AMER. ZOOL., 20:69-77 (1980)
Structure of Annelid High Molecular Weight
Hemoglobins (Erythrocruorins)1
ROBERT LEE GARLICK
Department of Zoology, The University of Texas at Austin,
Austin, Texas 78712
SYNOPSIS. The extracellular vascular hemoglobins (erythrocruorins) of annelids are polymeric oxygen carriers with molecular weights of approximately 3 x 106, or about 46 times
the molecular weight of a vertebrate hemoglobin tetramer. The molecule appears as a
dodecamer of 12 large submultiples arranged at the vertices of two regular hexagons, one
on top of the other in electron micrographs. The dimensions are about 250 A across the
face of the hexagon, and about 170 A in height. The molecular weight of a one-twelfth
submultiple is approximately 250,000. Biochemical studies suggest that each submultiple
contains 16 to 18 subunits and that the intact hemoglobin molecule contains approximately
200 subunits. Unlike vertebrate hemoglobin which contains one heme moiety for each
polypeptide chain, the annelid hemoglobins apparently contain one heme per 1.5 to 2.0
chains. The reasons for this lack of a 1:1 hemexhain stoichiometry are not known at
present. One possibility may be that it is the result of insufficient purification of the
hemoglobin. Alternatively, more than one globin chain might share a heme, certain globin
chains might lack the heme moiety and have a non-hemoglobin function, or certain globin
chains may lose their heme during purification of the hemoglobin. We are presently
determining the amino acid sequence of one globin chain of Lumbricus terrestris hemoglobin. This information should be helpful in understanding the structure of these interesting
polymers.
two alpha and two beta polypeptide chains,
of molecular weight ca. 15,500 each (Antonini and Brunori, 1971).
The invertebrate hemoglobins are either
extra- or intracellular. They vary enormously in molecular weight (Terwilliger,
1979). For example certain annelid hemoglobins from coelomic cells are monomeric
with molecular weights ca. 15—16,000
(Padlan and Love, 1968; Terwilliger and
Koppenheffer, 1973; Garlick and Terwilliger, 1974). In striking contrast are the
polymeric extracellular hemoglobins found
in certain annelids, arthropods, and molluscs which can have molecular weights in
the millions. The molecular weights of
most annelid extracellular hemoglobins
are ca. 3,000,000 (Svedberg and Hedenius, 1934; Antonini and Chiancone,
1977; Terwilliger, 1979).
INTRODUCTION
Hemoglobin is a heme-containing respiratory protein which binds molecular
oxygen at a respiratory surface (e.g., surface skin, gills, or lungs) where oxygen is
relatively plentiful and transports the oxygen to the inner tissues where it is utilized
in metabolism. In contrast with the vertebrate hemoglobins which have been extensively studied with regard to structure and
function, the invertebrate hemoglobins remain relatively unstudied. In this paper I
shall summarize experimental work on the
structures of some of the vascular hemoglobins of high molecular weight from annelids. These are also called erythrocruorins (Keilin and Hartree, 1951).
Vertebrate hemoglobin, carried within
circulating erythrocytes, is roughly spherical or tetrahedral in shape with dimensions of 64 A x 55 A x 50 A for horse
DISCUSSION
hemoglobin (Perutz et al, 1968). Most vertebrate hemoglobins have molecular Structure of the native hemoglobin
weights near 65,000 and are composed of
Svedberg and Eriksson (1933) were the
first to show that the annelid extracellular
vascular hemoglobins are very large mol1
From the Symposium on Respiratory Pigments presented at the Annual Meeting of the American So- ecules. They found that the hemoglobins
ciety of Zoologists, 27-30 December 1978, at Richmond, Virginia.
69
of Lumbricus terrestris and Arenicola marina
have molecular weights of 2,730,000 and
70
ROBERT LEE GARLICK
FIG. 1. Electron micrographs of Thelepus crispus and Pisla pacifica extracellular hemoglobins. A. T. crispus
hemoglobin. B. P. pacifica hemoglobin, face view. C. P. pacifica hemoglobin, edge view. D. One-twelfth submultiples of P. pacifica hemoglobin. The molecular dimensions for both native hemoglobins are 250 A across
the face of the molecule, and 170 A in height. Conditions of preparation are in the text.
2,850,000 respectively, and that these pigments have isoelectric points (pH 5.3 and
4.6) which are lower than those for vertebrate hemoglobins, which have isoelectric
points near pH 6.7 or higher. Svedberg
and Eriksson also found that the native 57
S hemoglobin from A. marina dissociates
at pH 8.9 to a mixture of 57 S and 10 S
aggregates.
Roche and co-workers (1960) examined
a number of annelid extracellular hemoglobins with the electron microscope. They
found that these pigments are composed
of 12 large spherical submultiples each
with diameters of 70 A, arranged at the
vertices of two regular hexagons, one on
top of the other. The two tiered hexagonal
structure apparently has a hollow central
channel. Figure 1 shows the fine structure
of hemoglobins from the marine polychaetes Pista pacifica and Thelepus crispus.
For a detailed description of this work see
Terwilliger et al. (1976). Figure 1A is a
view of T. crispus hemoglobin at pH 7.0
negatively stained with 1.5% phosphotungstic acid. Figure IB shows a six-fold
optical rotation of P. pacifica hemoglobin
in face view. Figure 1C is a four-fold optical rotation of an edge view of a single P.
pacifica hemoglobin molecule. The molecules in Figures IB and 1C were negatively
stained with 1% ammonium molybdate,
pH 4.5. The molecular dimensions for
both hemoglobins are 250 A across the
face of the molecule and 170 A in height.
The molecules shown in Figure 1 are char-
71
ANNELID HEMOGLOBIN STRUCTURE
-70A*
•240 A-
f
soA-
100A
•90A-
FIG. 2. Interpretations of electron micrographs of annelid extracellular hemoglobins, a. Arenicola marina
hemoglobin, face view. b. A. marina hemoglobin, edge view. c. A. marina hemoglobin one-twelfth submultiple.
d. Eumenia crassa hemoglobin one-twelfth submultiple. e. One-twelfth submultiple of Spirographis spallanzanii
chlorocruorin and of Lumbricus terrestris hemoglobin, f. Top view of Limnodrilus gotoi hemoglobin one-twelfth
submultiple. g. Edge view of L. gotoi hemoglobin submultiple. References are in the text.
acteristic of most annelid extracellular
hemoglobins (Roche et al., 1960; Levin,
1963; Roche, 1965). Figure ID illustrates
one-twelfth submultiples of P. pacifica
hemoglobin which were prepared by dialyzing the native protein against 0.1 M
Tris, 10 mM ethylene-diamine-tetraacetic
acid (EDTA), pH 7.0, prior to staining
with 1.5% phosphotungstic acid, pH 7.0.
Interpretations of electron micrographs of
annelid extracellular hemoglobins
Figure 2 illustrates ways in which various
workers have interpreted their electron
micrographs of annelid extracellular
hemoglobins. Figures 2a and 2b depict the
top view (2a) and edge view (2b) of Arenicola marina hemoglobin (Roche et al.,
1960; Roche, 1965). The one-twelfth submultiples of A. marina hemoglobin are
seen as spheres (Fig. 2c) of diameter 70 A,
which retain their spherical shape after
dissociation of the native hemoglobin at
alkaline pH (Roche, 1965).
On the other hand, Eumenia crassa
hemoglobin submultiples appear to Levin
(1963) as kite-shaped particles (Fig. 2d)
which are 100 A long by 80 A wide.
The submultiples of Spirographis spallanzanii chlorocruorin (Guerritore et al.,
1965) and Lumbricus terrestris hemoglobin
(Rossi-Fanelli et al., 1970) are described as
72
ROBERT LEE GARLICK
triangles (Fig. 2e) designated "A" submul- globin of Nephthys sp. (Wells and Dales,
tiples which are divisible into three "B" 1976) have electron dense material in the
subunits which are in turn tetrameric. central channel which may represent an
Thus the "A" submultiple would be a do- extra submultiple or submultiples. The exdecamer (12 subunits), and the intact pig- tra submultiple(s) would increase the numment would contain 144 polypeptide ber from 12 to 13 or 14. Van Bruggen and
chains. Waxman (1971), citing electron Weber (1974) suggest that the extra submicroscopy and biochemical evidence, sug- multiple gives 0. fulgida hemoglobin a
gests that the one-twelfth submultiple of slightly higher sedimentation coefficient
Arenicola cristata hemoglobin is tetrahedral than Arenicola marina hemoglobin, which
rather than triangular, and that the fourth lacks the dense central channel.
corner is hidden beneath the plane of the
Not all hemoglobin molecules from Euphotograph. Waxman's model states that zonus mucronata contain the electron dense
the A. cristata hemoglobin submultiple central core; those which do comprise 10—
contains four tetramers whose structures 15% of the total hemoglobin (Terwilliger
are stabilized by disulfide linkages (Wax- et al., 1977). They appear in the electron
man, 1971, 1975), and that the intact microscope like a dimeric stack of the mahemoglobin molecule contains 192 poly- jor fraction {i.e., four stacks of hexagons)
peptide chains.
and have an apparent molecular weight
6
The submultiple of Limnodrilus gotoi (6-7 x 10 daltons) twice that of6 the major
hemoglobin (Fig. 2f, g) is thought to be a hemoglobin fraction (3.2 X 10 daltons).
nonomer (nine subunits) which consists of Terwilliger et al. (1977) suggest that either
three stacks of three subunits each, in a there is indeed an additional submultiple
staggered array (Yamaghishi et al., 1966). (or two), or that the electron dense center
Figure 2f shows a top view of the proposed is caused by the trapping of stain between
L. gotoi submultiple, and Figure 2g illus- the two erythrocruorin monomers. Antrates the side view. According to the Ya- other possibility is that it is an optical armaghishi model the intact hemoglobin tifact caused somehow from looking at a
molecule contains 108 globin chains. In a dimeric stack rather than at a monomeric
later work Vinogradov et al. (1975) pro- unit.
pose that the subunit structure of this
Physical and chemical evidence for annelid
hemoglobin is more complex.
Terwilliger et al. (1976) report that the hemoglobin subunit structure
In addition to evidence from electron
one-twelfth submultiples of Eudistylia vancouveri chlorocruorin and of the hemoglob- microscopy there is additional physical eviins of Pista pacifica and Thelepus crispus ap-dence that annelid extracellular hemoglopear sometimes as kite-shaped particles and bins are composed of 12 major submultisometimes as isosceles triangles (Fig. 1), and ples which in turn are made up of smaller
have a central area of low electron density subunits. Svedberg and Eriksson (1933)
suggesting that the submultiple itself may first discovered that the 57 S hemoglobin
be hollow. The submultiples of P. pacifica from Arenicola marina dissociates to a 10 S
hemoglobin retain their shape after dis- submultiple at alkaline pH. Levin (1963)
sociation of the native molecule (Terwilli- and Weber (1970) later confirmed this
ger etai, 1976).
finding.
The majority of annelid extracellular
Table 1 summarizes physical and chemhemoglobins show an apparently hollow ical data on the subunit structures of three
central channel when viewed in the elec- extracellular hemoglobins and one chlotron microscope (Fig. 1A, IB; Fig. 2a). In rocruorin. The pigments in Table 1 range
contrast, the hemoglobins from the poly- in molecular weight from 2.6 x 106 for
chaetes Oenone fulgida (Van Bruggen and Abarenicola pacifica hemoglobin (Garlick
Weber, 1974) and Euzonus mucronata (Ter- and Terwilliger, 1977) to 3.4 X 106 for
williger et al., 1977), and the high molec- that of Pista pacifica (Terwilliger et al.,
ular weight extracellular coelomic hemo- 19756). We determined the apparent mo-
ANNELID HEMOGLOBIN STRUCTURE
lecular weights of native pigments by analytical ultracentrifugation and/or column
chromatography on Sepharose 4-B using
0.1 M Tris-HCl, pH 7.0, 10 mM in MgCl2
as a buffer. The presence of divalent cations is needed to prevent dissociation of
most annelid extracellular hemoglobins.
We determined the sizes of one-twelfth
submultiples after dialyzing each pigment
against an alkaline buffer (pH 10) containing 10 mM EDTA. The pigments were
then chromatographed on Sephadex G200 or BioGel A-1.5 M, or were subjected
to analytical ultracentrifugation. The apparent molecular weights for submultiples
range from 235,000 daltons for A. pacijica
hemoglobin to 280,000 daltons for P. pacifica hemoglobin.
We determined the apparent molecular
weights of the polypeptide chains by several methods. Thelepus crispus hemoglobin
(Garlick and Terwilliger, 1975) dissociates
to a 16,000 dalton protein on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The size of the subunit of this hemoglobin is also 16,000 from
Sephadex G-200 gel chromatography in 6
M urea plus 0.1 M 2-mercaptoethanol.
Mercaptoethanol (2-ME) disrupts disulfide
linkages within and between protein
chains. SDS-PAGE produces three protein
bands, corresponding to 15,700, 28,800,
and 59,000 daltons for Abarenicola pacijica
hemoglobin (Garlick and Terwilliger,
1977). When we made the SDS gels 8 M in
urea and included 2-ME, we saw only one
protein band corresponding to 15,800 daltons.
Pista pacijica extracellular hemoglobin
(Terwilliger et ai, 19756) and Eudistylia
vancouveri chlorocruorin (Terwilliger et al.,
1975a) each show two protein bands on
SDS-PAGE, corresponding to ca. 15,000
daltons and 30,000 daltons. Terwilliger
and co-workers (1976) also observed the
15,500 and 30,000 dalton bands for P. pacijica hemoglobin using the following procedures prior to SDS-PAGE: incubation of
the globin in 2% SDS plus 2% 2-ME or
dithiothreitol, or incubation of the globin
with 8 M urea plus 2% 2-ME and dialysis
against 2% SDS plus 2% 2-ME prior to
electrophoresis. Subunit heterogeneity was
73
present when P. pacijica hemoglobin was
subjected to sedimentation equilibrium ultracentrifugation in 6 M guanidine-HCl
plus 0.1 M 2-mercaptoethanol. Recently
Terwilliger (1980) dissociated P. pacijica
hemoglobin at neutral or alkaline pH in
the presence of 10 mM EDTA prior to
SDS-PAGE, and then he finds only the
15,500 dalton subunit on SDS-PAGE. Terwilliger (1978) has observed the same phenomenon for subunit dissociation of the
high molecular weight pigment of the
polychaete Serpula vermicularis.
Eudistylia vancouveri chlorocruorin, from
measurements in the analytical ultracentrifuge, dissociates to a 14,000 dalton subunit in 6 M guanidine-HCl plus 0.1 M 2ME. This pigment chromatographs as a
15,000 dalton protein on Sephadex G-200
in 8 M urea plus 0.1 M 2-ME (Terwilliger
etal., 1975a).
We have found that under the proper
conditions each of the four proteins in Table 1 will dissociate to a subunit of 1516,000 daltons, which is about the size of
a vertebrate hemoglobin polypeptide chain.
From these studies we have estimated that
the number of subunits in a one-twelfth
submultiple is probably 16-18, and that
the number of subunits in the intact hemoglobin molecule is 192-216. More accurate
subunit molecular weights will be obtained
from amino acid sequence analysis, which
is currently underway for earthworm
hemoglobin.
Other workers have studied the subunit
structures of a number of annelid high
molecular weight hemoglobins, usually
employing SDS-PAGE (Vinogradov et al.,
1976; Antonini and Chiancone, 1977). We
have found that SDS-PAGE can be a very
useful tool in attempting to determine subunit molecular weights, yet one might be
misled if SDS-PAGE is the only method
used.
The heme content oj annelid hemoglobins
The globin chain of each of the four pigments in Table 1 is of apparent molecular
weight 15-16,000, yet the measurements
indicate that each pigment contains one
mole heme per 22-30,000 grams pigment.
This lack of a one-to-one heme-polypep-
74
ROBERT LEE GARLICK
tide chain stoichiometry has been noted by
a number of workers (Antonini and Chiancone, 1977). For the vertebrate hemoglobins there is one heme moiety per polypeptide chain, which would correspond to
one mole heme per 15,500 grams hemoglobin. The annelid extracellular hemoglobins and chlorocruorins apparently
contain on the average 1 heme per 1.5-2.0
polypeptide chains. This phenomenon can
be explained in a number of ways. One
possibility is that this is an artifact of purification (Antonini and Chiancone, 1977).
For example, nearly all of the protein in
mammalian erythrocytes is hemoglobin.
After the cells are washed and lysed over
90% of the intracellular protein is hemoglobin. A one- or two-step purification
scheme ensures a pure hemoglobin preparation which contains one mole heme per
ca. 15,500 grams hemoglobin (i.e., one
heme per polypeptide chain). To obtain
annelid extracellular hemoglobins most
workers cut the worms into pieces in a neutral buffer solution, usually containing a
proteolytic enzyme inhibitor. The hemoglobin is extracted from the mixture usually by preparative ultracentrifugation or
by column chromatography. Both procedures separate molecules by size only. All
materials, including proteins and polynucleotides, in the three million dalton size
range would be assayed for heme content
along with the hemoglobin. The values for
heme content in Table 1 were obtained
after the pigments were purified on Sepharose 4-B, which separates molecules by
size.
Perhaps the apparent lack of stoichiometry between the number and molecular weights of globin chains and the actual
heme content is not an artifact of purification. Two polypeptide chains could
share one heme (Waxman, 1971, 1975).
The subunit sizes of Arenicola cristata
hemoglobin are 13,000 and 14,000 daltons, yet apparently there is one mole
heme per 26,000 grams hemoglobin.
Another hypothesis is that some globin
chains have a nonhemoglobin function,
such as helping to hold the aggregate together, and lack a heme. Perhaps the heme
is loosely held by the globin and some of
the heme separates from the globin during
hemoglobin purification. Terwilliger (1978)
notes that when dissociated subunits of the
high molecular weight respiratory pigment of Serpula vermicularis (contains both
protoheme and chlorocruoroheme) are
electrophoresed on polyacrylamide gels, a
colored material thought to be heme migrates with the tracker dye.
Certain human hemoglobin variants
have altered structures which affect the
binding of heme to globin. Hemoglobin
Koln (Wajcman et ai, 1971), for example,
has a single amino acid substitution (/3 98,
FG5, valine to methionine) in the heme
pocket which causes a loss of heme from
the mutated beta chains, accompanied by
intracellular precipitation of hemoglobin.
In whole fresh hemolysates from humans
heterozygous for Hb Koln approximately
10% of the heme is lost from the hemoglobin. Heme depletion reaches 40% in
the purified hemoglobin.
Waxman (1975) suggests that the different polypeptide chains of annelid hemoglobins may not all bind heme with equal
affinity, or perhaps an inefficient mechanism exists for inserting hemes into newly
synthesized globin chains. The problem of
the heme-to-globin stoichiometry is unanswered at present, and remains a truly
stimulating problem.
Current and future research
In the laboratory of Dr. Austen Riggs we
are very nearly finished with determining
the complete amino acid sequence of one
globin chain of earthworm hemoglobin.
We have separated Lumbricus terrestris
hemoglobin into a major (90-95%) and a
minor fraction by preparative isoelectric
focusing. The major component chromatographs on Sephacryl S-200 in 6 M
guanidine-HCl as a single peak corresponding to ca. 15,000 daltons, and has
been separated into three fractions by ion
exchange chromatography in 8 M urea
and 0.05 M 2-ME (Garlick and Riggs,
1978). We have nearly finished the determination of the entire amino acid sequence of one globin chain, called chain
AIII. This chain has no methionine, no
glucosamine or galactosamine, and no neu-
57 S
chlorocruorin
hemoglobin
Eudistylia vancouveri*
Abarenicola pacificaA
Native
15,700,28,800,
and 59,000
15,800
14,500
hemoglobin
Abarenicola pacifica^
c
b
Terwilliger el at., 19756, 1976; Terwilliger, 1979.
Garlick and Terwilliger, 1975.
Terwilliger et at., 1975n.
d
Garlick and Terwilliger, 1977.
a
14,000
15,000
15,000 and
30,000
chlorocruorin
Eudistylia vancouveric
SDS-PAGE in 8 M urea
Sephadex G-200 in 8 M urea + 0.1 M 2-ME
SDS-PAGE
SDS-PAGE after dissociation ;in EDTA
SDS-PAGE; Sephadex G-200 in 6 M urea
(carboxymethylated globin)
sed. equil. in 6 M GuHCl + 0.1 M 2-ME
Sephadex G-200 in 8 M urea + 0.1 M 2-ME
SDS-PAGE
235,000
—
270,000
280,000
Method
—
24,700 (iron)
26,300 (iron)
21,760 (iron)
24,461 (iron)
30,514
(heme)
Mill
Mol wt
BioGel A-1.5M
column, chrom.
sed. vel.
sed. vel. and
Sephadex G-200
Sephadex G-200
Submultiple
Mol wt
15,500
16,000
hemoglobin
Thelepus crispusb
Method
—
10 S
—
11 S
S2o,w
SDS-PAGE; sed. equil. in 6 M[ GuHCl
+ 0.1 M 2-ME
Subuniu
sed. vel.
sed. equil.
Sepharose 4-B
column, chrom.
sed. vel.
sed. equil.
Sepharose 4-B
column, chrom.
Method
15,500 and
30,000
hemoglobin
Mol wl
2,600,000
3,100,000
3,300,000
3,400,000
Mol wt
Pista pacifica"
Pigment
—
hemoglobin
Thelepus crisptts"
Species
58 S
hemoglobin
Pista pacifica"
—
Sio.w
Pigment
Species
TABLE 1. Subunit structures of three annelid hemoglobins and one chlorocruorin.
a
X
pi
•INELI
OGLOBIN STRL CTURE
76
ROBERT LEE GARLICK
tral hexose. Chain AIII shares little se- Welch Foundation Grant F-213 to Austen
quence homology with other hemoglobin Riggs. This paper was presented at a symchains. Of the first 35 amino acid resi- posium on respiratory pigments at the andues of the NH2-terminus of AIII, only nual meeting of the American Society of
six are identical with the beta chain of Zoologists in Richmond, Virginia in Dehuman hemoglobin, and only two are cember 1978. The symposium was aridentical with those of the monomeric ranged by Stephen C. Wood and supportcomponent of Glycera dibranchiata coelomiced by the National Science Foundation. I
cell hemoglobin (Garlick and Riggs, 1978). would like to thank Dr. Wood and the NSF
The entire amino acid sequence of G. for taking care of my expenses for those
dibranchiata monomeric hemoglobin is only meetings.
23% homologous with sperm whale myoREFERENCES
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Harrington, J. P., E. R. Pandofelli, and T. T. HerACKNOWLEDGMENTS
I wish to thank Robert C. Terwilliger,
Nora B. Terwilliger, and Austen Riggs for
critical review of the manuscript. Also I
would like to thank Florence Waddill and
Marie Ervin for help with the sequence
work. Work done in R. C. Terwilliger's laboratory was aided by PHS Physiology
Training Grant GM00336 to R.L.G., and
NSF Research Grant GB-34292 to R.C.T.
Work done at the University of Texas was
supported by NIH Grant GM 21314, NSF
Grant PCM 76-06719, and Robert A.
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ANNELID HEMOGLOBIN STRUCTURE
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