Biochimica et Biophysica Acta 829 (1985) 135-143
Elsevier
135
BBA 32191
P h y s i c o c h e m i c a l and structural properties of the extracellular h a e m o g l o b i n of
Ophelia bicornis *
V i n c e n z a M e z z a s a l m a b, L u c i o di S t e f a n o b, S a n t o Piazzese b, M i c h e l a Z a g r a b,
B e n e d e t t o S a l v a t o a, G i u s e p p e T o g n o n a a n d A n n a G h i r e t t i - M a g a l d i a , * *
a Department of Biology, University of Padova and Centro C.N.R. Fisiologia e Biochimica delle Emocianine e altre metallo
proteine, Padova, and b Institute of Histology, University of Palermo, Palermo (Italy)
(Received December 27th, 1984)
Key words: Hemoglobin structure; Erythrocruorin; Electron microscopy; Subunit composition; (O. bicornis, Annelid)
The physical, chemical and structural properties of the extracellular haemoglobin (erythrocruorin) from the
polychaete annelid Ophelia bicomis have been investigated. The structure of this protein is similar to that of
other annelid erythrocruorins with the exception of an additional subunit in the central cavity of the double
hexagonal prism. The hemolymph contains also a dimeric form of the protein. Dissociation in different media
has been studied and subunits of 60, 30 and 15 kDa have constantly been obtained. By reduction after
alkaline dissociation and denaturation, three classes of polypeptide chains, of 14, 15 and 16.5 kDa, are
produced from both the monomeric and the dimeric forms. A model for the fine structure of the main subunit
is proposed. It shows a great similarity to that suggested for the chlorocruorin of Spirographis spallanzanil.
Introduction
Annelid extracellular hemoglobins (erythrocruorins) are proteins of high molecular weight made
by twelve main subunits arranged at the vertices of
a double hexagonal prism. They are all about the
same size: 26-28 nm in diameter and 18 nm in
height [1].
Erythrocruorins from many species of annelids
have been studied by several authors. Very similar
chemical and physicochemical properties have been
found for these proteins, but as for the fine structure, no agreement has been attained even as to
whether the models proposed are all based on a
repetition of twelve main subunits.
* This paper is dedicated to the memory of Eraldo Antonini.
** To whom correspondence should be sent: Department of
Biology, University of Padova, via Loredan, 10, 35131
Padova, Italy.
When studying large molecules, the major problem arises from the difficulty in measuring precisely their relative molecular mass. As for erythrocruorins, the values reported range from 2.5 • 106
to 4.1.10 6 Da. Such great discrepancy concerns
not only erythrocruorins from different species but
also the protein obtained from the same species,
depending on the methods used a n d / o r the inferences made when evaluating the same parameters.
A second problem arises from the elusive nature
of the main (1/12th) subunit of erythrocruorins.
Only in a few cases has this subunit been obtained
in monodisperse solution: it dissociates under very
mild conditions, giving products of low molecular
weight. The adjective 'putative' has been rightly
given to the 1/12th subunit [2]. Its relative molecular mass therefore, has been deduced either from
the whole molecular mass (i.e., from a very unreliable value) or from the sum of the dissociation
products.
0167-4838/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
136
Three classes of subunit are generally obtained
by mild dissociation from most erythrocruorins of
different species: 50-60 kDa, 25-37 kDa and
13-18 kDa.
From an evaluation of their relative concentration, different models of the main subunit have
been inferred as far as the number and the organization of the polypeptide chains is concerned:
three tetramers [3,4], four tetramers [5,6], six trimers [7,8] or a combination of two tetramers and
one hexamer [9-11].
When studying the dissociation of the chlorocruorin from Spirographis spallanzanii [12], we
succeeded in preparing monodisperse solutions of
the 1/12th subunit (257 kDa) and of a tetramer of
polypeptide chains of about 62 kDa. The model
we proposed in which the main subunit is made by
four tetramers was strongly supported by electron
microscopy, image analysis and reconstruction [13].
In this paper we report the results of a study of
the erythrocruorin from the polychaete annelid
Ophelia bicornis.
The chemical, physical and structural properties
of this protein indicate that, in most aspects, it is
very similar to the other annelid haemoglobins.
The molecular weights and the relative concentrations of the dissociation products suggest
that the main subunit (1/12th) is a tetrameric
assembly of polypeptide chains.
Materials and Methods
Preparation and purification of erythrocruorin
Living specimens of O. bicornis (a small, 2-3
cm long, marine worm) collected from Sicilian sea
shores, were washed with filtered sea water and
homogenized with 3 vol. of cold 0.1 M Tris buffer
(pH 7.5)/20 mM CAC12/5 mM phenylmethylsulfonyl fluoride.
The clear supernatant obtained after centrifugation at 10000 x g was layered over 5 ml 20%
sucrose and centrifuged at 131000 x g for 7 h in a
swing-out rotor at 4°C. The red sediment was
redissolved in Tris buffer saturated with CO and
stored in the cold. For prolonged storage the protein was frozen in the presence of 20% ( w / v )
sucrose.
Before use, samples of the stock solution were
dialyzed against the 0.1 M Tris buffer and further
purified by gel filtration on a Bio-Gel A-5m column (2.6 X 140 cm). Some colourless, non-proteic
contaminants are eliminated, within the void
volume.
Apoprotein was prepared using acid acetone
[14]. The dried protein was redissolved in 0.1 M
NaOH, in 20% formic acid or in 1% SDS.
Chemical, physical and structural characterization
The heme content was determined by the pyridine haemochromogen method of De Duve [15],
using human haemoglobin as standard.
For the amino acid analysis, the apoprotein was
hydrolyzed for 21, 40 or 70 h with methansulfonic
acid according to Inglis [16]. Norleucine and L-aamino-fl-guanidopropionic acid were employed as
internal standards.
Measurements of circular dichroism in the far
ultraviolet were made using a Cary 61 dichrograph
in 0.05 cm cells on solutions containing 0.2 mg
protein per ml in 0.1 M phosphate buffer (pH 7).
The estimate of the apparent helix content was
based on the equation described by Chen et al.
[17].
The isoelectric point was measured with a LKB
instrument using LKB Ampholine in the pH range
3.5-10.
Sedimentation velocity measurements were carfled out with a Model E Beckman ultracentrifuge
equipped with the electronic speed control and
using the An-D rotor. The temperature was controlled at 20°C; a Leitz filter K 570 was positioned
along the light path to reduce the deep red protein
colour effects on the Schlieren pattern photographs. Samples containing from 1 to 8 mg protein
per ml in Tris buffer (pH 7) were used for s o
determinations.
The diffusion coefficient was determined by
laser light scattering with a 60-channel Malvern
autocorrelator K 7023 at 752.5 nm and 20°C on
samples containing 1.5 m g / m l protein in Tris
buffer (pH 7.0).
The partial specific volume was calculated from
the amino acid composition.
The molecular weight of the whole molecule
was measured also by gel filtration using a Sepharose CL-6B column (1.5 x 140 cm) equilibrated
with 0.1 M Tris buffer (pH 8)/0.4 M NaC1. The
column was calibrated with soybean trypsin in-
137
hibitor, bovine serum albumin, human ~-globulin,
ferritin monomer and ferritin dimer.
The spectra and the extinction coefficients of
oxy-, deoxy-, CO- and met-derivatives of erythrocruorin were measured using protein solutions of
appropriate concentrations in 0.1 M phosphate
buffer (pH 7.0) with a Perkin-Elmer 550-S spectrophotometer. The extinction coefficient of the
apoprotein was measured in 0.1 M NaOH and in
20% formic acid.
Structural observations were done with the
Hitachi H-600 electron microscope using the negative-staining technique. The native protein dissolved in 0.1 M phosphate buffer (pH 7) was
diluted with distilled water to a final concentration
of about 50 /Lg/ml, laid on a thin (less than 10
nm) carbon film supported on holey Formvar
membrane and stained with 1% (w/v) unbuffered
uranyl acetate. The dimensions of the molecules
were measured with a Nikon microcomparator.
Dissociation and molecular weight determination of
the subunits
Alkaline dissociation of erythrocruorin was obtained by prolonged dialysis against 0.1 M
carbonate-bicarbonate buffer at pH 9.6 in the
cold.
Dissociation by acylation was achieved within 1
h by stepwise addition of cytraconic (or succinic or
maleic) anhydride. A 6-fold molar excess of
cytraconic anhydride, relative to the lysine content, was used. The acyl groups were removed by
dialysis against 0.1 M acetate buffer (pH 5.6) for
at least 48 h in the cold [18].
The products from both dissociation procedures, alkaline pH and acylation, were analyzed
by gel filtration on Sephacryl S-300.
Dissociation by denaturation was performed on
apoprotein samples in 6 M guanidine-HC1 or in
1% (w/v) SDS at 100°C for 2-5 min.
Disulfide groups were reduced by adding 2%
(v/v) 2-mercaptoethanol to the denatured samples. Complete reduction was obtained when samples of native erythrocruorin were previously dialyzed against the pH 9.6 buffer for 24 h and then
cyanoethylated with acrylonitrile [19], before the
SDS treatment.
The dissociation products were separated on a
Sephacryl S-200 column equilibrated with 0.03 M
Tris buffer (pH 8.0)/2 mM EDTA/0.2% (w/v)
SDS.
SDS-polyacrylamide gel electrophoresis (T =
12.83, C=2.60) was carried on according to
Laemmli [20] on 14 cm slabs in a Pharmacia
apparatus. Staining and destaining were done
according to Weber et al. [21]. The Bio-Rad 'low
molecular weight' standard mixture (phosphorylase b, bovine serum albumin, egg albumin,
carbonic anhydrase, soybean trypsin inhibitor and
lysozine) was used for calibration.
Densitometric scanning of the gels was made
with an LKB Ultroscan apparatus. Band areas
were calculated according to Pionetti and Pouyet
[22].
Results
Characterization of the native protein
When subjected to chromatography on a BioGel A-5m column, a large peak is obtained which
is preceded by a small shoulder (Fig. 1). The
native erythrocruorin of O. bicornis, therefore, appears to be a mixture of two components, the
major one amounting to 88% of the total protein.
Fractions of these components have been separately collected and analyzed.
The amino acid composition of the major component is reported in Table I, the absorption maxima and the extinction coefficients of oxy-, deoxy-,
CO- and met-derivatives in Table II. The isoelectric point of this component was found to be 4.65.
3'
N
2,
/k
Fig. 1. Bio-Gel A-5m filtration. Fractions containing the major
and minor forms were pooled as indicated by arrows.
138
TABLE I
A M I N O A C I D C O M P O S I T I O N (RESIDUES PER 100+0.3)
O F T H E M A J O R C O M P O N E N T OF O. BICORNIS
Lys
His
Arg
Trp
Asp
Glu
Thr
Ser
Gly
5.08
7.1
6.74
1.03 (1.42 ~)
11,83
10.36
3.41 b
5.63 b
6.37
Ala
Pro
Val
Ile
Leu
Tyr
Phe
12-Cys
3.55
3.15
7.22
4.89
7.56
1.09
5.88
2.16
c
c
(1.17 a)
a
Spectrophotometric determination [35].
b Extrapolated to zero time.
c Extrapolated to infinite time.
d Determined as S-sulphocysteine.
From the haem content (2.45 + 0.2%) a minimal
relative molecular mass of 25 kDa has been calculated. The apparent a-helix content amounts to
about 58%.
Very similar results are found for the minor
component; they are not reported here because
this component has not so far been purified.
Sedimentation analysis of the native erythrocruorin and of the two isolated fractions shows
that the major and minor components have 55 S
and 95 S, respectively. In Fig. 2 the sedimentation
patterns of the original mixture (a), of the minor
T A B L E II
A B S O R P T I O N M A X I M A A N D E X T I N C T I O N COEFFICIENTS OF T H E M A J O R C O M P O N E N T O F O. BICORNIS
ERYTHROCRUORIN
Derivatives
Xmax
A 1~
Derivatives
Xmax
A 1~
Oxy-
278
415
540
575
17
41
4.81
4,75
Met-
278
396
500
630
15.25
33.06
4.17
1.04
Deoxy-
430
560
41,1
4,11
Apo-
278
CO-
418
537
567
58,18
4.49
4.34
a Dissolved in 20% formic acid.
b Dissolved in 0.1 M N a O H .
8.04 a
7.50 b
Fig. 2. Sedimentation analysis of the whole preparation (a) and
of the minor (b) and major (c) fractions obtained by Bio-Gel
filtration.
(b) and the major (c) components are presented.
Identical patterns are observed after keeping the
material for several days in the cold.
The s o of the major component at 20°C is
55.12 + 0.13 S; its diffusion coefficient (measured
on solutions containing 1.5 m g / m l at 20°C) is
1.84 + 0.026). 10-TcmZ/s. From the D and s values measured on identical protein solutions at
20°C (for 1.5 m g / m l s = 54.77 S), using a partial
specific volume of 0.73 m l / g , a molecular mass of
2.7- 106 Da has been calculated.
By gel filtration an M r of 3.2.106 Da was
obtained.
139
The electronmicrographs of O. bicornis erythrocruorin show that the 95 S fraction is an end-toend dimer. Both dimers and monomers possessing
a central subunit have been found. This subunit,
however, is present only in a small proportion of
the molecules (Fig. 3). The molecular dimensions
are the following: 27.5 nm from vertex to vertex,
26 nm from side to side in the top projection; 17.5
nm height and 25 nm side in the lateral projection.
We have observed that in the conditions used for
negative staining, the central subunit easily dissociates.
In Fig. 3, images of the monomeric (a) and
dimeric (b) components are shown. The dimeric
axial projections are easily recognized because of
the deeper stain deposition along the sides. High
magnifications of the axial projections of monomers with and without central subunit and of a
Fig. 3. Electron micrographs of
the major (a) and the minor (b)
fractions. The insets are higher
magnifications (650 000 × ):
from top to bottom, monomer
without central subunit, same
with central subunit, lateralview
of monomer, axial projection of
dimer, lateral view of dimer.
140
dimer, together with the relative lateral views are
presented in the insets of Fig. 3. While the typical
hexagonal s y m m e t r y is evident in the top view of
the monomer, the dimer appears to be less ordered.
Its lateral projection shows that its four stacked
discs are not in register.
70
A:
C
80
go
Dissociation products
All dissociating conditions - alkaline pH,
acylation, treatment with 6 M guanidine-HC1 or
1% SDS - produce only three types of subunit.
Their relative molecular masses as measured by gel
filtration or SDS-polyacrylamide gel electrophoresis (Table III) correspond to 60 (subunit A), 30
(subunit B) and 15 k D a (subunit C). Relatively
higher masses obtained for the acylated subunits
m a y be explained by a swelling of the protein
molecules induced by a higher electrostatic potential.
Alkaline p H dissociation is a very slow process
(Fig. 4a and b). Acylation with citraconic
anhydride (as well as succinic and maleic) dissociates the protein completely in a short time (Fig.
4c).
The products of both dissociation procedures
all contain haem usually as a mixture of Fe 2÷ and
Fe 3+ derivatives, which can be easily reduced to
the Fe 2+ form with dithionite at neutral pH.
Apoprotein subunits belonging to the same three
classes have been obtained after denaturation with
1% SDS, as controlled by Sephacryl S-200 filtration (Fig. 5). They can be prepared also by treatment with 6 M guanidine-HC1.
AI
b
AI
S 6o
A
C
o 70
oo
--
II,
91 .
¢
A
60.
70'
80,
S0,
IL
,
,r,o
z~)o
a;o ml
Fig. 4. Sephacryl S-300 filtration of the dissociation products
obtained by alkaline pH treatment for 3 days (a), 9 days (b)
and by acylation with citraconic anhydride for 1 h (c).
In Fig. 6 the results obtained by SDS-polyacrylamide gel electrophoresis are presented. Alkaline p H treatment followed by reduction dissoci-
TABLE II1
RELATIVE MOLECULAR MASSES OF SUBUNITS AND POLYPEPTIDE CHAINS OF THE MAJOR COMPONENT OF O.
BICORNIS ERYTHROCRUORIN
All relative molecular masses are quoted in kilodaltons.
Gel chromatography
Alkaline dissociation (kDa)
A2 = 190
A1=129
A = 63
B = 36
C =15.7
SDS-electrophoresis
Acylated
Deacylated
Unreduced
Reduced
A=87
A=63
A=55.7
al =16.6
all = 14.9
B = 40
C =19
B = 34.5
C =17.5
B = 30.8
C =13.6
B = 29.8 a
C =14.1
a Disappears when sample is treated with carbonate buffer (Fig. 6e).
141
15
TABLE IV
PROPOSED SUBUNIT COMPOSITION OF O.
ERYTHROCRUORIN
The percentage of the total M r was determined according to
the following equation: % =IO0.Ai.Mil/3/y~iA.M 1/3, where
A i is the densitometric area of stained band. SDS-polyacrylamide gel electrophoresis of unreduced native protein as in Fig.
6e.
10
c
0
co
/
0.5
0
i
:.
s'o
4O
BICORNIS
6"o
fractions
Subunit
Mr
(kDa)
Number
of
copies
A
B
C
63
29.8
14.1
1
1
2
Contribution to
M r of protein
(kDa)
63
29.8
28.2
Percentage
of
total M r
50.8
23.0
26.2
121
Fig. 5. Dissociation by SDS denaturation of the reduced and
cyanoethylated protein. Sephacryl S-200 filtration.
' W
ates the whole molecule into polypeptide chains.
Both the monomeric (a) and the dimeric (b) components give the same three classes of polypeptides. Reduction without alkaline treatment (d)
does not dissociate the protein completely: a residual band corresponding to subunit B (30 kDa) is
still present. From the unreduced protein (e) subunits A, B and C are obtained.
Subunit B appears to be resistant to reduction
and dissociates only when the protein is previously
treated at alkaline pH. Subunit C does not change
after reduction.
In Table IV are reported the relative contents of
subunits A, B and C, as calculated from the
densitometric trace of the SDS-polyacrylamide gel
electrophoresis of the unreduced protein.
¸
Discussion and Conclusions
W m
•
b
c
d
•
!
Fig. 6. SDS-polyacrylamide gel electrophoresis of denaturation
products of: (a) dimer and 9b) monomer after alkaline pH
dissociation and reduction; (d) reduced monomer; (e) alkalinepH-dissociated unreduced monomer. (c) and (f) are low-molecular-weight standard mixtures.
The erythrocruorin of O. bicornis is very similar
to the other annelid extracellular hemoglobins in
many aspects: the amino acid composition, the
haem-to-protein ratio, the spectral properties, the
chromatographic and electrophoretic patterns of
the subunits and the gross quaternary structure.
The presence of an additional subunit has also
been observed in the erythrocruorins of some other
annelids: Nephtys incisa [23], Oenone fulgida [24],
Nephtys hombergi [10] and Euzonus mucronata [25].
In O. bicornis, since the shape of the lateral
projections is identical to that of chlorocruorins
142
and other erythrocruorins, the additional subunit
must be deeply embedded into the central hole.
This has also been suggested by Van Bruggen and
Weber for O. fulgida [24]. The central subunit
appears, in fact, smaller than the external ones,
probably because it is partially masked by the
negative stain.
The pigment of E. mucronata (another Ophelid)
is the only one that shares with O. bicornis
haemoglobin the presence of a minor fraction
which, by sedimentation-and electron microscopy,
is seen to be a dimer of the major one. According
to Terwilliger et al. [25], in this erythrocruorin the
central subunit is present only in the dimeric form
and the dimer is formed from two stacked basic
units, one of which is rotated in comparison with
the other. The axial projections of the dimer appear, therefore, to have a dodecameric symmetry.
On the contrary, in O. bicornis erythrocruorin,
the central subunit is present also in the monomer
[26]. Under the conditions for negative staining it
dissociates easily so that the axial projections in
which it can be seen are relatively few.
From sedimentation studies it is evident that
the dimer is a stable form. The relative concentration of the components does not change even after
prolonged storage in the cold. If there is a dimermonomer equilibrium, this must be extremely slow.
As indicated by the haem content, the spectral
properties and the distribution of the polypeptide
chains and of the subunits, the two components
seem to be identical.
The relative molecular mass of the monomeric
molecule, as measured by two independent methods, is about 3 • 106 Da. This value is in very good
agreement with those found by different methods
for several extracellular haemoglobins in many
laboratories [1].
Much higher values (up to about 4.1 • 106 Da)
have been reported either on the basis of sedimentation equilibria or from small-angle X-ray diffraction studies [2,27-29]. Most authors, however, believe that all erythrocruorins, being so similar in
many physicochemical properties, cannot differ so
much in their molecular weight.
In order to check the internal consistency of the
methods used in this study, we have determined
the chromatographic elution volumes, the D and s
coefficients of the extracellular hemoglobins from
other annelid species such as Lumbricus terrestris,
Octodrilus complanatus, Eisenia foetida, AIlolobophora caliginosa (oligochaetes) and Spirographis spallanzanii (polychaete). In all cases the relative molecular mass was found to be about 3 • 106
Da.
Dissociation of O. bicornis erythrocruorin at
alkaline pH does not produce monodisperse solutions of the main subunit (1/12th). Evidently,
milder dissociation conditions are required to obtain this subunit.
Under all the conditions used: alkaline pH,
acylation, 6 M guanidine-HCl and 1% SDS,
erythrocruorin of O. bicornis behaves in surprisingly uniform manner. Three types of subunit are
produced in constant proportions: A (60 kDa), B
(30 kDa) and C (15 kDa).
Cytraconic anhydride seems the most convenient acylating agent, being rapid, highly reproducible and reversible.
When applied to other erythrocruorins, these
procedures give very similar results. It appears,
therefore, that acylation and mild reduction [30,31]
can be considered general methods for obtaining
functional subunits from extracellular haemoglobins.
As shown in Fig. 6, subunits A and B are both
made by polypeptide chains of about 15 kDa
linked by disulphide bridges. Subunit B (30 kDa)
is not a single polypeptide, but must contain some
hardly accessible disulphide bond, as found in
Pista pacifica erythrocruorin [32].
The apparent molecular mass of the subunit A
(Table III) as measured by SDS-polyacrylamide
gel electrophoresis, is underestimated because of
the presence of interchain disulphide bridges [33].
The 63 kDa value obtained by gel filtration for the
same subunit in the native form indicates the
presence of four polypeptides; subunit A, therefore, is a tetramer and not a trimer.
The densitometric scanning of the SDS°polyacrylamide gel electrophoresis pattern of the unreduced protein reveals a ratio of 1 : 1 : 2 for the
relative concentration of subunits A, B and C. The
main subunit (1/12th), therefore, could be made
by an assembly of four tetramers. Two tetramers
are subunits A and the other two are an association of a subunit B with two subunits C. Supporting evidence for this model has been obtained by
143
image analysis and reconstruction of electron micrographs of two dimensional crystals [26].
Different structures have been proposed for the
main subunit of the erythrocruorins in other annelid species. It has also been claimed that the tetrameric structure is peculiar to chlorocruorins only
[34]. Since the composition, the molecular structure and the function of all the annelid
haemoglobins are so similar that they can be considered members of a family of phylogenetically
related proteins, we have confidence that their fine
structures, too, are very similar.
Acknowledgements
The work was supported by a grant from M.P.I.,
Italy. Thanks are due to B. Filippi for CD spectra,
F. Madonia for diffusion coefficients determinations, R. Carbone for sedimentation analysis and
M.G. Cantone and P. Omodeo for the taxonomic
identification of the annelids used.
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