Lack of conventional oxygen-linked proton and anion binding sites does not impair allosteric regulation of oxygen binding in dwarf caiman hemoglobin

Lack of conventional oxygen-linked proton and anion binding sites does notimpair 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*1Zoophysiology, Department of Bioscience, Aarhus University, Aarhus, Denmark; 2School of Biological Sciences, Universityof 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 conven-tional oxygen-linked proton and anion binding sites does not impairallosteric regulation of oxygen binding in dwarf caiman hemoglobin. AmJ Physiol Regul Integr Comp Physiol 305: R300–R312, 2013. Firstpublished May 29, 2013; doi:10.1152/ajpregu.00014.2013.—In contrastto other vertebrate hemoglobins (Hbs) whose high intrinsic O2 affin-ities are reduced by red cell allosteric effectors (mainly protons, CO2,organic phosphates, and chloride ions), crocodilian Hbs exhibit lowsensitivity to organic phosphates and high sensitivity to bicarbonate(HCO3

�), which is believed to augment Hb-O2 unloading duringdiving and postprandial alkaline tides when blood HCO3

� levels andmetabolic rates increase. Examination of �- and �-globin amino acidsequences of dwarf caiman (Paleosuchus palpebrosus) revealed aunique combination of substitutions at key effector binding sitescompared with other vertebrate and crocodilian Hbs: �82Lys¡Gln,�143His¡Val, and �146His¡Tyr. These substitutions delete posi-tive charges and, along with other distinctive changes in residuecharge and polarity, may be expected to disrupt allosteric regulation ofHb-O2 affinity. Strikingly, however, P. palpebrosus Hb shows astrong Bohr effect, and marked deoxygenation-linked binding oforganic phosphates (ATP and DPG) and CO2 as carbamate (contrast-ing with HCO3

� binding in other crocodilians). Unlike other Hbs, itpolymerizes to large complexes in the oxygenated state. The highlyunusual properties of P. palpebrosus Hb align with a high content ofHis residues (potential sites for oxygenation-linked proton binding)and distinctive surface Cys residues that may form intermoleculardisulfide bridges upon polymerization. On the basis of its singularproperties, P. palpebrosus Hb provides a unique opportunity forstudies on structure-function coupling and the evolution of compen-satory mechanisms for maintaining tissue O2 delivery in Hbs that lackconventional effector-binding residues.

allosteric interaction; Bohr effect; carbon dioxide; crocodilians; oxy-gen-binding

THE ROLE OF HEMOGLOBIN (Hb) in transporting O2 to respiringtissues is governed by its intrinsic O2 affinity, its sensitivity tothe effectors that modulate Hb-O2 affinity, levels of thoseeffectors in the red blood cells, and the O2 tensions for loadingand unloading O2 at the respiratory surfaces and in tissues,respectively (91). Each of these factors may vary amongdifferent species and may also undergo reversible changeswithin the same individual animals in response to changes inenvironmental conditions, metabolic requirements, and modeof life.

In vertebrate Hbs that comprise two � chains and two �chains, O2 binding at the heme groups triggers a transition of

the protein molecules from the low-affinity, tense (T) state tothe high-affinity, relaxed (R) state, which is basic to coopera-tivity in O2 binding. Hb-O2 affinity is modulated by allostericeffectors, chiefly protons (low pH) and CO2 that decrease O2

affinity (increase O2 unloading in the tissues via the Bohreffect), and organic phosphate and chloride ions that com-monly reduce Hb-O2 affinity by preferential binding at specificsites of the molecules in the T state (62, 91). Thus, whereasprotons mainly bind at �146His (the COOH-terminal histidinesof the � chains), the organic phosphates [typically 2,3-diphos-phoglycerate (DPG) in mammals, and ATP in ectothermicvertebrates] interact with seven amino acid residues in thecavity between the � chains (�1Val of one chain; and �2His,�82Lys, and �143His of both � chains), CO2 binds at theunprotonated 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 characteris-tics. In contrast to the vast majority of vertebrates, they showlittle or no response to organic phosphates, Cl�, or CO2 (8, 9,39). The insensitivity to phosphates correlates with the replace-ment of three phosphate binding residues. Thus, compared tohuman Hb, �143His is replaced by Ala and �1Val-�2His byacetylated-Ala-Ser in the Nile crocodile (Crocodylus niloticus)and the American alligator (Alligator mississippiensis) and bySer-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 HCO3� ions (8), which is

considered to play an important role in unloading O2 andmaintaining aerobic metabolism when crocodilians dive anddrown their prey (8, 95), compensating for their low myoglobinO2 stores (46). The HCO3

� effect may also play a vital role inunloading O2 from the blood during postprandial alkaline tides(95) when increased blood HCO3

� concentrations (resultingfrom HCl secretion into the stomach to digest bone) coincideneatly with the increased demand for O2 (the specific dynamicaction of food) (19). This view aligns with the fact thatcrocodilians consume large amounts of bone (one alligatorstomach contained remnants of up to 12 turtles) (20) and thatthe postfeeding metabolic peak in C. porosus was 70% higherwhen fed bone-rich chicken necks than when fed homogenizedchicken (30).

The mechanisms basic to allosteric regulation of Hb-O2

affinity in crocodilians remain controversial. Although model-ing indicates deoxygenation-linked binding of HCO3

� at threeresidues in the central cavity between the two � chains; viz.,�82Lys, �144Glu of one � chain, and the NH2-terminalresidue of the partner � chain (Ser in C. crocodilus) (63),mutagenic replacement of �82Lys did not change the HCO3

�

* 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., DK1000Aarhus, Denmark (e-mail: [email protected]).

Am J Physiol Regul Integr Comp Physiol 305: R300–R312, 2013.First published May 29, 2013; doi:10.1152/ajpregu.00014.2013.

0363-6119/13 Copyright © 2013 the American Physiological Society http://www.ajpregu.orgR300

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sensitivity of Nile crocodile (C. niloticus) Hb, and replace-ments of 12 other amino acid residues clustered at the �1�2

interface were required to transplant the bicarbonate sensitivityinto human HbA (16, 46, 47). Also, in contrast to the reportedabsence of oxygenation-linked binding of CO2 in Cai. croc-odilus Hb (8), carbamate (carbamino) formation in crocodil-ians is reflected by the much (twofold to fivefold) larger CO2

Bohr effect than fixed-acid Bohr effect (measured when pH ischanged by adding CO2 and buffers, respectively) in Cai.crocodilus and A. mississippiensis blood (39, 95). Finally,contrary to previous reports [cf. (46)], crocodilian red cellsmay contain a wide spectrum of organic phosphate effectors,albeit at low concentrations. In fact Cai. crocodilus red cellscontain ATP and DPG, as well as inositol pentaphosphate (IPP,found in avian red cells), inositol hexaphosphate (IHP), andguanosine triphosphate (GTP, which is commonly encounteredin fish red cells) (86).

Aiming to elucidate molecular adaptations and structure-function relationships in crocodilian Hbs, we determined theamino acid sequences of the � and � chains of the Hb of thedwarf caiman, Paleosuchus palpebrosus. This species differsfrom larger alligators and crocodiles in that it frequents stonycreeks with clean, fast-running and cooler water, and mainlyfeeds on small vertebrates (e.g., tadpoles, frogs, fish, smallmammals) and a variety of insects and snails (18). Finding aunique combination of amino acid exchanges at highly con-served effector binding sites, we investigated the O2 bindingproperties of the Hb and its sensitivities to chloride ions, ATP,DPG, and IHP, and to CO2 and temperature, over a wide pHrange to discern possible alternative allosteric regulatory mech-anisms. On the basis of the aggregation of deoxygenated Hbsto octamers and larger polymers (that may occur in vivo)observed in a diverse array of sauropsid taxa (birds andnonavian reptiles) (67, 71, 80), we also assessed polymeriza-tion 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 controlsby the veterinarian at Cologne Zoological Gardens in accordance withexisting regulations, was a generous gift from the zoo. Red cellsseparated by centrifugation were lysed in five times their volumes of20 mM Tris·HCl, 40 mM DTE, pH 8.5 buffer. The hemolysateresolved into three electrophorectic bands by native alkaline discelectrophoresis, each of which was identified by NH2-terminal se-quences as a mixture of the same �A and � chains without any hint ofthe presence of additional subunits (not shown). This indicates that P.palpebrosus Hb consists of a single component whose �A/� chainsubunits show a strong tendency to form aggregates that involvedisulfide 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 digestfollowed by size-exclusion chromatography (Sephadex G-25 fine)with acidic elution (0.1 N acetic acid), which resolved the digest into10 fractions of variably sized peptides. Next, we employed reverse-phase HPLC with a LiChrospher 60 RP select B column and differenttrifluoroacetic acid (TFA)/acetonitrile gradients to separate the 10fractions into a total of 281 peptide-containing peaks, including all �A

and � chain tryptic peptides (Tp). The complete primary sequences ofthe �A and � subunits were then determined by means of automated

Edman degradation in conjunction with amino acid analyses of thesetryptic and some additional chymotryptic peptides. NH2-terminal andpeptide sequencing schemes of either subunit and tables of the aminoacid 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 andpyridyl-ethylated globin, whose reactive cysteinyl SH-groups couldno longer form disulfide-based aggregates, were NH2-terminally se-quenced up to �A position 24 and � position 58. This approachallowed 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. ThisGlu/Gln ambiguity might be explained either by a true allelic differ-ence (heterozygosity) at site 73 or by the spontaneous deamidation ofthe original amide (Gln). Proof of the critically important replacementof 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 (position136–141) and Tp 14b�15II (position 142–146), which providedevidence for a single His existing in Tp 14b�15I, whereas two Tyrresidues and no His residues were recovered for Tp 14b�15II; and 2) thesequence of the entire Tp 14b�15 that demonstrated a sole His residue inposition 1 (�136) along with Tyr phenylthiohydantoin (PTH)-derivativepeaks of almost equal yields for �145 and �146.

For comparisons of Paleosuchus globins with orthologous chainsof other species, percentage similarities and identities of amino acidresidues (Table 1) were calculated using the EMBOSS tool (http://www.ebi.ac.uk/Tools/psa/emboss_needle/) based on the global align-ment Needleman-Wunsch algorithm operating with the Blosum62matrix.

O2 Binding Measurements

Hemoglobin preparation. Hb solutions obtained by lysing washedred blood cells were centrifuged for 10 min at 400 g to remove cellulardebris, and were stripped of organic phosphates on a 38 � 2.1 cm(height � diameter) column of Sephadex G25 Fine gel and dialysedfor 24 h against 0.01 mol/l HEPES buffer containing 5 � 10�4 mol/lEDTA, as earlier described (92). The Hb showed no oxidation asjudged from equal absorption values at 539 and 569 nm after briefequilibration with carbon monoxide, and was frozen at �80°C in150-�l aliquots that were thawed individually for O2 equilibrium andother measurements.

O2 equilibria. Hb-O2 equilibria were measured in the presence of0.05 mol/l HEPES buffer (89) unless otherwise specified, using amodified gas diffusion chamber coupled to two cascaded Wösthoffpumps (Bochum, Germany) for mixing of air and pure (�99.998%)N2 (88, 93). P50 and n50 (respectively, O2 tension and Hill’s cooper-ativity coefficients at half-saturation) values were interpolated fromlinear 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 stepsbetween 30% and 70% O2-Hb saturation. The effects of ATP (diso-dium salt), DPG (pentacyclohexyl-ammonium salt), IHP (inositolhexaphosphate, sodium salt), and Cl� (KCl) were investigated byadding accurate volumes of standard, �100 mM solutions of theseeffectors to the stripped Hb solutions. ATP was assayed using Sigmatest chemicals, and Cl� was assayed using a CMT10 chloride titrator(Radiometer, Copenhagen, Denmark). The pH values were measuredin oxygenated Hb samples at the same temperature and CO2 tensionas the O2 equilibria using thermostatted pH electrode units (BMS2Blood Micro System; Radiometer).

The allosteric parameters [the allosteric constant, L, and the asso-ciation equilibrium constants of the T and R structures (KT and KR,respectively)] were assessed by analyzing precise O2 equilibrium datafor a wide range of O2 saturations (extended Hill plots) in terms of thetwo-state Monod-Wyman-Changeux (MWC) equation (57) that was

R301ALLOSTERIC REGULATION OF DWARF CAIMAN HEMOGLOBIN

AJP-Regul Integr Comp Physiol • doi:10.1152/ajpregu.00014.2013 • www.ajpregu.org

http://www.ebi.ac.uk/Tools/psa/emboss_needle/

http://www.ebi.ac.uk/Tools/psa/emboss_needle/

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 fittingthe number of binding sites, q, along with the other parameters toobtain the best possible fit. Additional fits were performed with q fixedat 4 (as applies to tetrameric Hb). Derived parameters, including themedian oxygen tension, Pm; the maximum slope of the Hill plot, nmax;and the free energy of heme-heme interaction, G; were evaluated aspreviously detailed (94). Heats of oxygenation were calculated fromthe van’t Hoff isochore [H 2.303 · R · log P50/(1/T)], where Ris the gas constant and T is the absolute temperature (°K). All quotedH values are exclusive of the heat of solvation of O2 (�12.6 kJ/mol).

Size Exclusion Chromatography

Hb quaternary structure and its oxygenation dependence wereinvestigated by gel filtration of Hb preparations that had not been incontact with CO on a 59.3 � 2.6 cm (height � diameter) column ofSephacryl 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)], whereVe 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 isexcluded by the gel)] of Hb fractions were compared with those ofhorse heart cytochrome c, oval albumin, and aldolase obtained fromBoehringer-Mannheim; myoglobin and bovine serum albumin ob-tained from Sigma (A-0380 and M-4503, respectively); and catalaseand ferritin from GE Health Care. Gel filtration of deoxygenated Hbwas carried out by adding Na-dithionite (1 mg/ml) to the Hb sampleapplied to the column and to the elution buffer after both solutions hadbeen equilibrated with gaseous N2. Hb fractions retrieved for O2

binding experiments were concentrated by ultrafiltration in Milliporeultrafree filter units (cutt-off molecular mass 10,000 Daltons). Frac-tions showing slight oxidation were briefly equilibrated with CO andreduced by dialysis (30 min) against N2-equilibrated 0.01 M HEPESbuffer (pH �7.6) containing freshly added Na-dithionite (0.1%)

A α-globin A1 A16 B1 B16 C11 5 10 15 20 25 30 35

Human HBA V L S P A D K T N V K A A W G K V G A H A G E Y G A E A L E R M F L S F PChicken HBA · · · A · · · N · · · G I F T · I A G · · E · · · · · T · · · · · T T Y ·P. palpebrosus HBA · · · E · · · S · · · G I · S · A C C · L E D · · · · T · · · L · F V Y ·C. crocodilus HBA · · · E E · · S H · · · I · · · · A G · L E · · · · · · · · · · · C A Y ·C. niloticus HBA · · · S D · · C · · · · V · S · · A G · L E · · · · · · · · · · · C A Y ·A. mississippiensis HBA · · · M E · · S · · · · I · · · A S G · L E · · · · · · · · · · · C A Y ·

C7 E1 E1940 45 50 55 60 65 70

Human HBA T T K T Y F P H F D L S H G S A Q V K G H G K K V A D A L T N A V A H V DChicken HBA P · · · · · · · · · · · · · · · · I · · · · · · · V A · · I E · A N · I ·P. palpebrosus HBA Q · · I · · · · · · · T · N · · · I R · · · · · · F L · · H D · · N · I ·C. crocodilus HBA Q · · I · · · · · · M · N · · · I R · · · · · · F A · · H D · · N · I ·C. niloticus HBA Q · · I · · · · · · · · · · · · · I R A · · · · · F A · · H E · · N · I ·A. mississippiensis HBA Q · · I · · · · · · M · · N · · · I R A · · · · · F S · · H E · · N · I ·

F1 F9 G175 80 85 90 95 100 105 110

Human HBA D M P N A L S A L S D L H A H K L R V D P V N F K L L S H C L L V T L A AChicken HBA · I A G T · · K · · · · · · · · · · · · · · · · · · · G Q · F · · V V · IP. palpebrosus HBA · L S G · · · R · · · · · · · N · · · · · · · · · · · · Q · V · · V F G VC. crocodilus HBA · L A G · · C R · · · · · · · N · · · · · · · · · F · · Q · I · · V F G VC. niloticus HBA · L · G · · C R · · E · · · · S · · · · · · · · · F · A Q · V · V V V · IA. mississippiensis HBA · L · G · · C R · · E · · · · S · · · · · · · · · F · A · · V · V V F · I

G19 H1 H21115 120 125 130 135 140

Human HBA H L P A E F T P A V H A S L D K F L A S V S T V L T S K Y RChicken HBA · H · · A L · · E · · · · · · · · · C A · G · · · · A · · ·P. palpebrosus HBA · H · G A L · · E · · · · · · · · · C A · · · · · · · · · ·C. crocodilus HBA · H · C S L · · E · · · · · · · · · C A · · A M · · · · · ·C. niloticus HBA · H · G S L · · E · · · · · · · · · C A · · S · · · · · · ·A. mississippiensis HBA · H · S A L S · E I · · · · · · · · C A · · A · · · · · · ·

B β-globin A1 A16 B1 B16 C11 5 10 15 20 25 30 35

Human HBB V H L T P E E K S A V T A L W G K V N V D E V G G E A L G R L L V V Y P WChicken HBB · · W · A · · · Q L I · G · · · · · · · A · C · A · · · A · · · I · · · ·P. palpebrosus HBB S P F S A H · E K L I L D · · A · · · · A A C · · D · · S · · · I I · · ·C. crocodilus HBB S P F S A H · E K L I V D · · A · · D · A S C · · D · · S · M · I I · · ·C. niloticus HBB A S F D · H · · Q L I G D · · H · · D · A H C · · · · · S · M · I · · · ·A. mississippiensis HBB A S F D A H · R K F · V D · · A · · D · A Q C · A D · · S · M · I · · · ·

C7 D1 D7 E140 45 50 55 60 65 70

Human HBB T Q R F F E S F G D L S T P D A V M G N P K V K A H G K K V L G A F S D GChicken HBB · · · · · A · · · N · · S · T · I L · · · M · R · · · · · · · T S · G · AP. palpebrosus HBB K R · Y · · H · · K M A · D Q D · L H · E · I Q E · · · · · · A S · G E AC. crocodilus HBB K R · Y · · H · · K · · · D Q D · L H · E · I R E · · · · · · A S · G E AC. niloticus HBB K R · Y · · N · · · I · N A Q · I · H · E · · Q · · · · · · · A S · G E AA. mississippiensis HBB K R · Y · · H · · K M C N A H D I L H · S · · Q E · · · · · · A S · G E A

E20 F1 F9 G175 80 85 90 95 100 105 110

Human HBB L A H L D N L K G T F A T L S E L H C D K L H V D P E N F R L L G N V L VChicken HBB V K N · · · I · N · · S Q · · · · · · · · · · · · · · · · · · · · D I · IP. palpebrosus HBB V K · · · · I Q · H · · H · · K · · Y E · F · · · C · · · K · · · · I I IC. crocodilus HBB V K · · · · I · · H · · H · · K · · F E · F · · · C · · · K · · · D I I IC. niloticus HBB V C · · · G I R A H · · N · · K · · · E · · · · · · · · · K · · · D I I IA. mississippiensis HBB V K · · · · I · · H · · N · · K · · · E · F · · · · · · · K · · · D I I I

G19 H1 H21115 120 125 130 135 140 145

Human HBB C V L A H H F G K E F T P P V Q A A Y Q K V V A G V A N A L A H K Y HChicken HBB I · · · A · · S · D · · · E C · · · W · · L · R V · · H · · · R · · ·P. palpebrosus HBB V · · G M · H P · · · · M E T H · · F · · L A R H · · A · · S V E · YC. crocodilus HBB V · · G M · H P · D · · L Q T H · · F · · L · R H · · A · · S A E · ·C. niloticus HBB I · · · A · Y P · D · G L E C H · · · · · L · R Q · · A · · · A E · ·A. mississippiensis HBB I · · · A · H P E D · S V E C H · · F · · L · R Q · · A · · A A E · ·

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 otherspecies are shown only where they differ from those in human Hbs. His residues highlighted in yellow and blue are titratable (surface) residues and nontitratableresidues, respectively, in human Hb (11, 53); those highlighted in green represent potential gains of titratable His residues compared with human Hb.

R302 ALLOSTERIC REGULATION OF DWARF CAIMAN HEMOGLOBIN


followed by extensive dialysis against N2/CO equilibrated HEPESbuffer without Na-dithionite.

RESULTS

Structural Analyses

Figure 1 shows the amino acid sequences of P. palpebrosusHb aligned with those for Cai. crocodilus, C. niloticus, A.mississippiensis, chicken, and human. The entire collection ofglobin 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 caima-nine member (Cai. crocodilus) than with those of C. niloticusand A. mississippiensis and with chicken than with human �

and � chains. Relative to the � chains, the � chains showedhigher sequence divergence in comparisons among species,which is consistent with previous reports of unusually highsubstitution rates in the � chain subunits of crocodilian Hbs(31, 36).

The primary structures of P. palpebrosus Hb chains reveal aunique combination of amino acid substitutions, including�82Lys¡Gln, �143His¡Val, and �146His¡Tyr (Fig. 1) thatdelete positively charged (anion-binding and proton-dissociat-ing) residues and thus may be expected to drastically impactthe sensitivities of the Hb to allosteric effectors. Anotherimpinging substitution of P. palpebrosus Hb is �93Cys¡Tyr,which removes a solvent-exposed cysteine residue that ishighly conserved among mammalian, avian, and reptilian Hbs.

Charge, Polarity, and Histidine Content

The sequence of P. palpebrosus Hb differs markedly fromthat of other crocodilian, avian, and human Hbs in charge,polarity, and hydropathy (hydrophobicity) indices of aminoacid residues, as well as the number and position of histidineresidues that may function as buffer groups and source of Bohrprotons and in stabilizing the Hb’s T-structure through theformation of internal salt bridges (11). P. palpebrosus Hbcontains 24 (11 � chain and 13 � chain) His residues in eachdimeric half-molecule (Fig. 1), which is considerably morethan in other vertebrates, including humans, who have 19(10 � 9) residues, but they align with the high numbers ofphysiological buffer groups in crocodilian Hbs (11).

Table 1. Identities and similarities in � and � chainsequences of P. palpebrosus hemoglobin compared withthose of other crocodilian, chicken and human hemoglobins

� Chains � Chains

Identity Similarity Identity Similarity

Cai. crocodilus 83.7 90.1 89.0 98.6C. niloticus 79.4 87.2 67.8 83.6A. mississippiensis 79.4 87.2 75.3 87.0Chicken 75.2 84.4 54.1 74.7Human 67.4 75.9 48.6 68.5

All values are %.

Table 2. Amino acid residues specifically encountered in � and � chains of P. palpebrosus hemoglobin compared with thoseof other species

Comparison*

Human P. palpebrosus Cai. crocodilus C. niloticus A. mississippiensis Charge Polarity

�12 Ala Gly Ala Ala Ala �18 Gly Cys Ala Ala Ser np (nnp)�19 Ala Cys Gly Gly Gly np (nnn)�23 Glu� Asp� Glu� Glu� Glu� �28 Ala Tyr Ala Ala Ala np (nnn)�32 Met Leu Met Met Met �34 Leu Phe Cys Cys Cys nn (ppp)�35 Ser Val Ala Ala Ala pn (nnn)�49 Ser Tyr Ser Ser Ser �64 Asp� Leu Ala Ala Ser � o (o o o) pn (nnp)�100 Leu Leu Phe Phe Phe �134 Tyr Tyr Ala Ser Ala pp (npn)�12 Tyr Leu Val Gly Val pn (nnn)�19 Asn Asn Asp� Asp� Asp� o o (- - -) �22 Asp� Ala Ser His� Gln �o (o � o) pn (ppp)�31 Leu Leu Met Met Met �49 Ser Ala Ser Ser Cys pn (ppp)�52 Asp� Glu� Gln Gln His� �� (o o �) �82 Lys� Gln Lys� Arg� Lys� �o (��) �93 Cys Tyr Phe Phe Phe pp (nnn)�108 Asn Asn Asp� Asp� Asp� o o (- - -) �121 Glu� Glu� Asp� Asp� Asp� �124 Pro Met Leu Leu Val �134 Val Ala Val Val Val �143 His� Val Ala Ala Ala �o (o o o) pn (nnn)�146 His� Tyr His� His� His� �o (� � �)

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: , samecharge/polarity in all species; �, positively charged side chains; �, negatively charged side chains, o, neutral side-chains. p, Polar side chain; n, nonpolar side chain.



Remarkably, the � and � chain sequences of P. palpebrosusexhibit distinctive features that are shared with human Hb, butnot with other hitherto investigated crocodilian Hbs (Table 2).Thus, both P. palpebrosus and human Hbs have neutral Asnresidues at �19 and at �108 compared with negatively chargedAsp in other crocodilians. Also both have negatively chargedresidues (Glu and Asp, respectively) at �52 compared withneutral Gln or weakly positive His in the crocodilians. Alsoboth have residues with opposite side-chain polarities thanother crocodilian Hbs at positions �34 and �93. However, P.palpebrosus Hb also shows distinguishing differences in resi-due polarity compared with human and other crocodilian Hbs(at positions �19, �28, �22, �49, and �143) that may contrib-ute 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 substantiallyreduced by Cl� ions, decreased pH, and increased temperature(Fig. 2; Table 3); at pH 7.4, P50 values at 10° and 25°C are 0.65and 2.69 mmHg, respectively, in the stripped Hb; and 1.53 and5.43 mmHg, respectively, in the presence of 0.1 M Cl�.Expressed as log P50,Cl � log P50,str, the chloride effectdecreases markedly at high pH where cationic binding sites areneutralized (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 Bohreffect. At pH 7.0–7.4, which characterizes physiological val-ues 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 effectswith increasing temperature accords with the temperature de-pendence of ionization of Bohr groups. Unlike in human andmost vertebrate Hbs (37, 73, 92) the Bohr effect is not in-creased by 0.1 M chloride, suggesting that allosteric protonbinding to this Hb is not enhanced by chloride. O2 equilibria ofstripped P. palpebrosus Hb in MES buffer (Fig. 2) reveal theabsence of a reverse (acid) Bohr effect (increasing O2 affinitywith decreasing pH) as found in human Hb below �6.5.

Phosphate sensitivities. Measurements of the interactiveeffects of organic phosphates and chloride at physiological pH(Fig. 3) show insensitivity of P. palpebrosus Hb to organicphosphates in the presence of chloride ions, as observed inother crocodilians (8, 9, 63). In contrast, pronounced sensitiv-ities to ATP and DPG are unmasked in the absence of chloride,as is also observed in A. mississippiensis and Cai. crocodilusHbs (96). Strikingly, however, the Hb-O2 affinity is virtuallyinsensitive 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 ofATP or DPG (Fig. 3B). Similar relative sensitivities to theseeffectors were observed in duplicate measurements at pH 7.0(data not shown).

Dose-response curves (Fig. 4) for the effects of increasingconcentrations of free phosphates calculated as [total phos-phate] � 0.5[Hb4], on the assumption that at P50 half of thetetrameric Hb molecules are phosphate-liganded (97), demon-strate virtual annihilation of the phosphate effects in the pres-ence of 0.1 M chloride. Strikingly, the double logarithmic plotsreveal slopes that markedly exceed 0.25 (i.e., the value ex-pected for one-to-one stoichiometry between phosphate andtetrameric Hb molecules if phosphate binding is limited to theT state). For DPG binding at pH 7.4 the slope of 0.37 (Fig. 4)indicates a stoichiometry of �1.5 DPG molecules bound perHb tetramer.

CO2 sensitivity. Admixture of CO2 in the equilibration gasesconspicuously lowers O2 affinity of the stripped P. palpebrosusHb at constant pH. This specific CO2 effect increases withincreasing pH and is markedly reduced by Cl� (Fig. 5). Thusat 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 consis-tent with carbamate formation at the (NH2-terminal) �-aminoresidues of the chains. Slopes of log P50 vs. log PCO2 plots (in

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) and25°C (full and half circles), in 0.05 M HEPES buffer (circles and diamonds) or0.05 M MES buffer (half-circles and triangles) and in the absence (opensymbols) and presence (closed symbols) of 0.1 M Cl�. Heme concentra-tion, 0.37 mM.

Table 3. P50 values and Bohr factors of strippedP. 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) 0.45 0.65 1.20 �0.67P50, str. � 0.1 M Cl (mmHg) 0.78 1.53 2.66 �0.61log P50,Cl � log P50,st. 0.23 0.37 0.35

25 P50, str. (mmHg) 2.02 2.69 4.26 �0.45P50, str. � 0.1 M Cl (mmHg) 3.09 5.43 8.61 �0.50log P50,Cl � log P50,str 0.18 0.31 0.32

�, Bohr factor; str., stripped.



the 7–30 mmHg PCO2 range) of �0.5 in the absence, and �0.4in the presence of Cl, indicate binding of one CO2 molecule pertwo O2 molecules released.

Allosteric control. Extended Hill plots (Fig. 6) reveal slopesof unity at extreme low and high O2 saturations consistent withnoncooperativity in binding the first and last O2 molecules tothe Hb, and good correspondence between Pm and P50 values(O2 tensions at, respectively, median and half-saturations),permitting assessment of allosteric effects from alterations inP50 values. As shown (Table 4), the n50 values are low, whichhampered 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 interactingbinding sites) floating and with the constraint that the allostericconstant (L) � 1. For the other curves, the fitted values of qwere close to 4, indicating that the functional units are tetram-ers, 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 loweringKT. Fits with q fixed at 4 (Table 4) show that the interactionenergy is increased in the presence of 0.1 M chloride and thatthis was due in part to a stabilizing effect of chloride on the Tstate (lowered KT). At pH 7.6 this was augmented by adestabilizing effect of chloride on the R state (increased KR),whereas this was not the case at pH 6.9. As evident from thestandard error on the fits, however, it is not safe to draw firmconclusions 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 Bohreffect (cf. Fig. 2), the overall oxygenation enthalpy (�56.3kJ/mol) corresponds closely with the intrinsic heat of oxygen-ation of human Hb (�59 kJ/mol) (4). The numerically lowervalue (�48.8 kJ/mol) found at pH 7.0 where the Bohr effect isoperative, is consistent with endothermic contributions fromBohr proton dissociation [�26 kJ/mol for imidazole groups ofhistidines (3, 4)]. Analogously, the reduced enthalpy valuesobserved 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) reflectendothermic contributions (�8.6, �7.7, and �6.5 kJ/mol,respectively) from oxygenation-linked chloride ion dissocia-tion. The semblance of these enthalpic effects with those inother (nonheterothermic) vertebrates (90) indicates the absenceof distinguishing adaptive traits related to body temperaturevariation in P. palpebrosus Hb.

Quaternary Structure and Effects

Unexpectedly, the size exclusion gel-filtration chromatogra-phy experiments showed that oxygenated P. palpebrosus Hbelutes with the void volume of the Sephacryl S-200 HRmedium, revealing an exceptionally high degree of polymer-ization (Fig. 7). In contrast, the elution pattern of Hb deoxy-genated with dithionite reveals the presence of three fractions

Fig. 3. A: P50 and n50 values of P. palpebro-sus Hb measured in 50 mM HEPES buffer at10° and 25°C in the absence (�) and pres-ence (�) of 0.1 M Cl�, and the absence(open symbols) and presence (closed sym-bols) of saturating levels [ATP/Hb and 2,3-diphosphoglycerate (DPG)/Hb ratio 6.2; ino-sitol hexaphosphate (IHP)/Hb ratio �28] ofthe polyanionic phosphate effectors (Pn�)ATP, DPG, and IHP. B: histograms showingthe P50 values of the stripped Hb (str., opencolumns) and the log P50 shifts (shaded col-umns) induced by chloride, ATP, DPG, andIHP at pH 7.4 and 10° and 25° C. Otherconditions as in Fig. 2.

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 oforganic phosphates (stars); and the presence of ATP (circles), DPG (triangles),and IHP (squares) at different phosphate/tetrameric Hb ratios, measured in0.05 M HEPES buffer. Fine dotted line shows a slope of 0.25 expected ifdeoxygenation of the Hb molecules were linked to binding of one phosphatemolecule. Heme concentration, 0.36 mM. (This figure replots earlier publishedP50 values (96) against the [free phosphate]/[Hb] ratios).



of smaller molecules, whose partition coefficients (Kav) indi-cate molecular masses of 31.6, 81.3, and 263 kDa. The value of38 kDa obtained for human Hb (Fig. 7, inset) is consistent withobservations that molecular mass estimates for tetrameric ver-tebrate Hbs on the basis of gel filtration are lower than

established values (64–68 kDa) due to reversible dissociationto dimers (17) and to elution volumes being proportional tomolecular Stokes radii of proteins rather than their molecularweights (1). In this light, the three fractions observed indeoxygenated Hb (labeled 4, 3, and 2 in Fig. 7) likely comprisetetramers, octamers (dimers of tetramers), and high-order poly-mers, respectively.

O2 equilibrium measurements of three major molecular massfractions obtained from gel filtration of deoxygenated Hb showsimilar O2 affinities and Bohr effects (Fig. 8) indicating thataggregation 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 affinitycombined with low cooperativity, suggesting that the allostericT-R equilibrium in this Hb is markedly shifted to the high-affinity 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 acidresidues compared with other Hbs. P. palpebrosus Hb providesa unique opportunity to reevaluate the roles of specific aminoacid residues implicated in the allosteric regulation of O2

affinity, and the possible compensatory mechanisms in natu-rally occurring Hbs that lack specific effector binding sites.

CO2 Effects

P. palpebrosus Hb binds molecular CO2. The distinct, spe-cific effect of CO2 on O2 affinity (Fig. 5) is at variance with thereported absence of carbamate formation in crocodilian Hbs

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), measuredat 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.

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.



(63). The reduction in the CO2 effect observed in the presence ofchloride and increased proton activity (pH decrease) (cf. Fig. 5) isconsistent with competition between these effectors for binding atthe NH2-terminal amino acid residues, and thus is analogous tothe antagonism between binding of CO2 on the one hand, and oflactate (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 perHb tetramer under physiological conditions, which indicatesthat CO2 binds to either the � or the � chains of P. palpebrosusHb as in human Hb, where O2-linked carbamate formation isconfined to the � chains (10). The identical NH2-terminalamino acid sequences (Fig. 1) of P. palpebrosus and Cai.crocodilus Hbs predict similar CO2 binding capacities in thesespecies. In C. niloticus and A. mississippiensis, however, the �

chain NH2-terminal residues are acetylated (75) and are there-fore not available for carbamate formation.

In A. mississippiensis Hb carbamate, free HCO3� and Hb-bound

HCO3� contribute approximately equally to the high CO2 carrying

capacity of deoxygenated red cells (41). Two observations indi-cate that HCO3

� binding to P. palpebrosus Hb is markedlyreduced compared with that of other crocodilians. First, the CO2

effect increases strongly with increasing pH (Fig. 5A), whereas theassociation constants for the reaction of Cai. crocodilus Hb withHCO3

� are essentially pH independent below pH 7.3 (8). Second,compared with other crocodilian Hbs, P. palpebrosus Hb exhib-its substitutions (viz., �34Cys¡Phe, �35Ala¡Val,�100Phe¡Leu, and �31Met¡Leu) (Fig. 1) that replace 4 ofthe 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 othercrocodilian Hbs, and confer a HCO3

� effect when engineeredinto human Hb (47). Among these the loss of polar �34Cys inP. palpebrosus Hb may be significant. If �82-Lys is a bicar-bonate-binding site as proposed by Perutz and his colleagues(63), its substitution by Gln would similarly contribute to lossof bicarbonate binding in P. palpebrosus Hb, but not in Cai.crocodilus and A. mississippiensis Hbs that have retained thisresidue, nor in C. niloticus Hb that shows a polar- and charge-neutral substitution (�82Lys¡Arg).1

The Bohr Effect

�146His¡Tyr substitution. The pronounced Bohr effect instripped Hb (� �0.50 at 25°C) (Fig. 2, Table 3) is remark-

1 The �82Arg residue in C. niloticus is incorrectly listed as �82Lys in Perutzet al. 1981 (63).

Table 4. MWC and derived parameters obtained by fitting the MWC two-state model to the data with four fixed interactingO2-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 0 3.58 3.53 1.29 1.66 0.333 �0.758 0.040 �0.146 0.075 2.446.964 0 8.02 7.62 1.21 3.49 1.593 �0.993 0.025 �0.0562 0.386 2.077.595 0.10 9.38 8.31 1.43 65.61 0.0026 �1.102 0.019 15.46 0.011 4.166.961 0.10 14.00 12.84 1.37 4.07 4.085 �1.279 0.063 �0.117 1.012 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, associationequilibrium constants of the T and R structures, respectively; G, free energy of heme-heme interaction. *Standard errors as estimated from the covariance matrixassociated with the fit.

Fig. 7. Elution profile of oxygenated (Œ) and deoxygenated (�) P. palpebrosusHb 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 withthose 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.) ofP. palpebrosus Hb isolated by gel filtration (cf. Fig. 7), measured at 25°C inHEPES (o, Œ, and �) and MES ({) buffers.



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. Inhumans where His residues account for almost 90% of the totalalkaline Bohr effect (11, 27, 53), the majority of this effect(63% at pH 7.4) (27) originates from proton release uponoxygenation-linked breakage of the T-state salt bridge between�146His and �94-Asp (61). The overriding role of �146His inexpression of the alkaline Bohr effect is supported by a host ofinvestigations. Enzymatic removal of the � chain COOH-terminal residues (145-Tyr and 146-His) from human HbAdrastically reduces the Bohr effect and cooperativity but in-creases O2 affinity, confirming its role in stabilizing the T staterelative to the R state (12, 43, 49, 56). Moreover, abnormalhuman Hb with �146His replaced by a wide range of otherresidues [viz., by Asp in Hb Hiroshima2 (38, 64), Pro in HbYork (48, 55), Tyr in Hb Bologna-St. Orsola (also calledHb Halamshire) (50, 52), Gln in Hb Kodaira (33), Leu in HbCowtown (76), and Arg in Hb Cochin-Port Royal (87)] show asimilar reduction in the Bohr effect, and generally decreasedheme-heme cooperativity and increased O2 affinities.

Available data indicate a similarly important role for�146His in ectothermic vertebrates. The Bohr effect of carpHb is halved following enzymatic cleavage of the � chainCOOH-terminal His residues (60), and that of Aethotaxismitopteryx Hb (�146His¡Val) is about half of that in otherAntarctic fishes (21). In conjunction with the pervasive corre-lation between �146His and Bohr effects in widely differentvertebrates, the strong Bohr effect in P. palpebrosus Hb indi-cates 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 crocodil-ians (11, 40), predicting high nonbicarbonate buffer values inP. palpebrosus blood that reduces arteriovenous pH changesand thus the in vivo contribution of the fixed-acid Bohreffect to tissue O2 release. In bicarbonate-sensitive Hbs, pHchanges are further dampened because proton binding by Hbis balanced by protons liberated upon O2-linked HCO3

�

binding (41).Apart from increasing the buffer capacity, the additional His

residues in crocodilian Hbs may take over the role of �146Hisin accounting for a strong fixed-acid Bohr effect expressed inP. palpebrosus Hb (Fig. 2). Although four of the His residuesfound in human Hb are substituted for other residues (i.e.,�2Pro, �116Met, �143Val, and �146Tyr), P. palpebrosus has10 His residues (at positions �67, �113, �6, �44, �56, �84,�87, �118, �127, and �136), which are not found in human Hband may be sources of Bohr protons (Fig. 1).

�143His¡Val substitution. Because �143His makes thelargest contribution to this reverse (acid) Bohr effect (71% atpH 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 substitu-tion, however, also deletes a phosphate binding site that woulddecrease phosphate binding. Indeed in humans, �143His¡Sersubstitution in the fetal � type chains increases the O2 affinity

of fetal blood via lesser DPG interaction. Analogously, abnor-mal human Hbs in which �143His is substituted by neutralresidues, including Hb Little Rock (�143His¡Gln), Hb Syr-acuse (�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 toorganic phosphates (8, 46, 63), ATP and DPG decrease O2

affinities of crocodilian Hb at low chloride levels (96) as mayoccur during the postprandial alkaline tide (20).

The observation that slopes of double logarithmic plots ofP50 against the free DPG and ATP concentrations (Fig. 4)clearly exceed the maximum value (0.25) expected for one-to-one stoichiometry between bound phosphate and Hb moleculesprovides evidence for an additional phosphate binding site(compared with that between the � chains), as observed forHbs of several other vertebrates including eel, billfish, anddromedary (2, 59, 97). Additional evidence for a secondbinding site derives from association/dissociation kinetics ofthe reaction of IHP with deoxygenated and carboxy human Hb(99) and molecular dynamic simulations of skua (seabirdCatharacta maccormicki) and pheasant Hbs (32, 68, 84). Thissecond site that comprises a cluster of positive charges betweenthe � chains may serve as an entry-leaving site, implying theexistence of a migration pathway for phosphates along thecentral cavity between the two phosphate binding sites (68). Inconjunction with the studies of fish, bird, and mammalian Hbs,our findings in P. palpebrosus provide further evidence of awide occurrence of an � chain phosphate binding site amongvertebrates.

Given the distinct effects of ATP and DPG, the insensitivityof P. palpebrosus Hb to IHP molecules (which carry sixnegative charges compared with four for ATP at physiologicalpH) indicates that binding of IHP molecules in the cavitybetween the � chains is impeded by steric hindrance or stereo-chemical mismatch with the positive charges lining the cavity.The observation that 0.1 M chloride decreases Hb-O2 affinityin the presence of IHP, but increases it in the presence of DPGand ATP indicates that chloride is a less potent effector thanATP or DPG but obstructs binding of these phosphates atshared cationic binding sites.

Collectively the �143His¡Val and �82Lys¡Gln substitu-tions in P. palpebrosus Hb delete four of seven phosphatebinding sites commonly found in vertebrates, which accordswith the loss of phosphate sensitivity in the presence of 0.1 MCl� (Fig. 4). Given that �82-Lys also is a Cl� binding site, itsreplacement by neutral Gln predictably reduces the chlorideeffect in P. palpebrosus, but not in C. niloticus, where it isreplaced 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 mayalso occur in vivo, as observed in the turtle Pseudemys scripta(81) and witnessed by inclusions resembling Hb crystals inerythrocytes of healthy iguanas (79). In vivo polymerizationmoreover may be associated with red cell sickling found iniguanas and several teleosts (45). In polymerizing to largeaggregates (Fig. 7), oxygenated P. palpebrosus Hb differs fromHbs of other reptiles, including Cai. crocodilus (9) and a large

2 The His¡Asp replacement in Hb Hiroshima originally reported at �143was later shown to be at �146.



majority of turtles (69, 82) in which polymerization is deoxy-genation-linked and does not proceed beyond octamers (dimersof tetramers).

Hb polymerization commonly involves Cys residues, asevident from the correlation between disulfide bridge forma-tion 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 incorpora-tion of a single Cys residue (6).

The dissociation of the large complexes of oxygenated P.palpebrosus Hb into smaller molecules upon deoxygenationcontrasts starkly with the deoxygenation-linked self-associ-ation encountered in Hbs of other ectothermic vertebrates(65), including hagfish and lampreys (monomer to dimer)(14, 26), snakes (dimer to tetramer) (29, 54), frogs Ranaesculenta and R. temporaria (tetramer to dimer of tetramers)(5, 23), and bullfrog R. catesbiana (tetramer to heterotrimerof tetramers) (83).

Given that dithionite (used to deoxygenate Hb) may reducedisulfide bonds (44), the possibility cannot be excluded thatsuch interaction contributed to the dissociation of the large Hbcomplexes upon deoxygenation. Nevertheless P. palpebrosusHb appears to be unique in polymerizing to giant complexes inthe oxygenated state (in the absence of dithionite).

The residues mediating the distinctive self-association re-main unknown. P. palpebrosus Hb lacks the only surface Cysresidue known in humans (�93Cys) (98), and �49Cys, whichcauses polymerization through intermolecular disulfide bridgesin a teleost fish (25), but has six Cys residues per dimer (at �18,�19, �104, �130, �23, and �100). Available data (36) showthat apart from the highly conserved �104Cys, all crocodilianHbs (as with chicken Hb) have Cys at �130 and �23 (Table 5).Strikingly, P. palpebrosus differs from other crocodilians inlacking �34Cys and �81Cys and uniquely possessing twoadjacent Cys residues at �18 and �19 (surface helical positionsA16 and AB1) (28) (Fig. 1), implying that these residues maybe involved in its unusual, oxygenation-linked polymerization.

The similar O2 affinities and Bohr effects in the three majormolecular mass fractions isolated in the deoxygenation state(cf. Figs. 7 and 8) indicates that polymerization does not affectthe O2 binding properties of Hb. This inference is supported byq values (the number of interacting O2 binding sites) near 4obtained in fitting the two-state allosteric model to the data. Italso aligns with observations that different quaternary assem-blies of C. porosus Hb exhibit the same CO2 effect (9) andthat 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 recombinanthuman Hb �83Gly¡Cys, which oligomerizes through di-

sulfide bonds, has similar CO binding properties as nativehuman HbA (24).

The lack of polymerization effects on O2 binding propertiesindicates that the highly variable number of Cys residues invertebrate Hbs has no consequence for O2 transport. Given thatCys (-SH) residues contribute to redox buffering during oxi-dative stress in periodic hypoxia and reoxygenation (66), itwould seem appropriate to compare Hb polymerization andred cell antioxidant defenses in crocodilians (in which Hbshave high Cys contents) and sauropsids such as the side-neck turtle, Podocnemis unifilis, whose Hbs completely lackCys residues (34).

Physiological and Evolutionary Implications

The singular structural and functional properties of P. pal-pebrosus Hb compared with other crocodilian (and vertebrate)Hbs may be either conserved ancestral traits or more recentadaptations related to specific differences in its habit andhabitat. In contrast to other large and predominantly aquaticcrocodilians in which HCO3

�-induced O2 unloading may sup-port aerobic metabolism during prolonged submergence andactivity (drowning of prey) and fuel the postprandial rise inmetabolic rate, this property is unlikely to retain utility in thesmall, semiterrestrial dwarf caiman that frequents runningstreams of clear and cool waters, feeding on snails, insects, andsmall vertebrates (18). It follows that P. palpebrosus may nothave been subjected to selection pressure that favored evolu-tion of the HCO3

� effect or that this trait may have beensecondarily lost to cope with changes in habitat and/or diet. Toplace these evolutionary events in perspective, the divergencebetween the alligator-caiman and the Paleosuchus-Caimanlineages, inferred from the genetic distances between the mi-tochondrial genome sequences of these taxa, occurred betweenapproximately 65–70 and 37–41 million years ago, respec-tively (74). Framed by these boundaries, recent fossil-basedstudies (15, 35) support the view of Paleosuchus evolving asthe most basal (plesiotypic) lineage among extant caimanines,indicating that it well may represent the ancestral trait ratherthan some specialized advanced condition. Either way, P.palpebrosus Hb exhibits allosteric control of O2 affinity on thebasis of reciprocating interaction between O2 binding andnonbicarbonate end products of oxidative metabolism (viz., asubstantial fixed-acid Bohr effect and the carbamino-mediatedCO2 effect) adopted by apparently novel compensatory mech-anisms. Accordingly, some key amino acid exchanges ob-served in dwarf caiman Hb (compared with other crocodilians)closely resemble those in human Hb, whereas other exchangesare distinct compared with human and other crocodilian Hbs.As with other crocodilians, dwarf caiman Hb is rich in titrat-able His residues that could serve as Bohr groups and com-pensate for replacement of �146His (the major Bohr group inother vertebrate Hbs). Similarly, a large number of reactivesulphhydryl groups would increase the tendency to form disul-fide bridges. The biological significance, if any, of the uniqueoxygenated-linked polymerization observed in P. palpebrosusis not clear.

Perspectives and Significance

P. palpebrosus Hb exhibits a unique combination of distinc-tive amino acid exchanges compared with other vertebrate and

Table 5. Positions of cysteine residues in the � and �chains of crocodilian, human, and chicken hemoglobins

� Chain � Chain

Human HbA 104 93, 112Chicken HbA 104, 130 23, 93, 126P. palpebrosus 18, 19, 104, 130 23, 100Cai. crocodilus 34, 81, 104, 115, 130 23, 100C. niloticus 8, 34, 81, 104, 130 23, 76, 93, 126A. mississippiensis 34, 81, 104, 130 23, 49, 93, 126



crocodilian Hbs that could potentially disrupt the allostericregulation of Hb function. The finding that Hb sustains markedsensitivities to allosteric effectors demonstrates the existenceof alternative, compensatory molecular mechanisms to main-tain O2 delivery to the tissues in this species. The functionalproperties of P. palpebrosus Hb disprove several publishedassumptions and tenets; notably: 1) that �146His is essentialfor expression of pronounced Bohr effects (P. palpebrosus Hblacks this residue but expresses a strong Bohr effect); 2) thatcrocodilian Hbs lack phosphate effects, that �82Lys is essen-tial for phosphate binding, and that IHP invariably is a morepotent 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 carbam-ino compounds (P. palpebrosus Hb binds CO2); and 4) thatoxygenated reptilian Hbs do not polymerize (the oxygenatedHb aggregates to large complexes). P. palpebrosus Hb thusprovides unique opportunity for further studies on structure-function coupling and the evolution of effector sensitivities invertebrate 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 ofCologne, Germany) for the blood samples. Primary structure determinationswere 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 Technol-ogy, Aarhus University grant to R. E. Weber, by Danish Council for Indepen-dent Research Natural Sciences Grant 10-084565 to A. Fago, by NationalHeart, Lung, and Blood Institute Grants R01 HL087216 and HL087216-S1 andNational Science Foundation Grant IOS-0949931 to J.F. Storz, and by personalfellowships from the Studienstiftung des Deutschen Volkes and Max PlanckSociety 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 re-search; R.E.W. and T.A.G. performed experiments; R.E.W., H.M., J.F.S., andT.A.G. analyzed data; R.E.W., A.F., J.F.S., and T.A.G. interpreted results ofexperiments; R.E.W., H.M., J.F.S., and T.A.G. prepared figures; R.E.W. andT.A.G. drafted manuscript; R.E.W., A.F., H.M., J.F.S., and T.A.G. approvedfinal version of manuscript; A.F., H.M., J.F.S. and T.A.G. edited and revisedmanuscript.

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Lack of conventional oxygen-linked proton and anion binding sites does not impair allosteric regulation of oxygen binding in dwarf caiman hemoglobin