Gene 398 (2007) 29 – 34

Gene 398 (2007) 29 – 34
www.elsevier.com/locate/gene

Crystal structures of two hemoglobin components from the midge larva
Propsilocerus akamusi (Orthocladiinae, Diptera)
Takao Kuwada a,b , Tomokazu Hasegawa c , Shu Sato d , Isamu Sato a,b ,Koichi Ishikawa e , Takashi Takagi d , Fumio Shishikura b,f,⁎baAdvanced Research Institute for the Sciences and Humanities, Nihon University, Chiyoda-ku, Tokyo 102-8251, Japan
Laboratory for Electron Beam Research and Application, Institute of Quantum Science, Nihon University, Funabashi 274-8501, Japanc
X-ray Research Laboratory, Rigaku Corporation, Akishima 196-8666, Japand
Graduate School of Life Sciences, Tohoku University, Sendai 980-8578, Japane
Department of Pharmacology, Nihon University School of Medicine, Itabashi-ku, Tokyo 173-8610, Japanf
Department of Chemistry, Nihon University School of Medicine, Itabashi-ku, Tokyo 173-8610, Japan

Received 24 October 2006; received in revised form 8 February 2007; accepted 13 February 2007
Available online 10 May 2007

Abstract
The polymorphic components of hemoglobin (Hb) of the midge larva Propsilocerus akamusi were classified into two distinct types dependent
on their spectroscopic properties, normal absorption (N) and low absorption (L). Analyses of the amino acid sequences of component VII (N-typeHb) and component V (L-type Hb) from P. akamusi indicated that one remarkable difference is the replacement of the distal histidine (His) withisoleucine (Ile) in component V. To clarify the structural differences between the two Hb components, we determined the crystal structures ofcomponents V and VII at resolutions of 1.64 Å and 1.50 Å, respectively. These crystal structures indicated a short additional helix comprising
three amino acid residues at the C-terminal region in component V, and a typical globin fold including eight helices in component VII.
Comparison of the heme regions of the Hb components suggests that the structural changes of the heme region in component V on ligation differ
from that of usual Hb.
© 2007 Elsevier B.V. All rights reserved.
Keywords: X-ray crystallography; Insect hemoglobin; Additional helix; Distal histidine; Amino acid replacement

1. Introduction
Both the biochemical nature and protein structure of
invertebrate hemoglobins (Hbs) are unique because they arepresent in the coelomic fluid or hemolymph as well as in thecellular components (Vinogradov, 1985; Riggs, 1998). Crystallographic analyses of invertebrate Hbs have revealed thecharacteristics of their tertiary and quaternary structures andstructural changes dependent on ligand binding (Bolognesi et al.,1997). The crystal structures of Hb from three species of Diptera,
Abbreviations: Hb, hemoglobin; L, low absorption; N, normal absorption;
PDB, Protein Data Bank; SAD, single-wavelength anomalous diffraction.
⁎ Corresponding author. Department of Chemistry, Nihon University Schoolof Medicine, 30-1 Oyaguchi-kami-machi, Itabashi-ku, Tokyo 173-8610, Japan.
Tel.: +81 3 3972 8111; fax: +81 3 3972 0027.
E-mail address: fshishi@med.nihon-u.ac.jp (F. Shishikura).
0378-1119/$ - see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.gene.2007.02.049

Chironomus thummi thummi (Chironomidae), Gasterophilus
intestinalis (Oestridae), and Drosophila melanogaster (Drosophilidae), were determined and the structural features of each Hbcomponent were characterized (Steingemann and Weber, 1979;
Pesce et al., 2005; de Sanctis et al., 2005, 2006). However,within one insect species, the structural differences between
polymorphic Hb components have not yet been analyzed by Xray crystallography techniques.
Propsilocerus akamusi (old species name: Tokunagayusurika
akamusi) is a common species of midge found in eutrophic lakes
in Japan that belongs to a different subfamily Orthocladiinae fromC. thummi thummi in Chironominae. The coelomic fluid of the
4th-instar midge larva contains polymorphic hemoglobin with as
many as eleven separable hemoglobin components, as discriminated on DEAE-cellulose column chromatography (Fukuda
et al., 1993). All the components are of low molecular mass,

30

T. Kuwada et al. / Gene 398 (2007) 29–34

17 kDa or less, and classified into two distinct types on the basis of
their spectroscopic properties: low absorbance (L) and normal (N)absorbance. Component V is representative of the six members of
the L type and component VII of the N type, they compriseapproximately 15% and 30% in the 4th larval instar coelomic
fluid, respectively. One of the most remarkable differences
between component V and component VII is the replacement of
the distal histidine by isoleucine, whereas component VII
contains the usual distal His residue. Component V also differsfrom the known midge larval hemoglobin components in having
an intra-molecular disulfide bridge bound by Cys 143 and Cys
148 (Fukuda et al., 1993).
Comparative studies of the tertiary structures of the two
distinct types of P. akamusi Hb components can help
understanding the structural differences among polymorphic
Hb components found in invertebrate hemolymph, in particular,
insect coelomic fluid (Shishikura et al., 2006). To clarify thetertiary structural differences among polymorphic Hb components of P. akamusi coelomic fluid, we performed X-ray
crystallographic analyses of the two components. Here we
report the crystallographic structures of components V and VII
at resolutions of 1.64 Å and 1.50 Å, respectively.
2. Materials and methods
2.1. Crystallization
The two distinct types of Hbs, components V and VII, fromthe 4th-instar midge larvae of P. akamusi were purified as
described by Fukuda et al. (1993), and crystallization was
carried out separately by hanging drop vapor diffusion
techniques at 20 °C. Crystals of component V were grown ina drop containing the protein solution (2.0 μl of 24 mg/ml)
mixed with an equal amount of a reservoir solution (25% PEG
3350, 200 mM ammonium sulfate, and 100 mM sodium acetate,
pH 4.6). The dimensions of the crystal of component V reached
0.7 × 0.4 × 0.05 mm in about 1 week.
At the same time, crystals of component VII were grown asclusters of thin plates in the drop mixed with equal volumes ofthe protein solution (2.0 μl of 15 mg/ml) and a reservoir solution(30% PEG 3350, 200 mM MgCl2, and 100 mM Tris–HCl, pH
8.5) within 1 week. Because these crystals were unsuitable for
X-ray diffraction analysis, we employed a seeding experiment.
The clusters of component VII thin plates were crushed andused as microseeds. Using a fine hair, the seeds of fine crystals
were transferred into drops of a protein solution mixed with thereservoir solution containing 25% PEG 3350, 150 mM MgCl2,
and 100 mM Tris–HCl, pH 8.5. Finally, component VII crystals
grew to about 0.2 × 0.2 × 0.05 mm within a day.
2.2. X-ray diffraction analyses
X-ray diffraction data of Hb components were collected fromflash-cooled crystals at 100 K using a Rigaku R-AXIS VII
image plate detector with CuKα radiation from rotating anodegenerators: Rigaku FR-ESuperBright for component V and Rigaku
RA-Macro7 for component VII (Rigaku Co., Tokyo, Japan). A

crystal of component V was immersed in cryoprotectant
solution containing 20% glycerol in the reservoir solution for
10–15 s, picked up with a loop, and rapidly transferred to a cold
stream. Following that, diffraction data were collected at 1.64 Å
resolution. A total of 140,766 observed intensities were
measured, 20,177 of which were unique reflections resulting
in an Rmerge of 0.027 and completeness of 97.3%. One crystal ofcomponent VII was transferred to a reservoir solution for 24 h
before flash-freezing and data collection. A total of 317,903
observed intensities were measured, 23,622 of which were theunique reflections. The resulting dataset was of 100.0%
completeness with an Rmerge of 0.041. Each set of diffraction
data was processed using the software program CrystalClear(version 1.3.5; Rigaku Co., Tokyo, Japan). Crystal data and datacollection statistics of components V and VII are summarized inTable 1.
2.3. Phasing and model building
The phases of component V were determined by the singlewavelength anomalous diffraction (SAD) method using theanomalous signal from heme irons. The SAD phases ofcomponent V were calculated and further refined by density
modification using the CNX program suite (MSI Inc., CA,
USA). The initial model of component V was built automatically using the program ARP/wARP (Morris et al., 2003) with
the SAD phases calculated in CNX. The high quality electron
density allowed tracing of the main- and side-chain atoms of
most residues except for residues in one N-terminal and one Cterminal. The model was rebuilt manually using the X-BUILD
Table 1
Data collection statistics, crystal data and structure refinement statistics

Data collection statistics
Resolution (Å)
No. of recorded
observations
No. of unique reflections
RmergeCompleteness (%)
Multiplicity
I/σI
Crystal parameters
Space group
a (Å)
b (Å)
c (Å)
Model statistics
R-factor/Rfree
No. of protein atoms
No. of heme atoms
No. of water atoms
rmsd bond lengths
rmsd bond angles
Ramachandram plot (%)
Most favored
Additional allowed

Component V

Component VII

49.14–1.64(1.70–1.64)
140,766

33.77–1.50(1.55–1.50)
317,903

20,1770.027 (0.068)
97.3 (94.2)
6.98 (6.91)
48.6 (15.8)

23,6220.041 (0.244)
100.0 (100.0)
13.46 (12.55)
31.0 (8.5)

P21212
65.14
74.85
33.42

C2221
42.01
69.11
99.65

0.194 / 0.2140
1213
43
226
0.006
1.024

0.194 / 0.227
1169
43
262
0.015
1.470

96.2
3.8

97.0
3.0

Note: Values in parentheses are for the highest resolution shell.

T. Kuwada et al. / Gene 398 (2007) 29–34

program (QUANTA, MSI Inc.), guided by a simulated
annealing omit map and/or 2Fo–Fc map. The model fitted tothe electron density map was refined by CNX functions: it
included simulated annealing, energy minimization, and Bfactor refinements. Following several rounds of manualintervention and CNX refinement, the heme and watermolecules were added to the model. The water molecules
were added using the X-SOLVENT program (QUANTA, MSI
Inc.), guided by Fo–Fc maps contoured at 3σ. The model
including water molecules was refined by energy minimization
and B-factor refinements. On the other hand, the SAD phases ofcomponent VII were determined using the anomalous signal
from heme irons. With the SAD phases, the initial model ofcomponent VII was built using the program ARP/wARP(Morris et al., 2003), it included most residues except for one
N-terminal and one C-terminal. The subsequent refinement ofcomponent VII model was conducted as described above.
The qualities of the final models were assessed using the
program PROCHECK (Laskowski et al., 1993). The atomic
coordinates and structure factors of P. akamusi Hb components

31

Fig. 2. The overall structure of Hb component VII from P. akamusi. The eighthelices are labeled A–H. The structural figure was drawn with Pymol (http://pymol.sourceforge.net/).

have been deposited with the RCSB Protein Data Bank with the
PDB codes of 1X3K for component V and 1X46 for componentVII.
3. Results and discussion
3.1. Refined models of Hb components from P. akamusi
The crystal of component V (L-type Hb) from P. akamusi
belongs to the orthorhombic space group P21212 with unit celldimensions of a = 65.14 Å, b = 74.85 Å, and c = 33.42 Å. The
VM value was calculated to 2.37 Å3/Da, suggesting that there isone monomeric molecule in an asymmetric unit. The model ofFig. 1. Sequence alignment of insect Hbs from P. akamusi, D. melanogaster, G.
intestinalis, and C. thummi thummi. The helices of the typical globin fold arelabeled A though H above the alignment. Amino acid residues in each helix are
highlighted in gray. Data on the primary and the secondary structures for each
Hb component were excerpted from the Macromolecular Structure Database(http://www.ebi.ac.uk/msd/). Sequence alignment was created with the help of
the ClustalX program (Thompson et al., 1997). DmHb, D. melanogaster Hb(PDB code 2BK9); GiHb1, G. intestinalis Hb1 (2C0K); PaHbV, P. akamusi Hb
component V; PaHbVII, P. akamusi Hb component VII; CttHbIII, C. thummi
thummi HbIII (1ECA).

Fig. 3. The overall structure of Hb component V from P. akamusi. The eighthelices are labeled A–H. An arrow indicates the C-terminal additional helix(post-H helix). The structural figure was drawn with Pymol (http://pymol.
sourceforge.net/).

32

T. Kuwada et al. / Gene 398 (2007) 29–34

ties of the final models of P. akamusi Hb components are
summarized in Table 1, showing that each was well refined withexcellent stereochemistry.
3.2. Overall structures of P. akamusi Hb components

Fig. 4. The electron density around the additional helix at C-terminal region ofcomponent V. Amino acid residues at the C-terminal, the F helix (yellow), andthe H helix (orange) are shown by sticks. Light green broken lines imply
hydrogen bonds between the amino acid residues at the C-terminal and that of
the F or H helices. The structural figure was drawn with DeepView (Guex andPeitsch, 1997). (For interpretation of the references to colour in this figurelegend, the reader is referred to the web version of this article.)component V was solved by the SAD method and refined at
1.64 Å resolution to an R-factor of 19.4% and an Rfree of 21.4%.
The final model of component V consisted of a complete
polypeptide chain (152 amino acid residues), one heme group,and 226 water molecules.
The crystal of component VII (N-type Hb) belongs to theorthorhombic space group C2221 with unit cell dimensions of
a = 42.01 Å, b = 69.11 Å, and c = 99.65 Å. The asymmetricunit contains one monomeric molecule corresponding to a VM
of 2.19 Å3/Da. The refined model of component VII solved by
the SAD method contained all 150 amino acid residues of thenative protein, one heme group, and 262 water molecules,
yielding an R-factor of 19.4% and an Rfree of 22.7% at 1.50 Å
resolution. Detailed refinement statistics and geometric proper-

In general, hemoglobin components comprise eight helices(named A–H from N to C terminus) and corner regions
connecting helices, and insect Hbs also possess this typicalglobin fold (Steingemann and Weber, 1979; Pesce et al., 2005;de Sanctis et al., 2005) (Fig. 1). Fig. 2 shows the crystal
structure of component VII. It has the typical globin fold basedon eight α helices except for residues 82–84 in the F helix andresidues 147–149 in the H helix which display a 310 helix
conformation. Fukuda et al. (1993) reported that component VII
showed higher sequence similarity to Chironomus Hb components than component V. When the secondary structures werecompared, component VII resembled component III of Chironomus Hb in the A, B, and C helices. The three helices, A, B,
and C in both component VII of P. akamusi and component III
of C. thummi thummi, differ from Hbs in other insects in thatthey are 3, 4, and 2 residues shorter (Fig. 1).
Fig. 3 shows the crystal structure of component V. It contains
the same α helices, A to H in which the only differences occur
in the C helix and residues 96–98 in the F helix which display a
310 helix conformation. The presence of an additional helix at
the C-terminal of component V is particularly remarkable. The
helix is a one-turn long 310 helix containing three residues(Ser149–Phe151). Fig. 4 shows the electron density in therefined model around the C-terminal region of component V. An
additional helix is next to the F and H helices dependent on
several noncovalent bonds and a disulfide bond between
Cys143 and Cys148; this additional helix does not provide
crystal contacts between neighbor molecules. The additional
helix located at the N-terminal region has been observed indimeric Hb (Hb I) from the blood clam, Scapharca inaequivalvis, and was named the pre-A helix (Royer, 1994). We call an

Fig. 5. Electron density maps (contoured at 1.5σ) corresponding to component V (A) and VII (B) heme regions. Distal His (Ile for component V), proximal His, andheme groups are shown by bolls and sticks. The structural figure was drawn with DeepView (Guex and Peitsch, 1997).

T. Kuwada et al. / Gene 398 (2007) 29–34

Fig. 6. Overlapping of component V (pink) and component VII (blue) based on
the F helix. Distal His (Ile for component V) (E7), proximal His (F8), and hemegroups are shown by sticks. Structure superimpositions and figure preparation
were performed with DeepView (Guex and Peitsch, 1997). (For interpretation of
the references to colour in this figure legend, the reader is referred to the web
version of this article.)

additional helix of P. akamusi Hb the post-H helix, and webelieve that it has no homologous relationships with any otherknown helix of Hb components. Its function cannot be
confirmed at this stage, still the modification of the post-H
helix associated with the heme state is remarkable because it
contacts to the heme region.
3.3. Comparison of heme region
As shown in Fig. 5, electron density maps for the heme
ligand are clearly evident in both components V and VII. Bothligands seem to be water molecules because of the shape andsize of the electron density, and because there is no attention toligand molecules on crystallization, which causes to form metforms by autooxidation from oxy-forms (Ladner et al., 1977).
Water molecules as ligand molecules are well fitted to electron
density and display B-factors of 12.4 A2 and 12.2 A2 forcomponents V and VII. The existence of these ligand moleculesin P. akamusi Hb components suggests some interesting
possibilities regarding the conformation of the heme region.

33

In vertebrate α2β2-type Hbs, the structural changes
dependent on ligand binding are remarkable, thus the heme
geometry of ligated Hb differs from that of unligated Hbs. Ourfindings indicate that both Hb components from P. akamusi arein the ligated form. However, overlapping the two components
from P. akamusi reveals the differences between them. Fig. 6
indicates the narrow heme region of component V due to theclose contact between the E and F helices: the distance between
the Cα atom of E7 and the Cα atom of F8 is 13.58 Å, 0.69 Åshorter than the value in component VII (14.27 Å, nearly 14.5 Å(± 0.2) in various Hb components (Riccio et al., 2002)); this isreminiscent of a scissors-like motion of the EF segment on
hemichrome formation in the Antarctic fish Hb, where thedistance between the Cα atoms of distal and proximal His is12.5 Å (Riccio et al., 2002). Difference of the coordinationgeometry of the heme region between components V and VII isalso significant.
The coordination geometry of the heme region in Table 2 isusual to illustrate the structural differences between ligated andunligated Hbs. Comparison of these parameters indicates thatthe heme geometry of component VII is similar to that of human
ligated Hbs. In component V, the location of the iron atom in theheme plate is almost the same as in average ligated Hbs, and the
distance between the heme iron and the ligand also is similar to
those of ligated human Hbs. At the same time, component V
closely resembles the unligated Hb components in the tilting of
the imidazole of the proximal His. In many Hbs, the heme ironmoves into the heme plate upon ligation, this brings about the
movement of the imidazole of the proximal His. This structuralfeature of proximal His was not observed in ligated component
V. It is possible that the structural changes in the heme on
ligation do not trigger the associated movement of the imidazoleof the proximal His in component V.
One of the most remarkable differences between component
V and many other Hbs is related to the substitution of the usual
distal His residue with Ile (Fukuda et al., 1993). Replacement of
the distal His might cause unusual structural changes upon
ligand binding. In fact, Hb components from a marine blood
worm and a hagfish, in which each distal His is replaced, show
small structural changes of heme region on ligation (Braden

Table 2The heme geometry of P. akamusi and human Hbs
Ligated Hb

Unligated (deoxy) Hb

HumanDistance (Å)
Fe-Ligand
Fe-Pn
Fe-F8Nε2
F8[Cδ-N3]-[Cε-N1]

P. akamusi

Human

Oxy-α

Oxy-β

CO-α

CO-β

VII

V

α1

β1

α2

β2

1.82
0.05
2.07
0.14

1.78
0.04
2.06
−0.01

1.74
−0.04
2.11
0.11

1.71
−0.07
2.12
0.02

2.08
0.01
2.07
−0.11

1.98
0.07
2.34
0.49

–
0.39
2.2
0.55

–
0.35
2.19
0.53

–
0.31
2.21
0.46

–
0.32
2.16
0.48

Fe-Ligand, the distance between the heme iron and the ligand molecule. For calculation of this parameter in P. akamusi Hbs, a water molecule was treated as the ligand
molecule. Fe-Pn, the distance between the heme iron and the center of the four porphyrin nitrogen atoms. This parameter represents the movement of the heme ironupon ligand binding. Fe-F8Nε2, the distance between the heme iron and the Nε atom of the proximal His. F8[Cδ-N3]-[Cε-N1], the difference between two distances
representing a tilting of the imidazole of the proximal His. One is between Cδ of the proximal His and the nitrogen of pyrrole 3 of porphyrin, and the other is between
Cε of the proximal His and the nitrogen of pyrrole 1. This tilting links the shift of the F helix toward the FG corner upon ligand binding (Mito et al., 2002). For
comparison, the parameters from the structures of human Oxy-Hb (PDB code 2DN1), CO-Hb (2DN3), and Deoxy-Hb (2DN2) (Park et al., 2006) are also included.

34

T. Kuwada et al. / Gene 398 (2007) 29–34

et al., 1994; Mito et al., 2002). Our comparative studies of thecrystal structure of P. akamusi Hb components revealed thecharacteristics of the tertiary structures of each component, andespecially the interesting heme region of component V. It seems
that the heme region of aquomet component V is in theintermediate state between a typical ligated (aquomet) andunligated or hemichrome form. Replacement of the distal His
might play a part in the formation of the equivocal state of theheme region in component V. Crystallographic analyses of P.
akamusi Hb in a variety of ligand state can prove helpful inunderstanding the details of the heme ligation and the associatedstructural changes.
Acknowledgements
This work was supported by the “Academic Frontier” Projectfor Private Universities: matching fund subsidy from MEXT
(Ministry of Education, Culture, Sports, Science and Technology), Japan, 2000–2004 and 2005–2007 and in part by Nihon
University Joint Research Grant (2005).
References
Braden, B.C., Arents, G., Padlan, E.A., Love, W.E., 1994. Glycera dibranchiata
hemoglobin. X-ray structure of carbon monoxide hemoglobin at 1.5 Å
resolution. J. Mol. Biol. 238, 42–53.
Bolognesi, M., Bordo, D., Rizzi, M., Tarricone, C., Ascenzi, P., 1997.
Nonvertebrate hemoglobins: structural bases for reactivity. Prog. Biophys.
Mol. Biol. 68, 29–68.
de Sanctis, D., et al., 2005. Bishistidyl heme hexacoordination, a key structuralproperty in Drosophila melanogaster hemoglobin. J. Biol. Chem. 280,
27222–27229.
de Sanctis, D., et al., 2006. Cyanide binding and heme cavity conformationaltransitions in Drosophila melanogaster hexacoordinate hemoglobin.
Biochemistry 45, 10054–10061.
Fukuda, M., Takagi, T., Shikama, K., 1993. Polymorphic hemoglobin from a
midge larva (Tokunagayusurika akamusi) can be divided into two different
types. Biochim. Biophys. Acta 1157, 185–191.

Guex, N., Peitsch, M.C., 1997. SWISS-MODEL and the Swiss-PdbViewer: anenvironment for comparative protein modeling. Electrophoresis 18,
2714–2723.
Ladner, R.C., Heidner, E.J., Perutz, M.F., 1977. The structure of horsemethaemoglobin at 2.0 Å resolution. J. Mol. Biol. 114, 385–414.
Laskowski, R.A., MacArthur, M.W., Moss, D.S., Thornton, J.M., 1993.
PROCHECK: a program to check the stereochemical quality of protein
structure. J. Appl. Crystallogr. 26, 283–291.
Mito, M., et al., 2002. Crystal structures of deoxy- and carbonmonoxyhemoglobin F1 from the hagfish Eptatretus burgeri. J. Biol. Chem. 277,
21898–21905.
Morris, R.J., Perrakis, A., Lamzin, V.S., 2003. ARP/wARP and automaticinterpretation of protein electron density maps. Methods Enzymol. 374,
229–244.
Park, S.Y., Yokoyama, T., Shibayama, N., Shiro, Y., Tame, T.J., 2006. 1.25 Å
resolution crystal structures of human haemoglobin in the oxy, deoxy andcarbonmonoxy forms. J. Mol. Biol. 360, 690–701.
Pesce, A., et al., 2005. Modulation of oxygen binding to insect hemoglobins: thestructure of hemoglobin from the botfly Gasterophilus intestinalis. Protein
Sci. 14, 3057–3063.
Riccio, A., Vitagliano, L., di Prisco, G., Zagari, A., Mazzarella, L., 2002. The
crystal structure of a tetrameric hemoglobin in a partial hemichrome state.
Proc. Natl. Acad. Sci. U. S. A. 99, 9801–9806.
Riggs, A.F., 1998. Self-association, cooperativity and supercooperativity ofoxygen binding by hemoglobins. J. Exp. Biol. 201, 1073–1084.
Royer Jr., W.E., 1994. High-resolution crystallographic analysis of a cooperative dimeric hemoglobin. J. Mol. Biol. 235, 657–681.
Shishikura, F., et al., 2006. Crystal structures of insect hemoglobin components
from Propsilocerus akamusi (Orthocladiinae, Deptera). XIV International
Conference on Dioxygen Binding and Sensing Proteins, Stazione Zoologica
Anton Dohr, Naples, September 3–7, p. 95.
Steingemann, W., Weber, E., 1979. Structure of erythrocruorin in different
ligand states refined at 1.4 Å resolution. J. Mol. Biol. 127, 309–338.
Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G.,
1997. The ClustalX windows interface: flexible strategies for multiple
sequence alignment aided by quality analysis tools. Nucleic Acids Res. 24,
4876–4882.
Vinogradov, S.N., 1985. The structure of invertebrate extracellular hemoglobins(erythrocruorins and chlorocruorins). Comp. Biochem. Physiol., B 82, 1–15.

Comparative Biochemistry and Physiology, Part B 142 (2005) 258 – 268
www.elsevier.com/locate/cbpb

Axolotl hemoglobin: cDNA-derived amino acid sequences of two a
globins and a h globin from an adult Ambystoma mexicanum
Fumio Shishikura a,*, Hiro-aki Takeuchi b, Takatoshi Nagai cbaDepartment of Biology, Nihon University School of Medicine, Itabashi-ku, Tokyo 173-8610, Japan
Department of Biology and Geosciences, Faculty of Science, Shizuoka University, Shizuoka 422-8529, Japanc
Department of Biology, Keio University School of Medicine, Hiyoshi, Yokohama 223-8521, Japan

Received 4 April 2005; received in revised form 3 July 2005; accepted 5 July 2005
Available online 6 September 2005

Abstract
Erythrocytes of the adult axolotl, Ambystoma mexicanum, have multiple hemoglobins. We separated and purified two kinds ofhemoglobin, termed major hemoglobin (Hb M) and minor hemoglobin (Hb m), from a five-year-old male by hydrophobic interaction
column chromatography on Alkyl Superose. The hemoglobins have two distinct a type globin polypeptides (aM and am) and a common hglobin polypeptide, all of which were purified in FPLC on a reversed-phase column after S-pyridylethylation. The complete amino acidsequences of the three globin chains were determined separately using nucleotide sequencing with the assistance of protein sequencing.
The mature globin molecules were composed of 141 amino acid residues for aM globin, 143 for am globin and 146 for h globin.
Comparing primary structures of the five kinds of axolotl globins, including two previously established a type globins from the same
species, with other known globins of amphibians and representatives of other vertebrates, we constructed phylogenetic trees for amphibian
hemoglobins and tetrapod hemoglobins. The molecular trees indicated that aM, am, h and the previously known a major globin were adulttypes of globins and the other known a globin was a larval type. The existence of two to four more globins in the axolotl erythrocyte ispredicted.
D 2005 Elsevier Inc. All rights reserved.
Keywords: Amphibia; Axolotl; Ambystoma mexicanum; Evolution; Globin; Hemoglobin; Nucleotide sequence; Primary structure

1. Introduction
Amphibian hemoglobins are of interest in the study ofthe evolution of vertebrate globin genes, gene expression
and gene switching during metamorphosis, and functional
properties of aquatic and terrestrial species. Althoughliving amphibians comprise three major orders, Apoda,
Urodela (Caudata) and Anura, most investigations havebeen carried out in anurans (frogs and toads): the bullfrog
Rana catesbeiana (Hamada et al., 1966; Moss and Ingram,
1968; Aggarwal and Riggs, 1969; Chauvet and Acher,* Corresponding author. Tel.: +81 3 3972 8111x2291; fax: +81 3 3972
0027.
E-mail address: fshishi@med.nihon-u.ac.jp (F. Shishikura).
1096-4959/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.cbpc.2005.07.003

1972; Baldwin and Riggs, 1974; Maruyama et al., 1980;
Watt et al., 1980; Dorn and Broyles, 1982; Maples et al.,1983) and the South African clawed toad Xenopus laevis(Hentschel et al., 1979; Jeffreys et al., 1980; Hosbach etal., 1983; Kay et al., 1983; Banville and Williams, 1985;
Knöchel et al., 1985; Weber et al., 1991). The Mexican
axolotl Ambystoma mexicanum, on the other hand,belonging to order Urodela, is an important animal in the
biological sciences because they become adult without
metamorphosing, a phenomenon termed neotenous adulthood. Although axolotls receive much attention as acounterpart of metamorphosing species, little is known
about hemoglobin components, population of globin
subunits and primary structures of globins. In ourreferences, there are only two reports on primary structuresof axolotl globin polypeptides (Boissel et al., 1980;

F. Shishikura et al. / Comparative Biochemistry and Physiology, Part B 142 (2005) 258 – 268

GenBank accession no. AF308869); both were assigned toan adult a type of globin and a larval a type of globin bycomputer analysis described later.
We describe here the separation of two kinds ofhemoglobin components, Hb M (major) and Hb m (minor),from a neotenous adult axolotl A. mexicanum and establish
the complete nucleotide sequences of the three kinds ofglobin chains, two of adult a type globins and adult h typeglobin common to both hemoglobin components.

2. Materials and methods
2.1. Blood cell collection
Blood of an adult axolotl A. mexicanum (five-year-old
male about 20 cm in body length and 600 g in mass)
was drawn from the second branchial artery into physiological saline (0.9% NaCl, 10 mM EDTA, 50 mM
Tris –HCl, pH 8.0). The blood was then centrifuged at1750 g for 15 min, and the packed cells were divided:
one half of the packed cells was lysed with 50 mM
Tris – HCl, pH 8.0, containing 10 mM EDTA for
preparation of hemoglobin solution, and the other half
was used for extraction of total RNA by Purescript RNA
Isolation Kit (Gentra Systems, Minneapolis, MN, USA)according to the manufacturer’s instructions. All procedures were done at 4 -C and the samples were stored at
80 -C.
2.2. Separation of hemoglobin components
A hemoglobin solution that had been saturated at 40% byadding 60% saturated ammonium sulfate was subjected toan Alkyl Superose column (Pharmacia Biotech, Uppsala,Sweden) equilibrated with 60% saturated ammonium sulfate(183 g/500 mL) in 50 mM ammonium bicarbonate, pH 8.0.
The experimental procedures were conducted as previously
described (Shishikura and Takami, 2001). Elution was
carried out with a gradient of 60 –0% saturated ammonium
sulfate in the 50 mM ammonium bicarbonate buffer, pH 8.0.
The flow rate was maintained at 0.5 mL/min, andfractions of the protein peaks were collected. The fractions
were monitored at 415 and 280 nm by two spectrophotometers (Model Uvidec-340Q, Jasco, Tokyo, Japan, andModel 116, Gilson Medical Electronics, Inc., WI, USA),
respectively.
2.3. Preparation of globin polypeptides
To separate a and h globin polypeptides, hemoglobin
components were S-pyridylethylated separately by themethod described previously (Friedman et al., 1970). Eachof the S-pyridylethylated hemoglobin components was thensubjected to a Resource column (Pharmacia Biotech) andeluted with a buffered gradient of 0.1% trifluoroacetic acid259

(TFA; Sigma-Aldrich, Inc., St. Louis, MO, USA) to 60%
acetonitrile in 0.08% TFA. All fractions were monitored at
214 and 280 nm. For further purification, re-chromatography on the Resource column was conducted undershallower gradient conditions, as described in our previous
report (Shishikura et al., 1987).
2.4. Protein sequencing
All three kinds of globin molecules modified by Spyridylethylation were separately digested with lysyl
endopeptidase (Achromobacter Protease I; Wako Pure
Chemicals, Tokyo, Japan) at an enzyme / substrate ratioof 1 : 30 (mol : mol) for 4 h at 37 -C in 0.1 M ammonium
bicarbonate, pH 8.2, containing 4 M urea. Peptide
fragments derived from each of the parent molecules
were separated using a reversed-phase column (Resource
RPC, Pharmacia Biotech.) in a buffered gradient of 0.1%
TFA to 60% acetonitrile in 0.08% TFA. Flow rates weremaintained at 0.3 mL/min. All fractions were monitored
at 214 and 280 nm. Re-chromatography of selected
peptides was conducted as previously described (Shishikura et al., 1987). Sequencing analyses of these fragments
as well as amino (N)-terminal amino acid residues ofparent molecules were performed using a gas phase
protein sequencer, PPSQ-10 (Shimadzu, Shiga, Japan),equipped with a class LC-10 amino acid analyzer(Shimadzu, Shiga, Japan). Phenylthiohydantoin (PTH)derivatives from the sequencer were separated andquantified. The peptides were aligned with the assistanceof sequence similarities toward the known globin structures of urodeles (Boissel et al., 1980; Kleinschmidt et al.,
1988; AF308869).
2.5. Isolation of total RNA and purification of mRNAs
Total RNA was extracted from the blood cells byPurescript Total RNA Isolation Kits (Gentra Systems),
and the mRNA fraction that included the three kinds ofglobin mRNAs was isolated with a Takara Oligotexi-dT30<Super>mRNA Purification Kit (Takara Bio, Shiga,
Japan).
2.6. Primer design
Degenerate primers were designed based on the aminoacid sequences of lysyl endopeptidase-digested fragments ofparent molecules as listed in Appendix 1. In order tosequence the PCR amplified fragments with a BigDye
Terminator v. 1.1 Cycle sequencing Kit (Applied Biosystems, Foster City, CA, USA), the degenerate oligo-nucleotide primers were tailed with the pUC/M13 forward or pUC/
M13 reverse sequencing primer tail (forward 17-mer: 5VGTAAAACGACGGCCAGT-3V, Sigma-Aldrich Japan,
Tokyo, Japan, and reverse 17-mer: 5V-CAGGAAACAGCTATGAC-3V, Promega, Tokyo, Japan).

260

F. Shishikura et al. / Comparative Biochemistry and Physiology, Part B 142 (2005) 258 – 268

2.7. Nucleotide sequencing
Single-strand cDNAs were synthesized with a Takara
RNA PCR Kit (v. 2.1) using the Oligo dT-Adaptor Primer(M13 primer M4, 17-mer: 5V-GTTTCCCAGTCACGACT15-3V), according to the manufacturer’s instructions(Takara Bio).
For PCR amplification of the 3V region of the cDNAs,
the primers used were the adaptor and a redundantoligomer based on the N-terminal amino acid sequence ofeach globin (see Appendix 1). The second PCR-amplification was conducted with a nested PCR primer (a redundantoligomer) and the adaptor (Appendix 1). One major
fragment was detected on agarose gel electrophoresis ineach PCR. Then, the amplified fragment was extracted by a
GenElutei Agarose Spin Column (Sigma-Aldrich), andsequenced directly with a BigDye Terminator v. 1.1 Cycle
Sequencing Kit. The rest of the unknown sequence of the
3V end was afterwards confirmed by 3V RACE (Frohman etal., 1988), with the adaptor and a gene-specific primer(Appendix 1).
For PCR amplification of the 5V region of cDNAs, genespecific primers with or without 5V monophosphate as listed
in Appendix 1, were designed in order to extend thesequences in the 5V ends using a Takara 5V-Full RACE Core
Set (Takara Bio) according to the manufacturer’s instructions. All forward and reverse primers, except for the Oligo
dT, listed in Appendix 1, were tagged with the pUC/M13
sequencing primers.

anion-exchange column chromatography on DEAE-Sephadex (Grasso et al., 1979) and by analytical isoelectrofocusing (Koussoulakos et al., 1986), respectively. However,
how many hemoglobin components are present inerythrocytes of the axolotl A. mexicanum, which belongs
to the Ambystomoidea, in the embryo or larval stages, oreven in the neotenous adult stage, has not been
investigated.
Fig. 1 shows two well-delimited peaks corresponding toa major (Hb M) and a minor hemoglobin (Hb m) fromerythrocytes of an adult axolotl. The two peaks, Hb M and
Hb m, were detected at 280 and 415 nm. The peaks wereseparately pooled as shown by bars, and then, each pooled
fraction was re-chromatographed on the same column
under the same conditions (insets of Fig. 1). They exist ina ratio of about 5 (Hb M) : 2 (Hb m) based on chromatogram area calculation. This value varied from 5 : 1 to 5 : 3
depending on sample preparation. Several small distinct
peaks (indicated by two arrows in Fig. 1) were still

A multiple alignment program, Clustal X v. 1.81
(Jeanmougin and Thompson, 1998), was used to align thethree kinds of adult axolotl globins, previously establishedknown sequences of amphibians and those from representative species of tetrapods. Two phylogenetic trees wereconstructed by the neighbor-joining method (Saitou andNei, 1987), and the molecular trees were drawn by the NJprot program stored in Clustal X.

0.1 au2.8. Computer analysis

415 nm
280 nm0.1 au280 nm

3. Results and discussion

Hb M

280 nmHb m

3.1. Two hemoglobin components of adult axolotl
Separation of an intact component from multiple
hemoglobins is a prerequisite to investigation of theproperties of individual hemoglobin component fromsources with such complicated life cycles as amphibians.
Several investigators have succeeded: in adult anurans(toads and frogs), four hemoglobin components have been
separated and characterized (Elli et al., 1970; MacLean andJurd, 1971; Tam et al., 1993). In adult urodeles, four andeight intact hemoglobin components were separated
from the newt Triturus cristatus (Salamandroidea) by

0 10

20

30

40
min

50

60

Fig. 1. Alkyl Superose HR5/5 column chromatography of intact hemoglobin of the axolotl, A. mexicanum. The hemoglobin solution was saturated at
40% by adding 60% saturated ammonium sulfate and applied to an Alkyl
Superose HR5/5 column equilibrated with 60% saturated ammonium
sulfate buffer. After washing with the buffer, the adsorbed proteins were
eluted with a decreasing concentration of ammonium sulfate, pH 8.0.
Concentration of ammonium sulfate and time: 60% (0 min) — 42% (10min) — 30% (40 min) — 0% (60 min). Insets show the purification of Hb
M and Hb m. Bars indicate the pooled (upper) and purified fractions (lower)of major and minor peaks, Hb M and Hb m, respectively.

F. Shishikura et al. / Comparative Biochemistry and Physiology, Part B 142 (2005) 258 – 268

detected in both purified and unpurified fractions, but nofurther purification was carried out because the otherhemoglobin peaks obtained were very poor and it was
impossible to characterize them further. Similar two peaks(Hb M and Hb m) were also detected in the case ofhemoglobin solution prepared from a five-year-old female(about 18 cm in body length and 500 g in mass: data notshown here).
The Alkyl Superose HR 5/5 column that we used first in
preparation of intact reptilian hemoglobin components(Shishikura, 2002; Kuwada et al., 2003) was used for
preparation of hemoglobin components from axolotl blood.
The separation pattern is superior to that obtained byisoelectric focusing (Hattingh and Bartels, 1973). The
chemistry of the separation is based on hydrophilicinteractions with the gel matrix and biological macromolecules. With the fraction, we are currently generating
crystals of the intact hemoglobin components, Hb M and Hbm, of the axolotl hemoglobin.
3.2. Three globin chains, sequences, and alignment
Like birds, reptiles and mammals, amphibians produce
different globin polypeptides during their life cycles(McCutcheon, 1936; McCutcheon and Hall, 1937; Gratzer
and Allison, 1960; Bunn and Forget, 1986). Among them,
the amphibians metamorphose dramatically, where thepattern of globin polypeptides of anurans (frogs and toads)
is replaced completely by that of adult globin polypeptides(Hentschel et al., 1979; Maples et al., 1988). Someurodeles, on the other hand, coexpress larval and adultglobin mRNAs in a single erythroid cell (Yamaguchi et al.,2000).

261

In contrast, Grasso et al. (1979) described at least 6– 7
globin polypeptides in the adult urodele T. cristatus, ofwhich Kleinschmidt et al. (1988) sequenced four chains
including two a globin chains and two h globin chains.
We purified one a and one h globin chain from theaxolotl Hb M and Hb m, respectively, and thensequenced some of the peptide fragments derived fromthe parent molecules digested with lysyl endopeptidase(chromatograms of peptide maps by lysyl endopeptidase
digestion are not shown here). Appendix 2 shows theresults of amino acid sequence analyses, in which thetwo a globin chains were distinct, but the two h globin
chains so far analyzed (only one sequence is shown here)
had identical sequences. This reminds us that the twohemoglobin components, B and C, of the adult R.
catesbeiana share a common h globin chain but havedifferent a globin chains (Tam et al., 1986; Smith et al.,1993). The number of globin polypeptides we found in
the adult axolotl erythrocytes confirmed the study ofDucibella (1974), who purified three globin subunits fromthe denatured hemoglobin solution of the neotenous adult,with apparent molecular weights of 15,000, 19,000 and
24,500 using sodium dodecyl sulfate-polyacrylamide gelelectrophoresis.
Using the primers listed in Appendix 1, cDNA-fragments were amplified by PCR. Fig. 2 shows one major
band and several very faint bands in each lane; each of
the major fragments was extracted from the agarose gelsand sequenced separately. Appendix 2 (A, B and C)
shows the results of nucleotide sequencing of twelve
cDNA-fragments providing enough information to determine complete nucleotide sequences of globin cDNAs.
The entire coding regions of the three kinds of adult A.

Fig. 2. Agarose gel electrophoresis (1.5% gels) of the PCR products amplified from three kinds of cDNAs using primers as listed in Appendix 1. A: RT-PCR,
B: Nested PCR, C: 3V RACE, and D: 5V cRACE. The major fragment in each lane, except for the M1 and M2 lanes, was extracted from the gel and sequenced.
PCR conditions: 30 cycles each consisting of 30 s at 94 -C for denaturation, 30 s at 60 -C for annealing, and 1 min at 72 -C for primer extension. M1 (100 bp
ladder) and M2 (pHY) are DNA markers.

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F. Shishikura et al. / Comparative Biochemistry and Physiology, Part B 142 (2005) 258 – 268

mexicanum globin genes have been deposited in the
GenBank/DDBJ/EMBL (under DDBJ accession nos:
AB185144 for aM globin, AB185145 for am globin,
AB185146 for h globin). The amino acid sequences
deduced from the cDNA nucleotide sequences wereidentical with those determined by peptide sequencing
analyses, whose complete primary structures are also shownin Appendix 2. Their sequences reinforce each other. The
nucleotide sequences of mature proteins are composed of
141 amino acid residues for aM globin, 143 for am globin,
and 146 for h globin. Boissel et al. (1980) reported an extra
residue for the a globin chain of the major component of A.
mexicanum compared with that of ordinary a globin chainsin tetrapods; however, this is not curious because one or
two longer residues were recently found in newts T.
cristatus a globin chain with 142 (Kleinschmidt et al.,1988) and Taricha granulosa a globin chain with 142

Residue No.

(Coates et al., 1977), and am of this study with 143 aminoacid residues.
Fig. 3 shows an alignment in which two previously
established globin sequences (Boissel et al., 1980;
AF308869) and three other globin chains of the present
study (aM, am and h) are comparable to those of human aand h globin chains. Hereafter we would like to use thetentative nomenclature of globins of the axolotl: a-1 for thea globin chain named by Boissel et al. (1980) and a-2 forthe a globin of AF308869. Compared with each other, fourgroups based on % homology can be detected: the firstgroup is 61– 70% homologous (am versus a-1; aM versus
am; aM versus a-1), the second group is 51 – 57%
homologous (am versus human a; aM versus human a; a1 versus human a), the third group is 47– 49% homologous(am versus a-2; aM versus a-2; a-1 versus a-2) and thefourth group is 28– 38% homologous (a-2 versus human a;1

αM globin
αm globin
α-1
α-2

31
*
*
* *
* *
**
*
--VLTAEDKANVKSAWDHIKGHEEAFGAEALCRMFDSEPVTKTYFGGK------DISEESMVPLSAGDKANVKAVWDHVKGHEEAFGADALHRCFKIDCQTQTYFPGK------DLNEGS-FKLSGEDKANVKAVWDHVKGHEDAFGHEALGRMFTGIEQTHTYFPDK------DLNEGS-MTLTAEDKALVVVLWGKIAGHTDALGGEALDRLFACFGQSRTYFSHF------DLSPGS

β globin-VHLTAEERKDVGAILGKVN--VDALGGQCLARLMCVYPWSRRYFPDFGDMSTCDAICHN

Helix position## ##o ooo$$ $$$ $--VLSPADKTNVKAAWGKVGAHAGEYDAEALERMFLSFPTTKTYFPHF------DLSHGS##o ooo$$ $ $-VHLTPEEKSAVTALWGKVN--VDEVGGEALGRLLVVYPWTQRFFESFGDLSTPDAVMGN
$$
NH1
B1
C1
CD1
D1
E1

Residue No.

61

Human αHuman βαM globin
αm globin
α-1
α-2

91
** **
* * *
*
*
* *** *SYLHSHGKKVMCALTNAVAHIDNIEACLDKLSDTHAHELMVDPTNFPRLGHNILLVIGIH
AFLHSHGKKVMSALTNAVAHIDDLEAALSKLIDKHAHDLMVDPANFVLLNHHILAVLAMH
FALHSHGKKVMGALSNAVAHIDDLEATLVKLSDKHAHDLMVDPAEFPRLAEDILVVLGFH
ADVKRHGGKVLNAVGEAAKHIDDLDMGLSKLSDLHAYNLRVDPGNFQLLSHCIQTVLAAH

β globin

ARVLAHGAKVMRSVCEATKHLDNLQEYYADLSSTHCLKLFVDPQNFKLFGRIVVVCLAQT

Human α

#

##

#

##

# #
## #
$$ $$$$oo oo
AQVKGHGKKVADALTNAVAHVDDMPNALSALSDLHAHKLRVDPVNFKLLSHCLLVTLAAH
#

#

#

##

#

##

Human βHelix position

# #
## #
$$$ $
$ o
o oo
PKVKAHGKKVLGAFSDGLAHLDNLKGTFATLSELHCDKLHVDPENFRLLGNVLVCVLAHH
EF1
F1
FG1
G1

Residue No.

121

αM globin
αm globin
α-1
α-2

149
*
*
*
MPQLLTCAMHCSLDKFLCQVAEVLTSKYR (This study, AB185144/DDBJ)LPQLFTPANHRSLDKFLHTVMRCLISKYR (This study, AB185145/DDBJ)LPAKFTYAVQCSIDKFLHVTMRLCISKYR (P02015/Swiss-Prot)
FPADFTPQAQAAWDKFLAAVSGVLTSKYR (Q90ZA5/Swiss-Prot, AF308869/DDBJ)

β globin

LQTEFTWHKQLAFEKLMRAVAHALSHSYH

Human αHuman βHelix position#oo ooo oo o$$
LPAEFTPAVHASLDKFLASVSTVLTSKYR#o oooo oo o$$
FGKEFTPPVQAAYQKVVAGVANALAHKYH
GH1 H1HC1(This study, AB185146/DDBJ))(Nagel, 1995)(Nagel, 1995)

Fig. 3. Alignment of the amino acid sequences of five kinds of globins from A. mexicanum. The 26 amino acid residues conserved in the fivekinds of globins are shown by asterisks in the upper part of the alignment. The residue number is an arbitrary number with the numbering
beginning from the N-terminal of the globins with the longest N-terminal extension. FHelix position_ refers to the helix position in human h globin(Nagel, 1995). The symbols refer to those residues functionally important in the human hemoglobin molecule (Fermi, 1975): #, residues involvedin contact with the heme group; o, residues involved in contacts between subunits a1 and h1; $, residues involved in contacts between subunits a1
and h2.

F. Shishikura et al. / Comparative Biochemistry and Physiology, Part B 142 (2005) 258 – 268

am versus h; aM versus h; a-1 versus h). Watt et al. (1980)
reported that the tadpole h chain of R. catesbeiana
differed greatly from that of the adult frog; only about
50% of the residues were identical. Based on theassumption of the overall rate of change in hemoglobin
as about 1% per 3.3 million years (Wilson et al., 1977),
they estimated a 50% difference corresponded to about
165 million years. Our findings, together with molecular
relationships shown in Fig. 4A and B, suggest that thethree a types of globins of the first group, aM, am anda-1, which are homologous globin polypeptides in thesame organism are caused by gene duplications of the263

a globin genes, and the second group, whose
homology was estimated to be about 50%, may haveoccurred at least as long ago as the common ancestor
to the axolotl and humans, dating about 165 million
years ago.
Of considerable importance in hemoglobin function are
the two contact regions between the subunits designateda1h1 and a1h2, and heme contact residues (Perutz et al.,
1968; Fermi, 1975). In mammalian hemoglobin, oxygenation is accompanied by large changes at the a1h2 contact
and relatively small changes at the a1h1 contact (Perutz,1970). To gain information on axolotl hemoglobin, we

Fig. 4. Phylogenetic tree based on primary structures of 22 globins of tetrapods (A) and 43 globins of amphibians (B), constructed from 1000 bootstrap
replications by the neighbor-joining method (Saitou and Nei, 1987). The scale shown in the upper left segment of the tree represents the evolutionary
distances, given as the average number of substitutions per site. The scores shown at each node represent bootstrap values. The scientific names andaccession numbers in data banks are as follows: African clawed frog: Xenopus laevis (larval h-1; P02137, larval h-2; P02133, a-1; P02012, a-2; P02013; aT3; P06636, a-T4; P06637, a-T5; P06638, h-1; P02132, h-2 (fragment); Q91702); Andean frog: Telmatobius peruvianus (a-2 (fragment); P83113; h-2;
P83114); Asian toad: Bufo bufo gargarizans (a; Q8AXX7); axolotl: A. mexicanum (aM; AB185144, am; AB185145, a-1; P02015, a-2; Q90ZA5, h;
AB185146); bullfrog: Rana catesbeiana (larval a-III; P02011, larval h-III; P02136, h; P02135, a-B; P51465, a-C; P55267, a (heart muscle); P02022);˙
chicken: Gallus gallus (a; P01994, kV; P02007, aD; P02001, h; P02112, q; P02128, U; P02127; edible frog: Rana esculenta (h; P02134); great crested newt:
Triturus cristatus (a-1; P10783, a-2; P10784, h-1; P10785, h-2; P10786); hagfish: Myxine glutinosa (P02209); human: Homo sapiens (a; P69905, ~; P02008,
h; P68871, y; P02042, q P02100, g A; P69891); Iberian ribbed newt: Pleurodeles waltlii (larval a; P11896, a-1; P06639, a-2 (fragment); P06640); Japanese˙salamander: Hynobius retardatus(larval a; Q9PVL9, larval h; Q9PVL8, adult h; Q9PVL7; Kenyan clawed frog: Xenopus borealis (a-1; P07430, a-2;
P07431, h-1; P07432, h-2; P07433); lamprey: Lampetra fluviatilis (P02207); roughskin newt: Taricha granulosa (a P02014); tortoise: Geochelone nigra (a;P83135, aD; P83124, h; P83123); Western clawed frog: Xenopus tropicalis (laval h-2; P08423, a; P07428, a-3; P08422, h-1; P07429). Axolotl globins arelabeled with a symbol (*).

264

F. Shishikura et al. / Comparative Biochemistry and Physiology, Part B 142 (2005) 258 – 268

compared the appropriate amino acid residues with those
involved in subunit interactions (a1h1 and a1h2) as wellas in heme contacts as compared with amino acidresidues responsible for subunit interactions and heme
contacts of human deoxyhemoglobin described by Fermi(1975). As shown in Fig. 3, seven and eight out of 27
amino acid residues were variant at the a1h2 contacts of
Hb M and Hb m, respectively, whereas many moredifferences (18 and 17 residues) were apparent at the
a1h1 contacts of Hb M and Hb m. Such a marked
difference in the a1h1 contacts compared to that at the
a1h2 contacts has been also reported for X. laevis
hemoglobin (Knöchel et al., 1985; Banville and Williams,
1985). The heme contact residues, on the contrary, were
well conserved compared with those detected in humanglobins (Fermi, 1975): 13 residues (81.3%) and 12 residues(75.0%) were invariant for aM and am or h globins,
respectively.
In general, the Bohr effect in human hemoglobin has
been postulated to involve the breakage of at least four salt
bridges on oxygenation (Perutz, 1970). On this basis, thetwo salt linkages between the protonated NH2-terminalgroup of the valine of one a chain and the COOH-terminalof the other a chain in the deoxyhemoglobin tetramer
carboxyl group and between the guanidino group at
HC3(141)a1 and the aspartic acid at position 9 of the H
helix of a2 globin chain, designated Asp H9(126)a2, areconserved. However, the other two salt linkages between
the HC3(146)h imidazole group and the Asp (94)h in thesame chain and between the carboxyl group of HC3(146)hand the q-NH3+ of the lysine at C6(40)a are replaced withother amino acid residues. These findings may account inlarge part for the decreased Bohr effect on the axolotl
hemoglobin compared with that of human hemoglobin(Amiconi et al., 1970; Hattingh and Bartels, 1973; Banvilleand Williams, 1985). It is worth noting that Lys C6(40) of
the aM chain is still conserved, compared with other atypes of axolotl globins. Although Boissel et al. (1980)
reported the replacement of histidine at H5(122)a byglutamine in axolotl, we detected that the amino acidresidue at H5(122) of the two a chains, aM and am, areunchanged, as reported in humans. Hence, it is advantageous to present multiple globins in situations where livingorganisms need to express a potential globin for adaptation
to changes in surroundings or changes of life cycles, inparticular, in amphibians (Hutchison et al., 1976; Perutz,1983).
In addition, Boissel et al. (1980) found a proline at
G6(107)a in the axolotl. Maruyama et al. (1980) notedthat the proryl residue at this position causes distortion of
the G helix of hemoglobin of Glycera dibranchiata, anannelid (Padlan and Love, 1974). We also detected aproline at G6 of aM globin rather than am globin of theaxolotl. To confirm the above estimation of changes inthe tertiary structures, a crystallographic investigation isrequired.

3.3. Molecular Trees
The gene family of X. laevis globin consists of twelve
genes in two clusters, each containing larval and adult a andh genes in a symmetrical arrangement (Hosbach et al.,1983). In the urodeles, we sequenced only three globin
cDNAs from an adult axolotl, A. mexicanum, and analyzed
their phylogenetic positions and relationships on molecular
trees including representatives of tetrapodes (Fig. 4A) and,in particular, amphibians (Fig. 4B).
Fig. 4A shows that a types of axolotl globins (aM, am
and a-1, but not a-2) make a group, which is more closely
related to the adult a type globins of amniotes (tortoises,
birds and mammals) than the aD type globins of some
reptiles and birds or embryonic globins of birds and humans,
kV and ~ (Chapman et al., 1980; Aschauer et al., 1981). The
aD globin is one of the globin subunits of the hemoglobin D
tetramer (aD
2 h2), which was first found in birds as a minor
component of the embryonic and adult definitive erythrocytes (Hagopian and Ingram, 1971), and also found in some
reptilians (Abbasi et al., 1988; Ruchnagel and Braunitzer,
1988; Gorr et al., 1998; Shishikura and Takami, 2001). On
the contrary, the a-2 globin (AF308869) was assigned to anancestral type of embryonic globins since human ~ and birdkV globins split off.
Fig. 4B shows the molecular relationship of 43 globin
sequences of amphibians, the members of which seem to bealmost all the known sequences of amphibians stored in the
Swiss-Prot data bank at the present time (release 45.5). It is
not unreasonable to predict that the split of the adult types ofglobins from the larval types or vice versa might besignificant for consideration of the evolution of vertebrate
globin genes since each of the ancestor genes of a and hglobins first duplicated itself into the four main clusters intotal, and thereafter further divergences occurred, eachbranching into anuran species and urodele species.
Finally, to compare the distribution pattern of the fiveglobin genes of the axolotl with that of X. laevis, R.
catesbeiana or other urodele species (Fig 4B), we canpredict that another two to four globin genes will be
expressed in either larval stages or neotenous adult stages.
However, it is necessary to investigate more currently
unknown globin genes that will be equivalent to the uniquegene arrangements reported in Xenopus and human globin
gene families.

Acknowledgements
We thank Indiana University Axolotl Colony for thecontinuous supply of axolotl. This work was supported inpart by Nihon University Joint Research Grant (2005) andthe ‘‘High-Tech Research Center’’ Project for Private
Universities and a matching fund subsidy from MEXT
(Ministry of Education, Culture, Sports, Science andTechnology), 2000 – 2004.

F. Shishikura et al. / Comparative Biochemistry and Physiology, Part B 142 (2005) 258 – 268

265

Appendix 1
Appendix 1A. Oligonucleotide primers used in the first PCR (RT-PCR) of globin cDNAs
αΜ –N# of amino acid residue
Amino acid sequence
Nucleotide sequence

1Vg

Forward# of amino acid residue
Amino acid sequence
Nucleotide sequence

1
M
a t

Forward# of amino acid residue
Amino acid sequence
Nucleotide sequence

6Eg

Forward
α

m –N

β –N

t

a

n

2
L
y

g

2
V
g t

r

7
E
g

t

a

n

3
T
a

c

4
A
n g

n

3
P
c

c

4
L
y

r

8
R
m g

n

9
K
n a

5
E
g

c

n

t

5
S
n w

a

r

a

s

10
D
g a

r

6
D
g

7
K
y a

n

6
A
g c

a

11
V
y g t

a

7
G
g g

n

12
G
n g g

Appendix 1B. Oligonucleotide primers used in 5' cRACE amplification of globin fragments

Reverse# of amino acid residue
Amino acid sequence
Nucleotide sequence

A consensus sequence for three kinds of globins; αM, αm and β
65
64
63
62
61
60
M
V
K
K/A
G
H
c a t c a c y t t s k y k c c r y

Reverse# of amino acid residue
Amino acid sequence
Nucleotide sequence

41
T
g t c

Reverse# of amino acid residue
Amino acid sequence
Nucleotide sequence

44
Y
a g

Reverse# of amino acid residue
Amino acid sequence
Nucleotide sequence

Phosphated

SP– αM

SP– αm

SP– β

t

t

40
K
t g

43
T
g t c

40
R
c t c

t

39
T
g t t
42
Q
g a

39
R
c g t

g

38
V
a c a

41
T
g t c
38
S
a c

t

37
P
g g c
40
Q
g g

37
W
c a g

g

36
E
c c

t

39
C
c a g

36
P
g g a

t

35
S
g a g

t

38
D
c g

37
I
t c

t

35
Y
a c

t

g

a

a

34
V
c a

34
D
c
36
K

c

Appendix 1C. An oligonucleotide primer used in 3' RACE amplification of globin fragments
3' RACE
Forward# of amino acid residue
Amino acid sequence
Nucleotide sequence

A consensus sequence for three kinds of globins; αM, αm and β
60
61
62
63
64
65
H
c

a

y

G
g

g m

K/A
r m

s

K
a a

r

V
g

t

g

M
a

All primers were synthesized with a tag pUC/M13 sequencing primer (forward 17-mer: 5'-GTAAAACGACGGCCAGT-3', or reverse 17-mer: 5'-CAGGAAACAGCTATGAC-3').

Appendix 2
Appendix 2A. Primary structure and nucleotide sequence of αM–globin of Ambrystoma mexicanum# of a. a. residue
Amino acid seq.
Nucleotide seq.

# of a. a. residue
Amino acid seq.
Nucleotide seq.

1
V L

5
T A E
N-terminal

10
D K A N V KS15A W D HI

20
K G H E

E

A

F

25
G A E

A L

30
C

k-1
k-2
gtg ctg act gct gag gac aag gcc aat gtg aag tcc gcg tgg gac cac atc aaa ggg cac gag gaa gcg ttt ggt gca gaa gcg ctt tgc
RT-PCR
5' cRACE
Nested PCR
35
40
45
50
55
60
S E P V T K T Y F G G K D I S E E S S Y L H S H G K
k-2
k-3
k-4
agg atg ttt gac tcg gag cct gta acc aag acc tac ttt ggc ggc aag gac atc tcc gaa gaa agc tct tac ctg cac agc cac ggc aag
RT-PCR
5' cRACE
Nested PCR
R M F

D

(continued on next page)266

F. Shishikura et al. / Comparative Biochemistry and Physiology, Part B 142 (2005) 258 – 268

Appendix 2 (continued)

Appendix 2A# of a. a. residue
Amino acid seq.
Nucleotide seq.

# of a. a. residue
Amino acid seq.

K

V M C

65
A L

T

70
N A V A H

I

75
D N I

E

A C

80
L D K

L

85
D T

S

H A H

90
E

aag gtg atg tgc gcg ctg acc aat gcc gtc gcc cac ata gac aac atc gaa gcc tgc ctg gac aaa ctc agc gac acg cac gcc cac gag
RT-PCR
3' RACEL M V D

95
P T

N

F

P

100
R L

G H N

105
I L

L

V

I

110
G I

H M P

115
Q L

L

T

C

120
A

Nucleotide seq.

ctg atg gtc gac ccc acc aac ttc cca agg ctg ggc cat aat atc ttg ctt gtt atc ggc atc cac atg cca cag ctg tta acc tgc gcc
RT-PCR
3' RACE# of a. a. residue
Amino acid seq.

135
140
V A E V L T S K Y R
k-5
atg cac tgc tcg ctg gac aag ttc ctg tgc cag gtg gca gaa gtg ctg acc agc aag tac cgt taa
RT-PCR
3' RACE

Nucleotide seq.

M H C

S

125
L D K

F

L

130
C Q

Appendix 2B. Primary structure and nucleotide sequence of α–globin of Ambrystoma mexicanum# of a. a. residue
Amino acid seq.
Nucleotide seq.

# of a. a. residue
Amino acid seq.
Nucleotide seq.

1
M V

5
10
15
20
25
30
P L S A G D K A N V K A V W D H V K G H E E A F G A D A
N-terminal
k-3
k-2
k-1
atg gtg ccg ctc tcg gca ggg gac aag gcg aac gtg aaa gcg gtc tgg gac cat gtg aag ggc cac gag gag gct ttc ggc gca gat gcg
RT-PCR
5' cRACE
Nested PCR
35
40
45
50
55
60
H R C F K I D C Q T Q T Y F P G K D L N E G S A F L H S H
k-3
k-4
tta cac agg tgt ttt aag atc gac tgc cag act cag acc tac ttt cca ggc aag gac ctg aac gaa ggc tcg gcc ttt ctg cac agt cac
L

RT-PCR
5' cRACE
Nested PCR# of a. a. residue
Amino acid seq.

G K K

65
V M S

A L

70
75
N A V A H I D D L

T

E

80
A A L

S

K

85
L I

90
D K H A

Nucleotide seq.

gga aag aag gtg atg agt gcc tta act aac gcc gtc gcc cat atc gac gac ctt gag gct gcc ctc agc aag ctg atc gac aaa cat gcc
RT-PCR
3' RACE# of a. a. residue
Amino acid seq.

95
H D L M V D

Nucleotide seq.

cat gac ctc atg gtg gat cct gca aac ttt gtt ctt ctt aat cat cat atc ctg gca gtc ctt gcc atg cac ctg ccc cag ctc ttc aca

P

A N

100
F V L

L

Nucleotide seq.

L

D K

130
F L

L

110
A V L

A M H

135
140
V M R C L I S K Y R
k-5
cct gca aac cac cgc tct ttg gac aag ttc ttg cat aca gtg atg cgt tgt ttg att agc aaa tac cgt taa
RT-PCR
3' RACEP125
A N H R S

I

RT-PCR

3' RACE# of a. a. residue
Amino acid seq.

105
N H H

H

T

115
L P

Q

L

F

120
T

F. Shishikura et al. / Comparative Biochemistry and Physiology, Part B 142 (2005) 258 – 268

267

Appendix 2 (continued)

Appendix 2C. Primary structure and nucleotide sequence of β–globin of Ambrystoma mexicanum# of a. a. residue
Amino acid seq.
Nucleotide seq.

# of a. a. residue
Amino acid seq.
Nucleotide seq.

# of a. a. residue
Amino acid seq.
Nucleotide seq.

# of a. a. residue
Amino acid seq.
Nucleotide seq.

# of a. a. residue
Amino acid seq.
Nucleotide seq.

1
V H

5
10
15
20
25
30
L T A E E R K D V G A I L G K V N V D A L G G Q C L A R
N-terminal
k-1
gtt cac ctg aca gcc gaa gaa cgc aag gac gtc ggt gcg att tta ggc aaa gtt aat gta gac gct ctc gga ggt caa tgc ctt gca agg
RT-PCR
5' cRACE
Nested PCR
35
40
45
50
55
60
V Y P W S R R Y F P D F G D M S T C D A I C H N A R V
N-terminal
ctg atg tgt gtg tat ccc tgg tca cgg aga tac ttc ccg gat ttc ggt gac atg tcc acc tgt gat gct atc tgc cat aac gcg agg gtc
RT-PCR
5' cRACE
Nested PCR
L M C

L

A H G

65
A K

V M R

70
75
80
85
90
S V C E A T K H L D N L Q E Y Y A D L S S
k-2
k-3
ctc gcc cat ggc gcc aaa gtg atg aga tcc gtc tgt gag gcc acc aag cac ctg gac aac ctc caa gaa tac tat gcc gac ctg agt agc
RT-PCR
3' RACE

T

H C

95
K L

F

L

V D

100
P Q

N

F

K

105
L F

G R

I

110
V V V C

L

115
A Q

T

L

Q

120
T

act cat tgc ctg aag ctc ttt gtg gac ccg caa aac ttc aag ctt ttt ggc cgc att gtg gtc gtg tgc ctg gcc caa acc ttg cag acc
RT-PCR
3' RACEE

F125
T W H K Q

L

A

130
F E

K

135
140
L M R A V A H A L

S

H

S

145
Y H

gaa ttt aca tgg cat aag cag ctg gcc ttc gaa aag ttg atg cgg gcc gtg gca cat gct ctg agc cac agc tac cac tga
RT-PCR
3' RACE

References
Abbasi, A., Wells, R.M.G., Brittain, T., Braunitzer, G., 1988. Primarystructure of the hemoglobin from Sphenodon (Sphenodon punctatus,
Tuatara, Rynchocephalia). Evidence for the expression of a D -gene.
Biol. Chem. Hoppe-Seyler 369, 755 – 764.
Aggarwal, S., Riggs, A., 1969. The hemoglobins of the bullfrog, Ranacatesbeiana: I. Purification, amino acid composition, and oxygen
equilibria. J. Biol. Chem. 244, 2372 – 2383.
Amiconi, G., Brunori, M., Antonini, E., Sorcini, M., Tentori, L., 1970. The
haemoglobin of amphibia IX. Functional properties of haemoglobinfrom Ambystoma tigrinum tigrinum and Mexican axolotl. Int. J.
Biochem. 1, 582 – 588.
Aschauer, H., Sanguansermsri, T., Braunitzer, G., 1981. EmbryonaleHämoglobine des Menschen: Die Primärstruktur der ~-Ketten. HoppeSeyler Z. Physiol. Chem. 362, 1159 – 1162.
Baldwin, T.O., Riggs, A., 1974. The hemoglobin of the bullfrog, Ranacatesbeiana. Partial amino acid sequence of the h chain of the major
adult component. J. Biol. Chem. 249, 6110 – 6118.
Banville, D., Williams, J.G., 1985. The pattern of expression of the
Xenopus laevis tadpole a-globin genes and the amino acid sequence ofthe three major tadpole a-globin polypeptides. Nucleic Acids Res. 13,5407 – 5421.
Boissel, J.-P., Wajcman, H., Labie, D., 1980. Hemoglobins of an amphibia,
the neotenous Ambystoma mexicanum. Complete amino-acid sequenceof the a chain of the major component using automatic solid-phase
Edman degradation. Eur. J. Biochem. 103, 613 – 621.
Bunn, H.F., Forget, B.G., 1986. Hemoglobin: Molecular, Genetic andClinical Aspects. Saunders, W.B., Co., Philadelphia.
Chapman, B.S., Tobin, A.J., Hood, L.E., 1980. Complete amino acidsequences of the major early embryonic a-like globins of the chicken.
J. Biol. Chem. 255, 9051 – 9059.
Chauvet, J.-P., Acher, R., 1972. Phylogeny of hemoglobis. h chain of frog
(Rana esculenta) hemoglobin. Biochemistry 11, 916 – 927.
Coates, M., Brimhall, B., Stenzel, P., Hermodson, M., Gibson, D., Jones,
R.T., Vedvick, T., 1977. a-chain sequence of newt haemoglobin(Taricha granulosa). Aust. J. Biol. Sci. 30, 1 – 19.
Dorn, A.R., Broyles, R.H., 1982. Erythrocyte differentiation during themetamorphic hemoglobin switch of Rana catesbeiana. Proc. Natl.
Acad. Sci. U. S. A. 79, 5592 – 5596.
Ducibella, T., 1974. The occurrence of biochemical metamorphic events
without anatomical metamorphosis in the axolotl. Dev. Biol. 38, 175 – 186.
Elli, R., Giuliani, A., Tentori, L., Chiancone, E., Antonini, E., 1970. The
hemoglobin of amphibia-X. Sedimentation behaviour of frog, triton andaxolotl hemoglobins. Comp. Biochem. Physiol. 36, 163 – 171.
Fermi, G., 1975. Three-dimensional Fourier synthesis of human deoxyhaemoglobin at 2.5 Å resolution: refinement of the atomic model.
J. Mol. Biol. 97, 237 – 256.
Friedman, M., Kruill, L.H., Cavins, J.F., 1970. The chromatographic
determination of cystine and cysteine residues in proteins as S-h-(4pyridylethyl)cysteine. J. Biol. Chem. 245, 3868 – 3871.

268

F. Shishikura et al. / Comparative Biochemistry and Physiology, Part B 142 (2005) 258 – 268

Frohman, M.A., Dush, M.K., Martin, G.R., 1988. Rapid production of fulllength cDNAs from rare transcripts: amplification using a single genespecific oligonucleotide primer. Proc. Natl. Acad. Sci. U. S. A. 85,
8998 – 9002.
Gorr, T.A., Mable, B.K., Kleinschmidt, T., 1998. Phylogenetic analysis of
reptilian hemoglobins: trees, rates, and divergences. J. Mol. Evol. 47,
471 – 485.
Grasso, J.A., Casale, G.P., Chromey, N.C., 1979. Multiple hemoglobins in
Triturus cristatus: their degradation by sulfhydryl compounds. Comp.
Biochem. Physiol. B 63, 93 – 101.
Gratzer, W.B., Allison, A.C., 1960. Multiple hemoglobins. Biol. Rev. 35,
459 – 495.
Hagopian, H.K., Ingram, V.M., 1971. Developmental changes of erythropoiesis in cultured chick blastoderms. J. Cell Biol. 51, 440 – 451.
Hamada, K., Sakai, Y., Tsushima, K., Shukuya, R., 1966. Biochemical
metamorphosis of hemoglobin in Rana catesbeiana: III. Molecularchange of hemoglobin during spontaneous metamorphosis. J. Biochem.
60, 37 – 41.
Hattingh, J., Bartels, H., 1973. The oxygen affinity of axolotl bloodand haemoglobin before and after metamorphosis. Respir. Physiol.
18, 1 – 13.
Hentschel, C.C., Kay, R.M., Williams, J.G., 1979. Analysis of Xenopus
laevis globins during development and erythroid cell maturation and theconstruction of recombinant plasmids containing sequences derived
from adult globin mRNA. Dev. Biol. 72, 350 – 363.
Hosbach, H.A., Wyler, T., Weber, R., 1983. The Xenopus laevis globin genefamily: chromosomal arrangement and gene structure. Cell 32, 45 – 53.
Hutchison, V.H., Haines, H.B., Engbretson, G., 1976. Aquatic life at highaltitude: respiratory adaptations in the Lake Titicaca frog, Telmatobius
culeus. Respir. Physiol. 27, 115 – 129.
Jeanmougin, F., Thompson, J.D., 1998. Multiple sequence alignment with
Clustal X. Trends Biochem. Sci. 23, 403 – 405.
Jeffreys, A.J., Wilson, V., Wood, D., Simons, J.P., 1980. Linkage of adult aand h-globin genes in X. laevis and gene duplication by tetraploidizaiton. Cell 21, 555 – 564.
Kay, R.M., Harris, R., Patient, R.K., Williams, J.G., 1983. Complete
nucleotide sequence of a cloned cDNA derived from the major adult aglobin mRNA of X. laevis. Nucleic Acids Res. 11, 1537 – 1542.
Kleinschmidt, T., Sgouros, J.G., Braunitzer, G., 1988. The primarystructures of the major and minor hemoglobin components of the great
crested newt (Triturus cristatus, Urodela, Amphibia). Biol. Chem.
Hoppe-Seyler 369, 1343 – 1360.
Knöchel, W., Meyerhof, W., Stalder, J., Weber, R., 1985. Comparative
nucleotide sequence analysis of two types of larval h-globin mRNAs of
Xenopus laevis. Nucleic Acids Res. 13, 7899 – 7908.
Koussoulakos, S., Kaparos, G., Stathakos, D., 1986. Multiple hemoglobins
in Triturus cristatus: their study by analytical isolelectrofocussing.
Comp. Biochem. Physiol. B 83, 475 – 481.
Kuwada, T., Hasegawa, T., Satoh, I., Ishikawa, K., Shishikura, F., 2003.
Crystallization and preliminary X-ray diffraction study of hemoglobin
D from the Aldabra giant tortoise, Geochelone gigantea. Prot. Peptide
Letters 10, 422 – 425.
MacLean, N., Jurd, R.D., 1971. The haemoglobins of healthy and anemic
Xenopus laevis. J. Cell Sci. 9, 509 – 528.
Maples, P.B., Dorn, A.R., Broyles, R.H., 1983. Embryonic and larval
hemoglobins during the early development of the bullfrog, Ranacatesbeiana. Dev. Biol. 96, 515 – 519.
Maples, P.B., Palmer, J.C., Broyles, R.H., 1988. Determination ofhemoglobin expression patterns in erythroid cells of Rana catesbeiana
tadpoles. Comp. Biochem. Physiol. B 91, 755 – 762.

Maruyama, T., Watt, K.W.K., Riggs, A., 1980. Hemoglobins of the tadpoleof the bullfrog, Rana catesbeiana. Amino acid sequence of the a chainof a major component. J. Biol. Chem. 255, 3285 – 3293.
McCutcheon, F.H., 1936. Hemoglobin function during the life history of thebullfrog. J. Cell. Comp. Physiol. 8, 63 – 81.
McCutcheon, F.H., Hall, F.G., 1937. Hemoglobin in amphibia. J. Cell.
Comp. Physiol. 9, 191 – 197.
Moss, B., Ingram, V.M., 1968. Hemoglobin synthesis during amphibian
metamorphosis: I. Chemical studies on the hemoglobins from larval andadult stages on Rana catesbeiana. J. Mol. Biol. 32, 481 – 492.
Nagel, R.L., 1995. Disorders of hemoglobin function and stability. In: Handin,
R.I., Lux, S.E., Stossel, T.P. (Eds.), Blood: Principles and Practice of
Hematology. J.B. Lippincott Co, Philadelphia, pp. 1591 – 1644.
Padlan, E.A., Love, W.E., 1974. Three-dimensional structure of hemoglobin from the polychaete annelid Glycera dibranchiata, at 2.5 A
resolution. J. Biol. Chem. 249, 4067 – 4078.
Perutz, M.F., 1970. The Bohr effect and combination with organic
phosphates. Nature 228, 734 – 739.
Perutz, M.F., 1983. Species adaptation in a protein molecule. Mol. Biol.
Evol. 1, 1 – 28.
Perutz, M.F., Muirhead, H., Cox, J.M., Goaman, L.C.G., 1968. Threedimensional Fourier synthesis of horse oxyhaemoglobin at 2.8 Å
resolution: the atomic model. Nature 219, 131 – 139.
Rüchnagel, K.P., Braunitzer, G., 1988. The primary structure of the major
and minor hemoglobin component of adult Western painted turtle(Chrysemys picta bellii). Biol. Chem. Hoppe-Seyler 369, 123 – 131.
Saitou, N., Nei, M., 1987. The neighbor-joining method: a new method forreconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406 – 425.
Shishikura, F., 2002. The primary structure of hemoglobin D from the
Aldabra giant tortoise, Geochelone gigantea. Zool. Sci. 19, 197 – 206.
Shishikura, F., Snow, J.W., Gotoh, T., Vinogradov, S.N., Walz, D.A., 1987.
Amino acid sequence of the monomer subunit of the extracellularhemoglobin of Lumbricus terrestris. J. Biol. Chem. 262, 3123 – 3131.
Shishikura, F., Takami, K., 2001. The amino acid sequence of the a- and hglobin chains of hemoglobin from the Aldabra giant tortoises, Geochelone gigantea. Zool. Sci. 18, 515 – 526.
Smith, D.J., Zhu, H., Kolatkar, P.R., Tam, L.-T., Baldwin, T.O., Roe, B.A.,
Broyles, R.H., Riggs, A.F., 1993. The hemoglobin of the bullfrog, Ranacatesbeiana. The cDNA-derived amino acid sequences of the a chainsof adult hemoglobins B and C: their roles in deoxygenation-induced
aggregation. J. Biol. Chem. 268, 26961 – 26971.
Tam, L.-T., Gray, G.P., Riggs, A.F., 1986. The hemoglobins of the bullfrog
Rana catesbeiana. The structure of the h chain of component C and therole of the a chain in the formation of intermolecular disulfide bonds.
J. Biol. Chem. 261, 8290 – 8294.
Tam, L.-T., Manning, D., Cox, D.J., Riggs, A.F., 1993. The hemoglobin of
the bullfrog, Rana catesbeiana. Deoxygenation-linked association oftetrameric components B and C to form the trimer BC2: sedimentation
analysis and oxygen equilibria. J. Biol. Chem. 268, 26972 – 26977.
Watt, K.W.K., Maruyama, T., Riggs, A., 1980. Hemoglobins of the tadpoleof the bullfrog, Rana catesbeiana. Amino acid sequence of the h chainof a major component. J. Biol. Chem. 255, 3294 – 3301.
Weber, R., Blum, B., Muller, P.R., 1991. The switch from larval to adultglobin gene expression in Xenopus laevis is mediated by erythoroid
cells from distinct compartments. Development 112, 1021 – 1029.
Wilson, A.C., Carlson, S.S., White, T.J., 1977. Biochemical evolution.
Annu. Rev. Biochem. 46, 573 – 639.
Yamaguchi, M., Takahashi, H., Wakahara, M., 2000. Erythropoiesis andunexpected expression pattern of globin genes in the salamander
Hynobius retradatus. Dev. Genes Evol. 210, 180 – 189.