority of vertebrates, they show little or no response to organic phosphates, Cl⫺, or CO2 (8, 9, 39). The insensitivity to phosphates correlates with the replacement of three phosphate binding residues. Thus, compared to human Hb, 143His is replaced by Ala and 1Val-2His by acetylated-Ala-Ser in the Nile crocodile (Crocodylus niloticus) and the American alligator (Alligator mississippiensis) and by Ser-Pro in the spectacled caiman (Caiman crocodilus) (51, 75). Perhaps the best known feature of crocodilian Hbs is that O2 affinity is drastically decreased by HCO⫺ 3 ions (8), which is considered to play an important role in unloading O2 and maintaining aerobic metabolism when crocodilians dive and drown their prey (8, 95), compensating for their low myoglobin O2 stores (46). The HCO⫺ 3 effect may also play a vital role in unloading O2 from the blood during postprandial alkaline tides (95) when increased blood HCO⫺ 3 concentrations (resulting from HCl secretion into the stomach to digest bone) coincide neatly with the increased demand for O2 (the specific dynamic action of food) (19). This view aligns with the fact that crocodilians consume large amounts of bone (one alligator stomach contained remnants of up to 12 turtles) (20) and that the postfeeding metabolic peak in C. porosus was 70% higher when fed bone-rich chicken necks than when fed homogenized chicken (30). The mechanisms basic to allosteric regulation of Hb-O2 affinity in crocodilians remain controversial. Although modeling indicates deoxygenation-linked binding of HCO⫺ 3 at three residues in the central cavity between the two  chains; viz., 82Lys, 144Glu of one  chain, and the NH2-terminal residue of the partner  chain (Ser in C. crocodilus) (63), mutagenic replacement of 82Lys did not change the HCO⫺ 3
0363-6119/13 Copyright © 2013 the American Physiological Society
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ALLOSTERIC REGULATION OF DWARF CAIMAN HEMOGLOBIN
sensitivity of Nile crocodile (C. niloticus) Hb, and replacements of 12 other amino acid residues clustered at the ␣12 interface were required to transplant the bicarbonate sensitivity into human HbA (16, 46, 47). Also, in contrast to the reported absence of oxygenation-linked binding of CO2 in Cai. crocodilus Hb (8), carbamate (carbamino) formation in crocodilians is reflected by the much (twofold to fivefold) larger CO2 Bohr effect than fixed-acid Bohr effect (measured when pH is changed by adding CO2 and buffers, respectively) in Cai. crocodilus and A. mississippiensis blood (39, 95). Finally, contrary to previous reports [cf. (46)], crocodilian red cells may contain a wide spectrum of organic phosphate effectors, albeit at low concentrations. In fact Cai. crocodilus red cells contain ATP and DPG, as well as inositol pentaphosphate (IPP, found in avian red cells), inositol hexaphosphate (IHP), and guanosine triphosphate (GTP, which is commonly encountered in fish red cells) (86). Aiming to elucidate molecular adaptations and structurefunction relationships in crocodilian Hbs, we determined the amino acid sequences of the ␣ and  chains of the Hb of the dwarf caiman, Paleosuchus palpebrosus. This species differs from larger alligators and crocodiles in that it frequents stony creeks with clean, fast-running and cooler water, and mainly feeds on small vertebrates (e.g., tadpoles, frogs, fish, small mammals) and a variety of insects and snails (18). Finding a unique combination of amino acid exchanges at highly conserved effector binding sites, we investigated the O2 binding properties of the Hb and its sensitivities to chloride ions, ATP, DPG, and IHP, and to CO2 and temperature, over a wide pH range to discern possible alternative allosteric regulatory mechanisms. On the basis of the aggregation of deoxygenated Hbs to octamers and larger polymers (that may occur in vivo) observed in a diverse array of sauropsid taxa (birds and nonavian reptiles) (67, 71, 80), we also assessed polymerization in dwarf caiman Hb, its dependence on oxygenation state, and its possible effects on Hb-O2 affinity. MATERIALS AND METHODS
Primary Structure Blood from two young (2–3 yr old) specimens of dwarf caiman, P. palpebrosus, sampled in connection with routine diagnostic controls by the veterinarian at Cologne Zoological Gardens in accordance with existing regulations, was a generous gift from the zoo. Red cells separated by centrifugation were lysed in five times their volumes of 20 mM Tris·HCl, 40 mM DTE, pH 8.5 buffer. The hemolysate resolved into three electrophorectic bands by native alkaline disc electrophoresis, each of which was identified by NH2-terminal sequences as a mixture of the same ␣A and  chains without any hint of the presence of additional subunits (not shown). This indicates that P. palpebrosus Hb consists of a single component whose ␣A/ chain subunits show a strong tendency to form aggregates that involve disulfide bonds. For a better yield of purified peptides we subjected a 1:1 mix (wt/wt) of lyophilized native and oxidized globin to a tryptic digest followed by size-exclusion chromatography (Sephadex G-25 fine) with acidic elution (0.1 N acetic acid), which resolved the digest into 10 fractions of variably sized peptides. Next, we employed reversephase HPLC with a LiChrospher 60 RP select B column and different trifluoroacetic acid (TFA)/acetonitrile gradients to separate the 10 fractions into a total of 281 peptide-containing peaks, including all ␣A and  chain tryptic peptides (Tp). The complete primary sequences of the ␣A and  subunits were then determined by means of automated
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Edman degradation in conjunction with amino acid analyses of these tryptic and some additional chymotryptic peptides. NH2-terminal and peptide sequencing schemes of either subunit and tables of the amino acid analyses of ␣A and  peptides are available upon request. For proper placement of tryptic peptides within the ␣A or  chain, comparison to the complete globins from the closely related Cai. crocodilus was crucial. In addition, enriched native subunits and pyridyl-ethylated globin, whose reactive cysteinyl SH-groups could no longer form disulfide-based aggregates, were NH2-terminally sequenced up to ␣A position 24 and  position 58. This approach allowed the unequivocal identification of alkylated cysteines at ␣A positions 18/19 and  position 23, respectively (see Fig. 1). Recovery of  Tp9a from the HPLC column occurred in two peaks, 70% of which contained Glu and 30% Gln at position 73. This Glu/Gln ambiguity might be explained either by a true allelic difference (heterozygosity) at site 73 or by the spontaneous deamidation of the original amide (Gln). Proof of the critically important replacement of the COOH-terminal His by Tyr in the  chain (i.e., of 146 Tyr) was obtained by: 1) amino acid analyses of Tp 14b⫹15I (position 136 –141) and Tp 14b⫹15II (position 142–146), which provided evidence for a single His existing in Tp 14b⫹15I, whereas two Tyr residues and no His residues were recovered for Tp 14b⫹15II; and 2) the sequence of the entire Tp 14b⫹15 that demonstrated a sole His residue in position 1 (136) along with Tyr phenylthiohydantoin (PTH)-derivative peaks of almost equal yields for 145 and 146. For comparisons of Paleosuchus globins with orthologous chains of other species, percentage similarities and identities of amino acid residues (Table 1) were calculated using the EMBOSS tool (http:// www.ebi.ac.uk/Tools/psa/emboss_needle/) based on the global alignment Needleman-Wunsch algorithm operating with the Blosum62 matrix. O2 Binding Measurements Hemoglobin preparation. Hb solutions obtained by lysing washed red blood cells were centrifuged for 10 min at 400 g to remove cellular debris, and were stripped of organic phosphates on a 38 ⫻ 2.1 cm (height ⫻ diameter) column of Sephadex G25 Fine gel and dialysed for 24 h against 0.01 mol/l HEPES buffer containing 5 ⫻ 10⫺4 mol/l EDTA, as earlier described (92). The Hb showed no oxidation as judged from equal absorption values at 539 and 569 nm after brief equilibration with carbon monoxide, and was frozen at ⫺80°C in 150-l aliquots that were thawed individually for O2 equilibrium and other measurements. O2 equilibria. Hb-O2 equilibria were measured in the presence of 0.05 mol/l HEPES buffer (89) unless otherwise specified, using a modified gas diffusion chamber coupled to two cascaded Wösthoff pumps (Bochum, Germany) for mixing of air and pure (⬎99.998%) N2 (88, 93). P50 and n50 (respectively, O2 tension and Hill’s cooperativity coefficients at half-saturation) values were interpolated from linear regressions (r2 ⬎ 0.99) of Hill plots [log Y/(1 ⫺ Y) vs. log PO2, where Y is the fractional saturation] of four or more equilibration steps between 30% and 70% O2-Hb saturation. The effects of ATP (disodium salt), DPG (pentacyclohexyl-ammonium salt), IHP (inositol hexaphosphate, sodium salt), and Cl⫺ (KCl) were investigated by adding accurate volumes of standard, ⬃100 mM solutions of these effectors to the stripped Hb solutions. ATP was assayed using Sigma test chemicals, and Cl⫺ was assayed using a CMT10 chloride titrator (Radiometer, Copenhagen, Denmark). The pH values were measured in oxygenated Hb samples at the same temperature and CO2 tension as the O2 equilibria using thermostatted pH electrode units (BMS2 Blood Micro System; Radiometer). The allosteric parameters [the allosteric constant, L, and the association equilibrium constants of the T and R structures (KT and KR, respectively)] were assessed by analyzing precise O2 equilibrium data for a wide range of O2 saturations (extended Hill plots) in terms of the two-state Monod-Wyman-Changeux (MWC) equation (57) that was
AJP-Regul Integr Comp Physiol • doi:10.1152/ajpregu.00014.2013 • www.ajpregu.org
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A α-globin
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Human HBA Chicken HBA P. palpebrosus HBA C. crocodilus HBA C. niloticus HBA A. mississippiensis HBA
1 V · · · · ·
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Human HBA Chicken HBA P. palpebrosus HBA C. crocodilus HBA C. niloticus HBA A. mississippiensis HBA
75 D · · · · ·
M I L L L L
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A K R R R R
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F L L L L L
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G19 Human HBA Chicken HBA P. palpebrosus HBA C. crocodilus HBA C. niloticus HBA A. mississippiensis HBA
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Fig. 1. Amino acid sequences of the ␣ and  globins of dwarf caiman (Paleosuchus palpebrosus) hemoglobin (Hb) compared with those for spectacled caiman (Caiman crocodilus), Nile crocodile (Crocodylus niloticus), the American alligator (Alligator mississippiensis) (51), chicken, and human. Residues in the other species are shown only where they differ from those in human Hbs. His residues highlighted in yellow and blue are titratable (surface) residues and nontitratable residues, respectively, in human Hb (11, 53); those highlighted in green represent potential gains of titratable His residues compared with human Hb.
fit to the data in the form log [Y/(1 ⫺ Y)] vs. log PO2 (end weighting) using the curve-fitting procedure previously described (94), and fitting the number of binding sites, q, along with the other parameters to obtain the best possible fit. Additional fits were performed with q fixed at 4 (as applies to tetrameric Hb). Derived parameters, including the median oxygen tension, Pm; the maximum slope of the Hill plot, nmax; and the free energy of heme-heme interaction, ⌬G; were evaluated as previously detailed (94). Heats of oxygenation were calculated from the van’t Hoff isochore [⌬H ⫽ 2.303 · R · ⌬log P50/⌬(1/T)], where R is the gas constant and T is the absolute temperature (°K). All quoted ⌬H values are exclusive of the heat of solvation of O2 (⫺12.6 kJ/mol). Size Exclusion Chromatography Hb quaternary structure and its oxygenation dependence were investigated by gel filtration of Hb preparations that had not been in contact with CO on a 59.3 ⫻ 2.6 cm (height ⫻ diameter) column of Sephacryl S-200 HR. The proteins were eluted with 0.025 M Tris
buffer (pH 7.40 at 5°C) containing 0.025 M NaCl and 0.003 M NaN3. The partition coefficients, Kav [(⫽ Ve ⫺ Vo/Vt ⫺ Vo) (cf. 49a)], where Ve is the elution volume, Vt is the total volume of the gel bed, and Vo is the void volume (the elution volume of Blue Dextran that is excluded by the gel)] of Hb fractions were compared with those of horse heart cytochrome c, oval albumin, and aldolase obtained from Boehringer-Mannheim; myoglobin and bovine serum albumin obtained from Sigma (A-0380 and M-4503, respectively); and catalase and ferritin from GE Health Care. Gel filtration of deoxygenated Hb was carried out by adding Na-dithionite (1 mg/ml) to the Hb sample applied to the column and to the elution buffer after both solutions had been equilibrated with gaseous N2. Hb fractions retrieved for O2 binding experiments were concentrated by ultrafiltration in Millipore ultrafree filter units (cutt-off molecular mass 10,000 Daltons). Fractions showing slight oxidation were briefly equilibrated with CO and reduced by dialysis (30 min) against N2-equilibrated 0.01 M HEPES buffer (pH ⬃7.6) containing freshly added Na-dithionite (0.1%)
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Table 1. Identities and similarities in ␣ and  chain sequences of P. palpebrosus hemoglobin compared with those of other crocodilian, chicken and human hemoglobins ␣ Chains
Cai. crocodilus C. niloticus A. mississippiensis Chicken Human
 Chains
Identity
Similarity
Identity
Similarity
83.7 79.4 79.4 75.2 67.4
90.1 87.2 87.2 84.4 75.9
89.0 67.8 75.3 54.1 48.6
98.6 83.6 87.0 74.7 68.5
All values are %.
followed by extensive dialysis against N2/CO equilibrated HEPES buffer without Na-dithionite.
and  chains. Relative to the ␣ chains, the  chains showed higher sequence divergence in comparisons among species, which is consistent with previous reports of unusually high substitution rates in the  chain subunits of crocodilian Hbs (31, 36). The primary structures of P. palpebrosus Hb chains reveal a unique combination of amino acid substitutions, including 82Lys¡Gln, 143His¡Val, and 146His¡Tyr (Fig. 1) that delete positively charged (anion-binding and proton-dissociating) residues and thus may be expected to drastically impact the sensitivities of the Hb to allosteric effectors. Another impinging substitution of P. palpebrosus Hb is 93Cys¡Tyr, which removes a solvent-exposed cysteine residue that is highly conserved among mammalian, avian, and reptilian Hbs.
RESULTS
Charge, Polarity, and Histidine Content
Structural Analyses
The sequence of P. palpebrosus Hb differs markedly from that of other crocodilian, avian, and human Hbs in charge, polarity, and hydropathy (hydrophobicity) indices of amino acid residues, as well as the number and position of histidine residues that may function as buffer groups and source of Bohr protons and in stabilizing the Hb’s T-structure through the formation of internal salt bridges (11). P. palpebrosus Hb contains 24 (11 ␣ chain and 13  chain) His residues in each dimeric half-molecule (Fig. 1), which is considerably more than in other vertebrates, including humans, who have 19 (10 ⫹ 9) residues, but they align with the high numbers of physiological buffer groups in crocodilian Hbs (11).
Figure 1 shows the amino acid sequences of P. palpebrosus Hb aligned with those for Cai. crocodilus, C. niloticus, A. mississippiensis, chicken, and human. The entire collection of globin peptides isolated from the 10 size exclusion fractions (see MATERIALS AND METHODS) was free of peptides that matched ␣D subunits of birds and nonavian reptiles, revealing that P. palpebrosus, like other adult crocodilians, does not express ␣D globin (31). The ␣ and  chain sequences of P. palpebrosus (Table 1) show higher identity with those of the other caimanine member (Cai. crocodilus) than with those of C. niloticus and A. mississippiensis and with chicken than with human ␣
Table 2. Amino acid residues specifically encountered in ␣ and  chains of P. palpebrosus hemoglobin compared with those of other species Comparison*
␣12 ␣18 ␣19 ␣23 ␣28 ␣32 ␣34 ␣35 ␣49 ␣64 ␣100 ␣134 12 19 22 31 49 52 82 93 108 121 124 134 143 146
Human
P. palpebrosus
Cai. crocodilus
C. niloticus
A. mississippiensis
Charge
Polarity
Ala Gly Ala Glu⫺ Ala Met Leu Ser Ser Asp⫺ Leu Tyr Tyr Asn Asp⫺ Leu Ser Asp⫺ Lys⫹ Cys Asn Glu⫺ Pro Val His⫹ His⫹
Gly Cys Cys Asp⫺ Tyr Leu Phe Val Tyr Leu Leu Tyr Leu Asn Ala Leu Ala Glu⫺ Gln Tyr Asn Glu⫺ Met Ala Val Tyr
Ala Ala Gly Glu⫺ Ala Met Cys Ala Ser Ala Phe Ala Val Asp⫺ Ser Met Ser Gln Lys⫹ Phe Asp⫺ Asp⫺ Leu Val Ala His⫹
Ala Ala Gly Glu⫺ Ala Met Cys Ala Ser Ala Phe Ser Gly Asp⫺ His⫹ Met Ser Gln Arg⫹ Phe Asp⫺ Asp⫺ Leu Val Ala His⫹
Ala Ser Gly Glu⫺ Ala Met Cys Ala Ser Ser Phe Ala Val Asp⫺ Gln Met Cys His⫹ Lys⫹ Phe Asp⫺ Asp⫺ Val Val Ala His⫹
⫽ ⫽ ⫽ ⫽ ⫽ ⫽ ⫽ ⫽ ⫽ ⫺ o (o o o) ⫽ ⫽ ⫽ o o (- - -) ⫺o (o ⫹ o) ⫽ ⫽ ⫺⫺ (o o ⫹) ⫹o (⫹⫹⫹) ⫽ o o (- - -) ⫽ ⫽ ⫽ ⫹o (o o o) ⫹o (⫹ ⫹ ⫹)
⫽ np (nnp) np (nnn) ⫽ np (nnn) ⫽ nn (ppp) pn (nnn) ⫽ pn (nnp) ⫽ pp (npn) pn (nnn) ⫽ pn (ppp) ⫽ pn (ppp) ⫽ ⫽ pp (nnn) ⫽ ⫽ ⫽ ⫽ pn (nnn) ⫽
Underlined residues have polar side chains; ⫹ and ⫺ denote residues whose side chains are positively and negatively charged, respectively, at pH 7.4. *Comparison between hemoglobins of, respectively, humans, P. palpebrosus, and (in brackets) Cai. crocodilus, C. niloticus,and A. mississipiensis: ⫽, same charge/polarity in all species; ⫹, positively charged side chains; ⫺, negatively charged side chains, o, neutral side-chains. p, Polar side chain; n, nonpolar side chain. AJP-Regul Integr Comp Physiol • doi:10.1152/ajpregu.00014.2013 • www.ajpregu.org
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Fig. 2. Oxygen tension (P50) and Hill’s cooperativity coefficients (n50) at half O2 saturation of P. palpebrosus Hb measured at 10°C (triangles and diamonds) and 25°C (full and half circles), in 0.05 M HEPES buffer (circles and diamonds) or 0.05 M MES buffer (half-circles and triangles) and in the absence (open symbols) and presence (closed symbols) of 0.1 M Cl⫺. Heme concentration, 0.37 mM.
Remarkably, the ␣ and  chain sequences of P. palpebrosus exhibit distinctive features that are shared with human Hb, but not with other hitherto investigated crocodilian Hbs (Table 2). Thus, both P. palpebrosus and human Hbs have neutral Asn residues at 19 and at 108 compared with negatively charged Asp in other crocodilians. Also both have negatively charged residues (Glu and Asp, respectively) at 52 compared with neutral Gln or weakly positive His in the crocodilians. Also both have residues with opposite side-chain polarities than other crocodilian Hbs at positions ␣34 and 93. However, P. palpebrosus Hb also shows distinguishing differences in residue polarity compared with human and other crocodilian Hbs (at positions ␣19, ␣28, 22, 49, and 143) that may contribute to distinctive functional properties. Oxygen Binding O2 affinity and its pH and chloride sensitivities. Stripped P. palpebrosus Hb exhibits a high O2 affinity that is substantially reduced by Cl⫺ ions, decreased pH, and increased temperature (Fig. 2; Table 3); at pH 7.4, P50 values at 10° and 25°C are 0.65 and 2.69 mmHg, respectively, in the stripped Hb; and 1.53 and 5.43 mmHg, respectively, in the presence of 0.1 M Cl⫺. Expressed as log P50,Cl ⫺ log P50,str, the chloride effect decreases markedly at high pH where cationic binding sites are neutralized (Table 3). Cooperativity of O2 binding was low (n50 ⫽ 1.2–1.5) under all conditions investigated (Fig. 2). Significantly, P. palpebrosus Hb exhibits a pronounced Bohr effect. At pH 7.0 –7.4, which characterizes physiological values in crocodilians (77), the Bohr factors ( ⫽ ⌬logP50/⌬pH) in the absence and presence of 0.1 M chloride were ⫺0.67 and ⫺0.61, respectively, at 10°C; and ⫺0.45 and ⫺0.50, respec-
tively, at 25°C (Fig. 2; Table 3). The decrease in Bohr effects with increasing temperature accords with the temperature dependence of ionization of Bohr groups. Unlike in human and most vertebrate Hbs (37, 73, 92) the Bohr effect is not increased by 0.1 M chloride, suggesting that allosteric proton binding to this Hb is not enhanced by chloride. O2 equilibria of stripped P. palpebrosus Hb in MES buffer (Fig. 2) reveal the absence of a reverse (acid) Bohr effect (increasing O2 affinity with decreasing pH) as found in human Hb below ⬃6.5. Phosphate sensitivities. Measurements of the interactive effects of organic phosphates and chloride at physiological pH (Fig. 3) show insensitivity of P. palpebrosus Hb to organic phosphates in the presence of chloride ions, as observed in other crocodilians (8, 9, 63). In contrast, pronounced sensitivities to ATP and DPG are unmasked in the absence of chloride, as is also observed in A. mississippiensis and Cai. crocodilus Hbs (96). Strikingly, however, the Hb-O2 affinity is virtually insensitive to polyanionic IHP. Thus in the absence of chloride, addition of ATP, DPG, and IHP increase log P50 by 0.49, 0.52, and 0.089, respectively, at 25°C and pH 7.4 (Fig. 3B). Also, whereas the effects of IHP and chloride are additive (i.e., chloride plus IHP depress O2 affinity more than IHP alone), addition of chloride increases Hb-O2 affinity in the presence of ATP or DPG (Fig. 3B). Similar relative sensitivities to these effectors were observed in duplicate measurements at pH 7.0 (data not shown). Dose-response curves (Fig. 4) for the effects of increasing concentrations of free phosphates calculated as [total phosphate] ⫺ 0.5[Hb4], on the assumption that at P50 half of the tetrameric Hb molecules are phosphate-liganded (97), demonstrate virtual annihilation of the phosphate effects in the presence of 0.1 M chloride. Strikingly, the double logarithmic plots reveal slopes that markedly exceed 0.25 (i.e., the value expected for one-to-one stoichiometry between phosphate and tetrameric Hb molecules if phosphate binding is limited to the T state). For DPG binding at pH 7.4 the slope of 0.37 (Fig. 4) indicates a stoichiometry of ⬃1.5 DPG molecules bound per Hb tetramer. CO2 sensitivity. Admixture of CO2 in the equilibration gases conspicuously lowers O2 affinity of the stripped P. palpebrosus Hb at constant pH. This specific CO2 effect increases with increasing pH and is markedly reduced by Cl⫺ (Fig. 5). Thus at pH 7.0 and 7.4, addition of 29.7 mmHg CO2 raises log P50 by 0.85 and 0.98 units, respectively, in the absence of chloride, compared with 0.49 and 0.64, respectively, in 0.1 M chloride. This CO2 effect and its pH and Cl⫺ dependencies are consistent with carbamate formation at the (NH2-terminal) ␣-amino residues of the chains. Slopes of log P50 vs. log PCO2 plots (in Table 3. P50 values and Bohr factors of stripped P. palpebrosus hemoglobin and their dependence on pH, [Cl⫺] and temperature °C
Property
pH 8.0
pH 7.4
pH 7.0
(7.0–7.4)
10
P50, str. (mmHg) P50, str. ⫹ 0.1 M Cl (mmHg) log P50,Cl ⫺ log P50,st. P50, str. (mmHg) P50, str. ⫹ 0.1 M Cl (mmHg) log P50,Cl ⫺ log P50,str
0.45 0.78 0.23 2.02 3.09 0.18
0.65 1.53 0.37 2.69 5.43 0.31
1.20 2.66 0.35 4.26 8.61 0.32
⫺0.67 ⫺0.61
25
, Bohr factor; str., stripped.
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Fig. 3. A: P50 and n50 values of P. palpebrosus Hb measured in 50 mM HEPES buffer at 10° and 25°C in the absence (⫺) and presence (⫹) of 0.1 M Cl⫺, and the absence (open symbols) and presence (closed symbols) of saturating levels [ATP/Hb and 2,3diphosphoglycerate (DPG)/Hb ratio 6.2; inositol hexaphosphate (IHP)/Hb ratio ⬃28] of the polyanionic phosphate effectors (Pn⫺) ATP, DPG, and IHP. B: histograms showing the P50 values of the stripped Hb (str., open columns) and the log P50 shifts (shaded columns) induced by chloride, ATP, DPG, and IHP at pH 7.4 and 10° and 25° C. Other conditions as in Fig. 2.
the 7–30 mmHg PCO2 range) of ⬃0.5 in the absence, and ⬃0.4 in the presence of Cl, indicate binding of one CO2 molecule per two O2 molecules released. Allosteric control. Extended Hill plots (Fig. 6) reveal slopes of unity at extreme low and high O2 saturations consistent with noncooperativity in binding the first and last O2 molecules to the Hb, and good correspondence between Pm and P50 values (O2 tensions at, respectively, median and half-saturations), permitting assessment of allosteric effects from alterations in P50 values. As shown (Table 4), the n50 values are low, which hampered fits of the MWC two-state model to the data. Thus, at high pH and in the presence of chloride, it was not possible
to fit the model to the data with q (the number of interacting binding sites) floating and with the constraint that the allosteric constant (L) ⬎ 1. For the other curves, the fitted values of q were close to 4, indicating that the functional units are tetramers, despite Hb polymerization (see below). As illustrated (Fig. 6), lowered pH decreases O2 affinity by reducing KT and KR, whereas chloride ions decrease O2 affinity mainly by lowering KT. Fits with q fixed at 4 (Table 4) show that the interaction energy is increased in the presence of 0.1 M chloride and that this was due in part to a stabilizing effect of chloride on the T state (lowered KT). At pH 7.6 this was augmented by a destabilizing effect of chloride on the R state (increased KR), whereas this was not the case at pH 6.9. As evident from the standard error on the fits, however, it is not safe to draw firm conclusions about KR at the low pH. Temperature sensitivity and enthalpic effects. At pH 7.5– 8.0, at which stripped P. palpebrosus Hb virtually lacks a Bohr effect (cf. Fig. 2), the overall oxygenation enthalpy (⫺56.3 kJ/mol) corresponds closely with the intrinsic heat of oxygenation of human Hb (⫺59 kJ/mol) (4). The numerically lower value (⫺48.8 kJ/mol) found at pH 7.0 where the Bohr effect is operative, is consistent with endothermic contributions from Bohr proton dissociation [⬃26 kJ/mol for imidazole groups of histidines (3, 4)]. Analogously, the reduced enthalpy values observed in the presence of 0.1 M Cl⫺ (⫺47.7, ⫺46.6, and ⫺42.3 kJ/mol at pH 8.0, 7.5, and 7.0, respectively) reflect endothermic contributions (⫹8.6, ⫹7.7, and ⫹6.5 kJ/mol, respectively) from oxygenation-linked chloride ion dissociation. The semblance of these enthalpic effects with those in other (nonheterothermic) vertebrates (90) indicates the absence of distinguishing adaptive traits related to body temperature variation in P. palpebrosus Hb. Quaternary Structure and Effects
Fig. 4. P50 and n50 of P. palpebrosus Hb at pH 7.4 and 25°C in the absence (open symbols) and presence (solid symbols) of 0.1 M Cl⫺; the absence of organic phosphates (stars); and the presence of ATP (circles), DPG (triangles), and IHP (squares) at different phosphate/tetrameric Hb ratios, measured in 0.05 M HEPES buffer. Fine dotted line shows a slope of 0.25 expected if deoxygenation of the Hb molecules were linked to binding of one phosphate molecule. Heme concentration, 0.36 mM. (This figure replots earlier published P50 values (96) against the [free phosphate]/[Hb] ratios).
Unexpectedly, the size exclusion gel-filtration chromatography experiments showed that oxygenated P. palpebrosus Hb elutes with the void volume of the Sephacryl S-200 HR medium, revealing an exceptionally high degree of polymerization (Fig. 7). In contrast, the elution pattern of Hb deoxygenated with dithionite reveals the presence of three fractions
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Fig. 5. A: pH dependence of P50 and n50 values of P. palpebrosus Hb in the presence of 0, 1, and 4% CO2 (PCO2 ⫽ 0, 7.4, and 29.4 mm, respectively), measured at 25°C in 50 mM HEPES buffer. B: the specific (pH-independent) effect of CO2 on Hb-O2 affinity at pH 7.0 and 7.4. Heme concentration 0.37 mM.
of smaller molecules, whose partition coefficients (Kav) indicate molecular masses of 31.6, 81.3, and 263 kDa. The value of 38 kDa obtained for human Hb (Fig. 7, inset) is consistent with observations that molecular mass estimates for tetrameric vertebrate Hbs on the basis of gel filtration are lower than
established values (64 – 68 kDa) due to reversible dissociation to dimers (17) and to elution volumes being proportional to molecular Stokes radii of proteins rather than their molecular weights (1). In this light, the three fractions observed in deoxygenated Hb (labeled 4, 3, and 2 in Fig. 7) likely comprise tetramers, octamers (dimers of tetramers), and high-order polymers, respectively. O2 equilibrium measurements of three major molecular mass fractions obtained from gel filtration of deoxygenated Hb show similar O2 affinities and Bohr effects (Fig. 8) indicating that aggregation state does not affect oxygenation properties of P. palpebrosus Hb. DISCUSSION
O2 Affinity and its Sensitivity to Allosteric Effectors P. palpebrosus Hb exhibits a high intrinsic O2 affinity combined with low cooperativity, suggesting that the allosteric T-R equilibrium in this Hb is markedly shifted to the highaffinity R state. Its O2 affinity is reduced by allosteric effectors (protons and CO2, and organic phosphate and chloride anions) despite replacements of functionally important amino acid residues compared with other Hbs. P. palpebrosus Hb provides a unique opportunity to reevaluate the roles of specific amino acid residues implicated in the allosteric regulation of O2 affinity, and the possible compensatory mechanisms in naturally occurring Hbs that lack specific effector binding sites. CO2 Effects Fig. 6. Extended Hill plots of P. palpebrosus Hb at pH ⬃7.60 (half circles) and ⬃6.96 (triangles) in the absence (open symbols) and presence (closed symbols) of 0.10 M Cl⫺. Heme concentration, 0.37 mM.
P. palpebrosus Hb binds molecular CO2. The distinct, specific effect of CO2 on O2 affinity (Fig. 5) is at variance with the reported absence of carbamate formation in crocodilian Hbs
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Table 4. MWC and derived parameters obtained by fitting the MWC two-state model to the data with four fixed interacting O2-binding sites pH
Cl mM
P50 mmHg
Pm mmHg
n50
log L
SE*
log KT
SE*
log KR
SE*
⌬G kJ·(M heme)⫺1
7.608 6.964 7.595 6.961
0 0 0.10 0.10
3.58 8.02 9.38 14.00
3.53 7.62 8.31 12.84
1.29 1.21 1.43 1.37
1.66 3.49 65.61 4.07
0.333 1.593 0.0026 4.085
⫺0.758 ⫺0.993 ⫺1.102 ⫺1.279
0.040 0.025 0.019 0.063
⫺0.146 ⫺0.0562 15.46 ⫺0.117
0.075 0.386 0.011 1.012
2.44 2.07 4.16 3.31
P50, O2 tension at half-saturation; Pm, median O2 tension; n50, Hill’s cooperativity coefficient at half-saturation; L, allosteric constant; KT and KR, association equilibrium constants of the T and R structures, respectively; ⌬G, free energy of heme-heme interaction. *Standard errors as estimated from the covariance matrix associated with the fit.
(63). The reduction in the CO2 effect observed in the presence of chloride and increased proton activity (pH decrease) (cf. Fig. 5) is consistent with competition between these effectors for binding at the NH2-terminal amino acid residues, and thus is analogous to the antagonism between binding of CO2 on the one hand, and of lactate (58) and DPG (7) on the other, in human HbA. The slope of ⬃0.5 for the log P50/log PCO2 relationship (Fig. 5) signifies a stoichiometry of two CO2 molecules bound per Hb tetramer under physiological conditions, which indicates that CO2 binds to either the ␣ or the  chains of P. palpebrosus Hb as in human Hb, where O2-linked carbamate formation is confined to the  chains (10). The identical NH2-terminal amino acid sequences (Fig. 1) of P. palpebrosus and Cai. crocodilus Hbs predict similar CO2 binding capacities in these species. In C. niloticus and A. mississippiensis, however, the 
chain NH2-terminal residues are acetylated (75) and are therefore not available for carbamate formation. In A. mississippiensis Hb carbamate, free HCO⫺ 3 and Hb-bound HCO⫺ 3 contribute approximately equally to the high CO2 carrying capacity of deoxygenated red cells (41). Two observations indicate that HCO⫺ 3 binding to P. palpebrosus Hb is markedly reduced compared with that of other crocodilians. First, the CO2 effect increases strongly with increasing pH (Fig. 5A), whereas the association constants for the reaction of Cai. crocodilus Hb with HCO⫺ 3 are essentially pH independent below pH 7.3 (8). Second, compared with other crocodilian Hbs, P. palpebrosus Hb exhibits substitutions (viz., ␣34Cys¡Phe, ␣35Ala¡Val, ␣100Phe¡Leu, and 31Met¡Leu) (Fig. 1) that replace 4 of the 12 amino acid residues (viz., 34Cys, 35Ala, 36Tyr, 38Gln, 41Ile, 100Phe, and 103Gln of the ␣ chain; and 29Ser, 31Met, 38Lys, 39Arg, and 41Tyr of the  chain) that are present in other crocodilian Hbs, and confer a HCO⫺ 3 effect when engineered into human Hb (47). Among these the loss of polar ␣34Cys in P. palpebrosus Hb may be significant. If 82-Lys is a bicarbonate-binding site as proposed by Perutz and his colleagues (63), its substitution by Gln would similarly contribute to loss of bicarbonate binding in P. palpebrosus Hb, but not in Cai. crocodilus and A. mississippiensis Hbs that have retained this residue, nor in C. niloticus Hb that shows a polar- and chargeneutral substitution (82Lys¡Arg).1 The Bohr Effect
146His¡Tyr substitution. The pronounced Bohr effect in stripped Hb ( ⫽ ⫺0.50 at 25°C) (Fig. 2, Table 3) is remark1 The 82Arg residue in C. niloticus is incorrectly listed as 82Lys in Perutz et al. 1981 (63).
Fig. 7. Elution profile of oxygenated (Œ) and deoxygenated () P. palpebrosus Hb on a column Sephacryl S-200 HR gel showing fractions pooled for analyses (open horizontal bars 1– 4). Inset: Kav values of these fractions compared with those of human Hb A and proteins of known molecular mass, viz., cyt.C (cytochrome c), Mb (myoglobin), oval albumin (O.A.), bovine serum albumin (B.S.A.), aldolase (Ald.), catalase (Cat.), and ferritin (Ferr.).
Fig. 8. P50 values and pH dependence of three molecular mass fractions (fr.) of P. palpebrosus Hb isolated by gel filtration (cf. Fig. 7), measured at 25°C in HEPES (o, Œ, and 䊐) and MES ({) buffers.
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able given the substitution of 146His by neutral Tyr in P. palpebrosus, and the prodigious evidence for the importance of 146His for expression of the Bohr effect in vertebrate Hbs. In humans where His residues account for almost 90% of the total alkaline Bohr effect (11, 27, 53), the majority of this effect (63% at pH 7.4) (27) originates from proton release upon oxygenation-linked breakage of the T-state salt bridge between 146His and 94-Asp (61). The overriding role of 146His in expression of the alkaline Bohr effect is supported by a host of investigations. Enzymatic removal of the  chain COOHterminal residues (145-Tyr and 146-His) from human HbA drastically reduces the Bohr effect and cooperativity but increases O2 affinity, confirming its role in stabilizing the T state relative to the R state (12, 43, 49, 56). Moreover, abnormal human Hb with 146His replaced by a wide range of other residues [viz., by Asp in Hb Hiroshima2 (38, 64), Pro in Hb York (48, 55), Tyr in Hb Bologna-St. Orsola (also called Hb Halamshire) (50, 52), Gln in Hb Kodaira (33), Leu in Hb Cowtown (76), and Arg in Hb Cochin-Port Royal (87)] show a similar reduction in the Bohr effect, and generally decreased heme-heme cooperativity and increased O2 affinities. Available data indicate a similarly important role for 146His in ectothermic vertebrates. The Bohr effect of carp Hb is halved following enzymatic cleavage of the  chain COOH-terminal His residues (60), and that of Aethotaxis mitopteryx Hb (146His¡Val) is about half of that in other Antarctic fishes (21). In conjunction with the pervasive correlation between 146His and Bohr effects in widely different vertebrates, the strong Bohr effect in P. palpebrosus Hb indicates that other titratable residues take over the role of 146His. Which residues might these be? The high His content of P. palpebrosus Hb (24 per ␣ dimer; Fig. 1) confirms the generality of this trait in crocodilians (11, 40), predicting high nonbicarbonate buffer values in P. palpebrosus blood that reduces arteriovenous pH changes and thus the in vivo contribution of the fixed-acid Bohr effect to tissue O2 release. In bicarbonate-sensitive Hbs, pH changes are further dampened because proton binding by Hb is balanced by protons liberated upon O2-linked HCO⫺ 3 binding (41). Apart from increasing the buffer capacity, the additional His residues in crocodilian Hbs may take over the role of 146His in accounting for a strong fixed-acid Bohr effect expressed in P. palpebrosus Hb (Fig. 2). Although four of the His residues found in human Hb are substituted for other residues (i.e., 2Pro, 116Met, 143Val, and 146Tyr), P. palpebrosus has 10 His residues (at positions ␣67, ␣113, 6, 44, 56, 84, 87, 118, 127, and 136), which are not found in human Hb and may be sources of Bohr protons (Fig. 1). 143His¡Val substitution. Because 143His makes the largest contribution to this reverse (acid) Bohr effect (71% at pH 5.1 in human Hb) (27), its replacement by neutral Val (Fig. 1) correlates deftly with the lack of a reverse Bohr effect in P. palpebrosus at low pH (Fig. 2). The 143His¡Val substitution, however, also deletes a phosphate binding site that would decrease phosphate binding. Indeed in humans, ␥143His¡Ser substitution in the fetal  type chains increases the O2 affinity 2 The His¡Asp replacement in Hb Hiroshima originally reported at 143 was later shown to be at 146.
of fetal blood via lesser DPG interaction. Analogously, abnormal human Hbs in which 143His is substituted by neutral residues, including Hb Little Rock (143His¡Gln), Hb Syracuse (143His¡Pro), and Hb Old Dominion (143His¡Tyr) all exhibit increased affinities in the presence of DPG [cf. (22)]. Phosphate Effects Contrary to the reported insensitivity of crocodilian Hbs to organic phosphates (8, 46, 63), ATP and DPG decrease O2 affinities of crocodilian Hb at low chloride levels (96) as may occur during the postprandial alkaline tide (20). The observation that slopes of double logarithmic plots of P50 against the free DPG and ATP concentrations (Fig. 4) clearly exceed the maximum value (0.25) expected for one-toone stoichiometry between bound phosphate and Hb molecules provides evidence for an additional phosphate binding site (compared with that between the  chains), as observed for Hbs of several other vertebrates including eel, billfish, and dromedary (2, 59, 97). Additional evidence for a second binding site derives from association/dissociation kinetics of the reaction of IHP with deoxygenated and carboxy human Hb (99) and molecular dynamic simulations of skua (seabird Catharacta maccormicki) and pheasant Hbs (32, 68, 84). This second site that comprises a cluster of positive charges between the ␣ chains may serve as an entry-leaving site, implying the existence of a migration pathway for phosphates along the central cavity between the two phosphate binding sites (68). In conjunction with the studies of fish, bird, and mammalian Hbs, our findings in P. palpebrosus provide further evidence of a wide occurrence of an ␣ chain phosphate binding site among vertebrates. Given the distinct effects of ATP and DPG, the insensitivity of P. palpebrosus Hb to IHP molecules (which carry six negative charges compared with four for ATP at physiological pH) indicates that binding of IHP molecules in the cavity between the  chains is impeded by steric hindrance or stereochemical mismatch with the positive charges lining the cavity. The observation that 0.1 M chloride decreases Hb-O2 affinity in the presence of IHP, but increases it in the presence of DPG and ATP indicates that chloride is a less potent effector than ATP or DPG but obstructs binding of these phosphates at shared cationic binding sites. Collectively the 143His¡Val and 82Lys¡Gln substitutions in P. palpebrosus Hb delete four of seven phosphate binding sites commonly found in vertebrates, which accords with the loss of phosphate sensitivity in the presence of 0.1 M Cl⫺ (Fig. 4). Given that 82-Lys also is a Cl⫺ binding site, its replacement by neutral Gln predictably reduces the chloride effect in P. palpebrosus, but not in C. niloticus, where it is replaced by another positively charged residue (Arg) (Fig. 1). Polymerization and Its Effect on Hb-O2 Binding Polymerization of Hbs may begin at hemolysis (69), but may also occur in vivo, as observed in the turtle Pseudemys scripta (81) and witnessed by inclusions resembling Hb crystals in erythrocytes of healthy iguanas (79). In vivo polymerization moreover may be associated with red cell sickling found in iguanas and several teleosts (45). In polymerizing to large aggregates (Fig. 7), oxygenated P. palpebrosus Hb differs from Hbs of other reptiles, including Cai. crocodilus (9) and a large
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ALLOSTERIC REGULATION OF DWARF CAIMAN HEMOGLOBIN
majority of turtles (69, 82) in which polymerization is deoxygenation-linked and does not proceed beyond octamers (dimers of tetramers). Hb polymerization commonly involves Cys residues, as evident from the correlation between disulfide bridge formation and polymerization in ectotherm Hbs (13, 25, 67, 69, 85). In human Hb mutants Mississippi (9Ser¡Cys) and Hb Ta-li (83Gly¡Cys), polymerization is induced by the incorporation of a single Cys residue (6). The dissociation of the large complexes of oxygenated P. palpebrosus Hb into smaller molecules upon deoxygenation contrasts starkly with the deoxygenation-linked self-association encountered in Hbs of other ectothermic vertebrates (65), including hagfish and lampreys (monomer to dimer) (14, 26), snakes (dimer to tetramer) (29, 54), frogs Rana esculenta and R. temporaria (tetramer to dimer of tetramers) (5, 23), and bullfrog R. catesbiana (tetramer to heterotrimer of tetramers) (83). Given that dithionite (used to deoxygenate Hb) may reduce disulfide bonds (44), the possibility cannot be excluded that such interaction contributed to the dissociation of the large Hb complexes upon deoxygenation. Nevertheless P. palpebrosus Hb appears to be unique in polymerizing to giant complexes in the oxygenated state (in the absence of dithionite). The residues mediating the distinctive self-association remain unknown. P. palpebrosus Hb lacks the only surface Cys residue known in humans (93Cys) (98), and 49Cys, which causes polymerization through intermolecular disulfide bridges in a teleost fish (25), but has six Cys residues per dimer (at ␣18, ␣19, ␣104, ␣130, 23, and 100). Available data (36) show that apart from the highly conserved ␣104Cys, all crocodilian Hbs (as with chicken Hb) have Cys at ␣130 and 23 (Table 5). Strikingly, P. palpebrosus differs from other crocodilians in lacking ␣34Cys and ␣81Cys and uniquely possessing two adjacent Cys residues at ␣18 and ␣19 (surface helical positions A16 and AB1) (28) (Fig. 1), implying that these residues may be involved in its unusual, oxygenation-linked polymerization. The similar O2 affinities and Bohr effects in the three major molecular mass fractions isolated in the deoxygenation state (cf. Figs. 7 and 8) indicates that polymerization does not affect the O2 binding properties of Hb. This inference is supported by q values (the number of interacting O2 binding sites) near 4 obtained in fitting the two-state allosteric model to the data. It also aligns with observations that different quaternary assemblies of C. porosus Hb exhibit the same CO2 effect (9) and that the O2 binding properties of turtle (Dermatemys mawi) Hb are not altered by mercaptoethanol-induced dissociation (80). Similarly, polymerization of teleost fish (25) and mouse (72) Hbs has no significant effect on O2 affinity, and recombinant human Hb 83Gly¡Cys, which oligomerizes through diTable 5. Positions of cysteine residues in the ␣ and  chains of crocodilian, human, and chicken hemoglobins ␣ Chain
Human HbA Chicken HbA P. palpebrosus Cai. crocodilus C. niloticus A. mississippiensis
104 104, 130 18, 19, 104, 130 34, 81, 104, 115, 130 8, 34, 81, 104, 130 34, 81, 104, 130
 Chain
93, 23, 23, 23, 23, 23,
112 93, 126 100 100 76, 93, 126 49, 93, 126
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sulfide bonds, has similar CO binding properties as native human HbA (24). The lack of polymerization effects on O2 binding properties indicates that the highly variable number of Cys residues in vertebrate Hbs has no consequence for O2 transport. Given that Cys (-SH) residues contribute to redox buffering during oxidative stress in periodic hypoxia and reoxygenation (66), it would seem appropriate to compare Hb polymerization and red cell antioxidant defenses in crocodilians (in which Hbs have high Cys contents) and sauropsids such as the sideneck turtle, Podocnemis unifilis, whose Hbs completely lack Cys residues (34). Physiological and Evolutionary Implications The singular structural and functional properties of P. palpebrosus Hb compared with other crocodilian (and vertebrate) Hbs may be either conserved ancestral traits or more recent adaptations related to specific differences in its habit and habitat. In contrast to other large and predominantly aquatic crocodilians in which HCO⫺ 3 -induced O2 unloading may support aerobic metabolism during prolonged submergence and activity (drowning of prey) and fuel the postprandial rise in metabolic rate, this property is unlikely to retain utility in the small, semiterrestrial dwarf caiman that frequents running streams of clear and cool waters, feeding on snails, insects, and small vertebrates (18). It follows that P. palpebrosus may not have been subjected to selection pressure that favored evolution of the HCO⫺ 3 effect or that this trait may have been secondarily lost to cope with changes in habitat and/or diet. To place these evolutionary events in perspective, the divergence between the alligator-caiman and the Paleosuchus-Caiman lineages, inferred from the genetic distances between the mitochondrial genome sequences of these taxa, occurred between approximately 65–70 and 37– 41 million years ago, respectively (74). Framed by these boundaries, recent fossil-based studies (15, 35) support the view of Paleosuchus evolving as the most basal (plesiotypic) lineage among extant caimanines, indicating that it well may represent the ancestral trait rather than some specialized advanced condition. Either way, P. palpebrosus Hb exhibits allosteric control of O2 affinity on the basis of reciprocating interaction between O2 binding and nonbicarbonate end products of oxidative metabolism (viz., a substantial fixed-acid Bohr effect and the carbamino-mediated CO2 effect) adopted by apparently novel compensatory mechanisms. Accordingly, some key amino acid exchanges observed in dwarf caiman Hb (compared with other crocodilians) closely resemble those in human Hb, whereas other exchanges are distinct compared with human and other crocodilian Hbs. As with other crocodilians, dwarf caiman Hb is rich in titratable His residues that could serve as Bohr groups and compensate for replacement of 146His (the major Bohr group in other vertebrate Hbs). Similarly, a large number of reactive sulphhydryl groups would increase the tendency to form disulfide bridges. The biological significance, if any, of the unique oxygenated-linked polymerization observed in P. palpebrosus is not clear. Perspectives and Significance P. palpebrosus Hb exhibits a unique combination of distinctive amino acid exchanges compared with other vertebrate and
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crocodilian Hbs that could potentially disrupt the allosteric regulation of Hb function. The finding that Hb sustains marked sensitivities to allosteric effectors demonstrates the existence of alternative, compensatory molecular mechanisms to maintain O2 delivery to the tissues in this species. The functional properties of P. palpebrosus Hb disprove several published assumptions and tenets; notably: 1) that 146His is essential for expression of pronounced Bohr effects (P. palpebrosus Hb lacks this residue but expresses a strong Bohr effect); 2) that crocodilian Hbs lack phosphate effects, that 82Lys is essential for phosphate binding, and that IHP invariably is a more potent effector than ATP and DPG (P. palpebrosus Hb lacks 82Lys and is sensitive to ATP and DPG but not to IHP); 3) that crocodilian Hbs bind bicarbonate and do not form carbamino compounds (P. palpebrosus Hb binds CO2); and 4) that oxygenated reptilian Hbs do not polymerize (the oxygenated Hb aggregates to large complexes). P. palpebrosus Hb thus provides unique opportunity for further studies on structurefunction coupling and the evolution of effector sensitivities in vertebrate Hbs. ACKNOWLEDGMENTS We thank Anny Bang (Aarhus) for technical assistance, and Prof. W. Böhme, Dr. O. Behlert, Dr. T. Ziegler, and U. Bott (Zoological Gardens of Cologne, Germany) for the blood samples. Primary structure determinations were carried out by T.A. Gorr under expert supervision of the late Prof. G. Braunitzer and Dr. T. Kleinschmidt, Max Planck Institute of Biochemistry, Munich-Martinsried, Germany. GRANTS Support for this study was provided by a Faculty of Science and Technology, Aarhus University grant to R. E. Weber, by Danish Council for Independent Research Natural Sciences Grant 10-084565 to A. Fago, by National Heart, Lung, and Blood Institute Grants R01 HL087216 and HL087216-S1 and National Science Foundation Grant IOS-0949931 to J.F. Storz, and by personal fellowships from the Studienstiftung des Deutschen Volkes and Max Planck Society to T.A. Gorr. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). AUTHOR CONTRIBUTIONS Author contributions: R.E.W. and T.A.G. conception and design of research; R.E.W. and T.A.G. performed experiments; R.E.W., H.M., J.F.S., and T.A.G. analyzed data; R.E.W., A.F., J.F.S., and T.A.G. interpreted results of experiments; R.E.W., H.M., J.F.S., and T.A.G. prepared figures; R.E.W. and T.A.G. drafted manuscript; R.E.W., A.F., H.M., J.F.S., and T.A.G. approved final version of manuscript; A.F., H.M., J.F.S. and T.A.G. edited and revised manuscript. REFERENCES 1. Ackers GK. Molecular exclusion and restricted diffusion processes in molecular-sieve chromatography. Biochemistry 3: 723–730, 1964. 2. Amiconi G, Bertollini A, Bellelli A, Coletta M, Condò SG, Brunori M. Evidence for two oxygen-linked binding sites for polyanions in dromedary hemoglobin. Eur J Biochem 150: 387–393, 1985. 3. Antonini E, Wyman J, Brunori M, Fronticelli C, Bucci E, RossiFanelli A. Studies on the relations between molecular and functional properties of hemoglobin. V. The influence of temperature on the Bohr effect in human and in horse hemoglobin. J Biol Chem 240: 1096 –1103, 1965. 4. Atha DH, Ackers GK. Calorimetric determination of the heat of oxygenation of human hemoglobin as a function of pH and the extent of reaction. Biochemistry 13: 2376 –2382, 1974. 5. Bårdgard A, Fago A, Malte H, Weber RE. Oxygen binding and aggregation of hemoglobin from the common European frog, Rana temporaria. Comp Biochem Physiol B Biochem Mol Biol 117: 225–231, 1997.
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