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Extracellular Hemoglobins from Annelids, and their Potential Use in Biotechnology
Franck Zal and Morgane Rousselot
Abstract
This chapter highlights the diversity of invertebrate hemoglobin structures, and gathers together up of 100 years of
research data. Among invertebrates, annelid hemoglobin
structures have been the most widely studied, probably due
to their typical symmetric shapes and sizes that allow direct
observations to be made using transmission electron
microscopy. Hemoglobin seems to be present in all phyla,
which suggests a very ancient origin. Using the hemoglobin
of the marine annelid Arenicola marina, the French biotechnology startup HEMARINA is developing medical
16.1
Introduction
All aerobic cells require oxygen and nutrients to fuel their
energetic and growth requirements. They find these elements
in their environment for biomolecule synthesis, and discard the
degradation products of their metabolism. However, multicellular organisms are not directly in contact with the external
environment where oxygen is available, and in this context two
physical mechanisms exist for the transportation of the respiratory gases (oxygen and carbon dioxide), namely diffusion and
convection. Diffusion mechanisms are effective only over small
distances (millimeters or less), and so the process plays an
important role only in unicellular organisms. If the distance
between the external and internal media is important, however,
then a convection process will be substituted in place of diffusion.
Yet, these two processes may intervene at either the same time or
separately during the circulation and respiration processes. The
primary function of the circulation process involves the requirements for oxygen and nutrients, as well as the elimination of
carbon dioxide and metabolic waste. Consequently, the circulating fluids establish important ties between distant specialized
cellular populations and the external environment via the intermediate of the exchange organs. In this context, blood has a key
position as it contains specialized molecules known as respiratory
pigments. These exist either intracellularly or extracellularly, and
are able to bind oxygen in a reversible fashion, which increases
considerably the ability of the blood to carry these gases.
products. The first generations of blood substitutes (hemoglobin oxygen carriers) were manufactured using intracellular hemoglobin (human and bovine) in order to
function outside the red blood cells. However, HEMARINA
technologies are based on a natural extracellular hemoglobin. This circulatory pigment, which is present in the blood
vessels of A. marina, has evolved over a million years and is
able to function extracellularly. Currently, two main products are under development at HEMARINA, namely
HEMO2life1 (for organ preservation) and HEMOXYCarrier1 (as a universal oxygen carrier).
During evolution, three different categories of oxygen carriers
were selected, each of which retained a characteristic color
(Table 16.1). Of these pigments, hemoglobin (Hb), with a typical
red color, is the most familiar and is present in almost all phyla,
whereas chlorocruorin, which is very similar to the Hb, is
characterized by a green color and exists in four families of
polychete annelids. Hemocyanin, a blue pigment, is currently
present in certain mollusks and arthropods, while hemerythrin,
which is pink in color occurs more sporadically among the
animal kingdom (Figure 16.1).
The heme-containing pigments are the most common among
life kingdom as they are found in 33% of zoological classes
(Toulmond, 1992); yet, surprisingly, these pigments are also
present in plants (Landsmann et al., 1986; Bogusz et al., 1988;
Fuchsman, 1992). Although the molecules are characterized by
an important diversity of molecular weight and structure (Royer,
1992; Terwilliger, 1992), the active site is always similar and is
constituted by a protoporphyrinic tetrapyrrolic ring (Mr ¼
616.5 Da) that has an iron atom at the center (Fe2þ) (Figure 16.2).
This Fe–protoporphyrin complex constitutes the heme molecule. The same active site is also found in other enzymes, such
as cytochromes, catalases and peroxidases. In the case of hemoglobin the iron atom is only bound by an amino acid from the
polypeptide chain, the proximal histidine. The complex constituted by the active site and the polypeptide chain forms the basic
functional unit that is able to bind oxygen only in a reversible
fashion. Two categories of heme-containing groups exist, but in
both cases the heme molecule is a chelate of iron associated with
Outstanding Marine Molecules: Chemistry, Biology, Analysis, First Edition. Edited by Stephane La Barre and Jean-Michel Kornprobst.
Ó 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2014 by Wiley-VCH Verlag GmbH & Co. KGaA.
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16 Extracellular Hemoglobins from Annelids, and their Potential Use in Biotechnology
Table 16.1 Classification and localization of the different respiratory pigment across the animal and plant kingdoms.
Pigments (localization)
Phylum
Mr (Da)
Active sites
Na)
Intracellular
Hemerythrin
Without heme
Sipunculida
Brachiopoda
Annelida
Priapula
40–110 103
2 Fe atoms
2
Arthropoda
Mollusca
4 105 to 3 106
3 106 to 9 106
2 Cu atoms
6–48
70–160
65 103
1 Fe atom
4
17–130 103
1 Fe atom
1–8
1 Fe atom
1 Fe atom
1 Fe atom
1 Fe atom
1
1
1
1
1
Extracellular
Hemocyanin
Intracellular
Hemoglobin stricto sensu
Hemoglobin of invertebrates dissolved in their body fluids
Hemoglobin cytoplasmic
Myoglobin
Hemoglobin
Leghemoglobin
Neurhemoglobin
Extracellular
Hemoglobins
Erythrocruorin
Chlorocruorin
With heme
Vertebrate
Mollusca
Echinodermata
Annelida
Echiuria
Vertebrate
Invertebrate
Bacteria
Leguminous plants
Annelida
Platyhelminthes
Nematyhelminthes
Nemerta
Mollusca
Arthropoda
Annelida
Vestimentifera
Pogonophora
Annelida
17 103
to
12 106
4 105
To
4 106
3 106 to 4 106
1 to >8
>8
>8
a) N ¼ no. of active sites capable of binding an oxygen molecule.
a protoporphyrin IX which is termed ferroprotoheme and
CH2); this complex is the
contains two vinyl groups (CH
most common (Falk, 1975), but in ferrochlorocruoroporphyrin
or chlorocruoroheme the vinyl group is replaced by a formyl
group (CH¼O) (Fischer and Seemann, 1936) (Figure 16.2). The
latter group is present only in the chlorocruorins. It is possible to
distinguish between five different categories of globin chains
(for a review, see Vinogradov et al., 1993):
Single-domain chains are the most common in the animal
kingdom. They comprise about 145 amino acids and have an
active site, and their existence is documented as three different Hbs, namely intracellular, extracellular, or cytoplasmic.
These chains can associate to create monomeric, dimeric,
tetrameric or polymeric Hbs.
Truncated single-domain chains that contain about 116–121
amino acids.
Chimeric single-domain chains have Mr 40 kDa, are cytoplasmic, and are characterized by an N-terminal region that is
able tobind a heme group and a C-terminal with several functions.
The chimeric bi-domain chains (40 kDa) are created by the
covalent association of two domain chains, one of which bears
a heme group.
Linear chimeric multidomain chains created by the covalent
association of several globin domains.
The main aim of this chapter is to describe the invertebrate
hemoglobins known to date, and briefly to review the extracellular Hbs obtained from annelids. The details also provided of
two therapeutical applications from Arenicola marina Hb.
16.2
Annelid Extracellular Hemoglobins
With numerous reviews having been produced on annelid
extracellular hemoglobins (EHbs) and chlorocruorins, the interest of the international “invertebrate hemoglobinist” community was directed towards this remarkable family of molecules
(Mangum, 1976a; Mangum, 1976b; Antonini and Chiancone,
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Figure 16.1 Occurrence of the respiratory pigments among the major phyla and classes (Toulmond and Truchot, 1993). The color dots indicate the type
of respiratory pigments eventually present. Red ¼ hemoglobins; green ¼ chlorocruorins; purple ¼ hemerythrins; blue ¼ hemocyanins. The absence of a dot
means that no respiratory pigment has yet been detected. However, the presence of dot does not mean that all of the group’s species possess the
respiratory pigment mentioned. With kind permission by La Recherche.
1977; Weber, 1978; Chung and Ellerton, 1979a; Garlick, 1980;
Vinogradov et al., 1980c; Vinogradov, Kapp, and Ohtsuki, 1982;
Vinogradov, 1985a; Vinogradov, 1985b; Gotoh and Suzuki,
1990; Riggs, 1990; Terwilliger, 1992; Lamy et al., 1996). The
EHbs are present in the three annelids classes, namely polychetes, oligochetes, and achetes. The molecules are very large
biopolymers with high molecular weights in the range of 3000–
4000 kDa, and corresponding sedimentation coefficients of
54–61 S, respectively. Each molecule consists of an assembly
of about 200 polypeptide chains that belong to between six
and eight different types that are usually grouped into two
categories:
Functional chains, which possess an active site that is able to
bind oxygen reversibly, and correspond to globin chains with
Mr-values of about 15 and 18 kDa.
Linker chains, which possess very few or no heme groups and
play an important role in the assembly of one-twelfth of the
molecule. These chains have Mr-values of between 22 and
27 kDa.
Some authors have revealed the importance of glycans on the
assembly of EHbs from Perinerreis aibuhitensis, and have termed
this mechanism “carbohydrate gluing” (Ebina et al., 1995;
Matsubara et al., 1996; Yamaki et al., 1996). However, this
mechanism cannot be universal as not all of these molecules
are glycosylated (e.g., Arenicola marina EHb; Zal et al., 1997a,
1997b). The EHbs of annelid are likewise characterized by an
acidic isoelectric point and low heme and iron contents, about
two-thirds of that observed for other hemoglobins, and corresponding to the presence of one mole of heme per 20000–
27000 Da of protein. This result indicated that not all of the
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16 Extracellular Hemoglobins from Annelids, and their Potential Use in Biotechnology
COO–
COO–
(a)
CH2
CH2
CH2
HC
C
C
C
C
C
C
C
C
H
C
CH3
C
H3 C
C
C
C
C
CH
N
N
CH2
C
Fe
C
C
C
C CH3
H2C
C
H
CH2
CH2
H
C
C
C
C
N
C
N
C
N
C
C
N
C
C
H
CH3
CH
Fe
C
C
C
HC
C
C
CH2
CH2
CH2
N
N
HC
H2C
H
C
COO–
COO–
(b)
C
C CH3
C
C O
CH3
H
H
Figure 16.2 Heme structure (Toulmond and Truchot, 1993). (a) Ferroprotoporphyrin or ferroprotoheme (porphyrin IX) corresponding to the hemoglobin
oxygen-binding site; (b) Ferrochlorocruoroporphyrin, corresponding to the chlorocruorin oxygen-binding site. One of the vinyl groups (CH
CH2) of
O). With kind permission by La Recherche.
molecule (a) takes over from a formyl group (CH
polypeptide chains possessed a heme group (Vinogradov, Kapp,
and Ohtsuki, 1982; Vinogradov, 1985a; Vinogradov, 1985b).
16.3
Architecture
Interest in the global structure of these molecules first emerged
during the 1960s, some 30 years after the molecular weights of the
pigments had been determined. The initial studies involved observations using transmission electron microscopy (TEM), and the
first micrographs were obtained from Arenicola marina (Roche,
Bessis, and Thiery, 1960a; Roche, Bessis, and Thiery, 1960b). The
images revealed hexagonal structures with diameters of 22–26 nm,
where each molecule was constituted by two superimposed hexagons with a height of 11–17 nm (Levin, 1963; Roche, 1965). This
structure was typically referred to as a hexagonal-bilayer (HBL).
Each hexagon was composed of an assembly of six elements with a
water-drop shape (Kapp and Crewe, 1984; Van Bruggen and Weber,
1974), and were also referred to as having a hollow globular
structure (HGS) (De Haas et al., 1996a, 1996b, 1996c, 1996d) or
one-twelfth. The molecule was built up by 12 of these subunits,
each of molecular weight ca. 250 kDa, being association one with
another. Subsequently, these observations were extended to other
molecules belonging to other annelid species, and revealed compounds of similar dimensions (Table 16.2).
Table 16.2 Dimension of several extracellular hemoglobins measured from TEM images.
Species
High (nm)
Polycheta
Nephthyidae
Nephtys incisa
Nereidae
Tylorrynchus heterochaetus
19.8 1.2
Perinereis aibuhitensis
Arenicolidae
Arenicola marina
Abarenicola affinis
Opheliidae
Euzonus mucronata
Ophelia bicornis
Travisia japonica
Eophilia tellinii
Eunicidae
Eunice aphroditois
a (nm)a)
18.2
19.2 1.2
20.0 1.8
7.5 0.8
16.0 0.8
16.0
14.8
19.7
17.0
25.5 0.8
24.0
20
27.5
25.5
17.0
17.5
16.0
19.0
25.5
26.0
23.5
28.0
17.9 0.3
26.3 0.3
b (nm)b)
Stainingc)
References
31.6 1.1
AU
Messerschmidt et al., 1983
28.4
30.1 0.8
29.4 0.8
30.0
30.4
28.4
30.0
27.5
Suzuki, Kapp, and Gotoh, 1988
Kapp et al., 1982
Matsubara et al., 1996
PT
PT
PT
AU
PT
Levin, 1963
Roche, 1965
Breton-Gorius, 1963
Toulmond et al., 1990
Chung and Ellerton, 1982
MA, PT
AU
PT
AU
Terwilliger et al., 1977
Mezzasalma et al., 1985
Fushitani et al., 1982
Cejka et al., 1989
AU
Bannister, Bannister, and Anastasi, 1976
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16.3 Architecture
Cirratulidae
Cirriformia sp.
Terrebelidae
Pista pacifica
Thelepus crispus
Ampharetidae
Amphitrite ornata
17.0
24.0
AU
Van Gelderen, Shlom, and Vinogradov, 1981
17.0 0.3
17.0 0.3
25.0 0.5
25.0 0.5
MA, PT
MA, PT
Terwilliger and Koppenheffer, 1973
Terwilliger and Koppenheffer, 1973
10.5
21.8
29.0
PT
AU
Mangum, 1976
Chiancone et al., 1980
30.4
MA, PT
AU
Terwilliger and Terwilliger, 1984
Toulmond et al., 1990
AU
St€ockel, Mayer, and Keller, 1973
Kapp and Crewe, 1984
Alvinellidae
Alvinella pompejana
19.7
24.5
27.0
21.1
16.0
31.2
Oligocheta
Tubificidae
Tubifex tubifex
Lumbricidae
Lumbricus terrestris
Eisenia fetida
Limnodrilus gotoi
Maoridrilus montanus
Megascolecidae
Pheretima communis
Pheretima hilgendorfi
Acheta
Hirudidae
Haemopis sanguisuga
j 365
20.0
16.0 0.7
16.0
23.7
20.0 0.9
21.5
17.5
16.5
14.0
16.0 0.7
30.0
26.5 0.0
26.5
16.7
16.7
15.2 1.4
26.0
AU
AU
PT
PT
AU
PT
Levin, 1963
Levin, 1963
Roche, 1965
Ben Shaul, 1974
Kapp et al., 1982
Kapp and Crewe, 1984
Frossard, 1982
Frossard, 1982
Ochiai and Enoki, 1981
Yamagishi et al., 1966
Ellerton, Bearman, and Loong, 1987
26.0
26.0
PT
PT
Ochiai and Enoki, 1979
Ochiai and Enoki, 1979
24.4 2
AU
Wood, Mosby, and Robinson, 1976
28.2 0.8
31.3
26.0
26.0
25.0
22 1.2
25.0
37.0
29.9 0.8
AU
PT
PT
AU
a) Distance between two parallel sides.
b) Maximal molecule diameter.
c) AU ¼ uranyl acetate; PT ¼phosphotungstate, MA ¼ ammonium molybdate.
The hexagon center is generally devoid of subunits and was
constituted by a hole of about 7–8 nm diameter; however, some
annelid EHbs were shown to possess a central subunit, such as
Oenone fulgida (Van Bruggen and Weber, 1974), Nepthys hombergii (Wells and Dales, 1976), Nepthys incisa (Wells and Dales,
1976; Messerschmidt et al., 1983; Vinogradov and Kapp, 1983),
Ophelia bicornis (Ghiretti-Magaldi et al., 1985; Mezzasalma et al.,
1985; Cejka et al., 1991, 1992), Maoridrilus montanus (Ellerton
et al., 1987), Glossoscolex paulistus (El Idrissi Slitine, Torriani,
and Vachette, 1990), Eophila tellinii (Cejka et al., 1989), Euzonus
mucronata (Terwilliger et al., 1977b), and Arenicola marina (Zal
et al., 1997a, 1997b) (Figure 16.3). The existence of a central
subunit suggested that this was similar or equivalent to another
one-twelfth of the native molecule, although to date no central
subunit has been isolated and/or characterized. GhirettiMagaldi et al. (1985) also postulated that the subunit for Ophelia
bicornis would differ from the other one-twelfth, while Messerschmidt et al. (1983) proposed that the central subunit for
Nephtys incisa was too small to correspond to one-twelfth of the
native molecule.
Figure 16.3 Three-dimensional cryomicroscopic structure of Arenicola marina EHb. Ludovic Jouan, 2003.
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16 Extracellular Hemoglobins from Annelids, and their Potential Use in Biotechnology
The use of TEM revealed a symmetric structure, and consequently several quaternary structure models were proposed.
However, in order to construct an accurate model, knowledge
is required of three parameters: (i) the molecular weight of the
biopolymer in its native condition; (ii) the number and relative
amounts of subunits and polypeptide chains that constituted the
molecule; and (iii) the accurate molecular weight of each component. Access to these parameters was gradually achieved due
to the emergence of new technologies such as mass spectrometry and multiangle light-scattering, which were first applied by
one of the present chapter authors (F.Z.).
A sixfold axis of symmetry was identified by using X-ray
diffraction with a resolution of 6 A (Royer and Hendrickson,
1988), although the existence of minor components was suggested without this symmetry. The same observation was made
several years later by Boekema and van Heel (1989), when using
electron microscopy with a resolution of 20 A. An analysis using
small-angle X-ray scattering of Lumbricus terrestris EHb – the
most extensively studied molecule – revealed the presence of a
central subunit that was not visible with TEM (Pilz, Schwarz,
and Vinogradov, 1980), in contrast to Tylorrynchus heterochaetus
where the subunit was visible (Pilz et al., 1988). The same
conclusion was drawn in the case of Arenicola marina and
Glossoscolex paulistus EHbs (Wilhelm, Pilz, and Vinogradov,
1980; El Idrissi Slitine, Torriani, and Vachette, 1990). A threedimensional reconstruction of Lumbricus EHb confirmed these
results when using scanning transmission electron microscopy
(STEM), and suggested that the mass could correspond to onetwelfth at the molecule center (Crewe, Crewe, and Kapp, 1984).
Alternatively, Vinogradov et al. (1986) suggested that the mass
could correspond to the linker chains. The same observation
was made via a three-dimensional reconstruction of the molecule by cryomicroscopy (Schatz et al., 1995; Taveau et al., 1999),
but these authors suggested that only immunolabeling could
confirm the exact nature of the central subunit. The major
conclusions deduced by Schatz et al. (1995) were the presence
of a sixfold axis of symmetry, in agreement with Royer and
Hendrickson (1988), and a threefold axis of symmetry at the
one-twelfth level. The latter level of symmetry was also confirmed by Martin et al. (1996a) after crystallization and observa
tion of the subunit at 2.9 A resolution.
16.4
Model of Quaternary Structures
Molecular weight determinations using the primary sequence
permitted the realization of several models of quaternary
structure for these EHbs. However, several approaches were
employed to understand the different component assemblages
inside the biopolymers (Table 16.3).
16.4.1
Electron Microscopy
Based on TEM images which showed molecular symmetry,
Rossi-Fanelli et al. (1970) proposed a model for Lumbricus
terrestris EHb (Figure 16.4). This model was inspired by the
Figure 16.4 First quaternary structure model proposed for Lumbricus
terrestris extracellular haemoglobin. Reused from Rossi-Fanelli et al., 1970
with kind permission from Elsevier.
Table 16.3 Main structural models proposed in the literature for the extracellular hemoglobins of annelids and Vestimentifera.
Species
No. of globin
chains
No. of linker
chains
Methods
Assembly for the
one-twelftha)
References
Tylorrynchus heterochaetus
Tylorrynchus heterochaetus
Perinereis aibuhitensis
Arenicola marina
Lumbricus terrestris
Lumbricus terrestris
Lumbricus terrestris
Macrobdella decora
Alvinella pompejana
Alvinella pompejana
Riftia pachyptila
192
144
192
156
192
144
144
144
144
120
144
24
36
24
42
24
36
36
42
60
72
36
HPLC
ESI-MS
HPLC
ESI-MS
HPLC
HPLC
ESI-MS
ESI-MS
ESI-MS
ESI-MS
ESI-MS
(T þ M)4 þ L2
(T þ M)3 þ L3
(T þ M)4 þ L2
(T þ M9) þ L3.5
(T þ M)4 þ L2
(T þ M)3 þ L3
(T þ M)3 þ L3
(D þ M2)3 þ L3.5
(T þ M)3 þ L5
(T þ M2)2 þ L6
(D þ M)4 þ L3
Suzuki et al., 1990a
Green et al., 1995
Matsubara et al., 1996
Zal et al., 1997a
Ownby et al., 1993
Vinogradov et al., 1991
Martin et al., 1996b
Weber et al., 1995
Zal et al., 1997b
Zal et al., 1997b
Zal et al., 1996a
a) M ¼ monomeric globin chain; D ¼ covalent dimer of globin chains; T ¼covalent trimer of globin chains; L ¼ linkers chains.
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Figure 16.5 Proposed model for Lumbricus terrestris extracellular
hemoglobin using transmission electron microscopy. (a) Face view;
(b) Internal bracelet constituted by D1 (central ring) and D2 (external ring)
subunits, where the twelfths of the molecule were fixed. With Courtesy by
J.C. Taveau, 1991.
findings of Guerritore et al. (1965), who were first to establish a
structure of chlorocruorine from Spirographis. In this model, the
polymer was constituted by an HBL structure with a maximum
diameter of 26.5 nm, where each hexagon was composed of six
identical triangular subunits (A in Figure 16.4), located at each
corner. Each of the subunits was built from three smaller
subunits (B in Figure 16.4), assembled in a triangle. The
molecular weights were estimated as 270 kDa for A and
92 kDa for B.
The model proposed for the Lumbricus terrestris EHb by
Vinogradov et al. (1986) is known as the “bracelet model,”
and is constituted by the assembly of monomers M and trimers
T with Mr-values of 16 and 50 kDa, respectively. The cohesion of
this structure was produced by a central ring constituted by two
other dimeric subunits that were devoid of heme, D1 and D2,
with Mr-values of 31 and 37 kDa, respectively. The assembly of
these subunits was modeled by Taveau (1991), based on TEM
images (Figure 16.5).
16.4.2
Estimation of Heme Number and Minimal Molecular Weight
The minimal molecular weight of the native EHb, using either
the heme or the number of iron atoms, revealed a value of about
one mole of heme per 27 000 Da of protein. This value was
higher than for the smaller subunit found on EHb, determined
either by electrophoresis or by using the primary sequences.
These observations confirmed that not all of the polypeptide
chains possessed a heme group, in contrast to vertebrate Hbs.
The mean mass of 27 000 Da corresponded to one heme for
about 1.5–2 globin chains.
Waxman (1971) suggested a model for the EHb of Arenicola
cristata in which two polypeptide chains of the three would be
able to bind a heme group. Some years later, Hendrickson and
Royer (1986) proposed a unique model for EHb molecules,
based on similar aspects of TEM images. In the latter model
the molecule was proposed to have been built by the assembly
of three different chains, of which two (a and b) would contain
a heme group, in agreement with the findings of Waxman
Figure 16.6 Proposed model for all annelid hexagonal-bilayer
hemoglobin. (a) Trion constituted by three polypeptide chains, two with
the similar tertiary structure of the myoglobin (a and b) and one chain
different (c); (b) Hexatrion built by the association of six trions; (c,d)
Localization of these chains on the whole hexagonal-bilayer hemoglobin
(c, profile view; d, face view). Reused from (Hendrickson, 1983) with kind
permission from Elsevier.
(1971). The molecule would be characterized by the same
folding as Mb but with the third (c) chain being different. The
three chains would be associated in trimer or “trion” fashion,
with six trions associated to a “hexatrion,” which itself would
be associated by 12 to create the EHb molecule (Figure 16.6).
This model described a biopolymer with Mr 3880 kDa, constructed from an association of 216 chains of which 144 would
possess heme.
The deficiency in the stoichiometry of one mole of heme per
polypeptide chain proved to be a major result, and allowed an
understanding of the quaternary structure of these huge molecules. However, the scientific community found this result very
difficult to accept, and both Antonini and Chiancone (1977) and
Garlick (1980) concluded that these results may have been due
to purification artifacts. Subsequently, the absence of stoichiometry was admitted without real proof, although Fushitani
et al. (1982a) rejected this hypothesis using high-performance
liquid chromatography (HPLC). In fact, these authors showed
that the polychete EHb from Travisia japonica was assembled
from five components with Mr-values ranging between 14 and
18 kDa, each of which contained one heme, although the
minimal molecular weight was about 23 000 Da (Fushitani
et al., 1982a; Fushitani et al., 1982b; Fushitani, Morimoto,
and Ochi, 1983) (Table 16.4).
When Suzuki et al. (1983) were able to isolate (by HPLC) only
six heme-components from the EHb of Perinereis brevicirris,
their result was in agreement with that of Fushitani et al.
(1982a). Subsequently, all of these results were challenged,
and following the determination of the primary sequences of
three globin chains involved in the trimer subunit of Lumbricus
EHb, Fushitani and Riggs (1988) recalculated the heme
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16 Extracellular Hemoglobins from Annelids, and their Potential Use in Biotechnology
Table 16.4 Minimum molecular weight (Da) obtained by iron or heme
content, or by electrophoresis (SDS–PAGE).
Minimal Mr
Species
Polycheta
Nephtys incise
Tylorrhynchus heterochaetus
Perinereis cultifera
Arenicola marina
Arenicola cristata
Abarenicola affinis
Abarenicola pacifica
Euzonus mucronata
Travisia japonica
Travisia fetida
Eunice aphroditois
Marphysa sanguinea
Pista pacifica
Thelepus crispus
Alvinella pompejana
Oligochaeta
Tubifex tubifex
Glossoscolex paulistus
Lumbricus terrestris
Maoridrilus montanus
Eisenia fetida
Pheretima communissima
Acheta
Haemopis grandis
Haemopis sanguisuga
Dina dubia
ND ¼ not determined.
Fe or Heme
SDS–PAGE
22 400
26 500
23 700
23 613
26 100
23 525
24 700
23 370
23 000
23 400
26 700
27 589
24 454
23 400
11 000
12 000
13 000
14 000
13 000
13 700
15 800
12 500
14 000
14 600
14 600
14 000
15 000
15 000
13 900
21 000
25 250
24 500
23 900
24 700
25 430
13 000
ND
12 400
13 500
14 800
14 500
24 000
24 800
21 200
13 000
12 600
12 000
percentage. Their results suggested that all three chains possessed heme and a proportion of about 32.5 kDa of protein
without heme per one-twelfth of the molecule that remained
(Fushitani and Riggs, 1988). Using the HPLC data, Gotoh and
Suzuki (1990) proposed a quaternary model for Tylorrynchus
heterochaetus EHb which, according to these results, would
consist of an assembly of four globin chains. This model
described a molecule built by assembling 192 globin chains.
Later, Suzuki et al. (1990a) sequenced two linker chains without
heme from the Tylorrynchus EHb, and confirmed that not all
polypeptide chains in EHb would contain a heme group, thus
rejecting in facto their previous model.
16.4.3
Small-Angle Light Scattering
The model proposed by El Idrissi Slitine (1991) for Arenicola
marina was obtained by the assembly of 186 basic spherical
units of 21 A radius. This radius was calculated as being the
distance between the next two spherical units, and was inferior
to the units’ own diameter. This model was based on three
different levels: (i) a “peripheral structure” made from 14
elemental spheres set out in three stairs (4/6/4); (ii) a “central
structure” formed by six spheres arranged in this way (1/4/1);
and (iii) a “contact structure” constituted by two spheres,
which occupied the free space between the peripheral and
central structures (Figure 16.7). This model described a molecule built by the assembly of 12 “peripheral structures” linked
together by 12 “contact structures”; this assemblage surrounded a protein material, the density of which could not
be attributed exactly to a complete one-twelfth (El Idrissi
Slitine, Torriani, and Vachette, 1990). This model described
Figure 16.7 Model proposed for the extracellular hemoglobin from Arenicola marina, based on small-angle X-ray scattering data. P ¼ peripheral structure;
C ¼ central structure; L ¼ contact structure. Courtesy of Fouzia El Idrissi Slitine, 1991.
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16.4 Model of Quaternary Structures
a molecule of 168 chains, of which 144 globin chains and 24
linker chains were devoid of heme.
16.4.4
Low- and High-Pressure Liquid Chromatography and
SDS–PAGE
To date, the details of two major quaternary models for
Lumbricus terrestris EHb have been reported. For both models,
each twelfth is constituted only by the globin chain, and the
twelfths were tied together by the intermediate of linker
chains that possessed no or little heme. The first of these
models was proposed by Vinogradov et al. (1986), and
described a molecule assembled from 12 subunits (“dodecamers”) and constituted 12 globin chains (Vinogradov et al.,
1991; Sharma et al., 1996). The molecule was composed of
144 globin chains, associated with 36 linker chains.
Although, the names of the different components have
changed with time, at this point the first nomenclature
will be used. For Vinogradov’s model, a monomer M
(i.e., d) with a Mr of 16 750 Da possessing a heme group
was associated with a trimer subunit T (i.e., a, b, c) with a Mr
of about 50 000 Da; together, this complex constituted a
tetramer. The trimer complex constituted the covalent assembly of three globin chains with Mr about 16 000 Da, and hence
each twelfth or dodecamer was constituted by the assembly of
three tetramers (3 (M þ T)). These twelfths were tied
together in the native molecule by two different monomers
D1 and D2 of Mr about 31 and 32 kDa, respectively. D1 and
D2 were suspected of being devoid of heme, and to have a
protein bracelet at the molecule centers.
The bracelet model was based on TEM images as well as
dissociation–reassociation experiments (Kapp et al., 1984,
1987; Mainwaring et al., 1986; Vinogradov, 1986; Vinogradov
et al., 1986), and contained 200 polypeptide chains with a
global Mr of 3800 kDa. In addition, the model could explain
the heterogeneity of the results observed with SDS–PAGE, as
the molecule could be cleaved randomly at different levels
(Figure 16.8).
The second model proposed for Lumbricus terrestris was
built by assembling 192 globin chains (i.e., a, b, c, d) and 24
linker chains (i.e., three majors L1, L2 and L3 and one minor
L4) (Fushitani and Riggs, 1988; Ownby et al., 1993; Zhu et al.,
1996). For this model, each twelfth would be constituted by a
dimer of tetramer [(a,b,c,d)2] that would be able to dimerize to
create a tetramer of [(a,b,c,d)4]. The molecule would be
constituted in the following way: [(a,b,c,d)4,L2] 12, where
a, b, c and d corresponded to globin chains, and L to linker
chains. This conceptual model would have a Mr of 3800 kDa,
as for the bracelet model (Fushitani and Riggs, 1988; Riggs,
1990).
16.4.5
Electrospray Ionization-Mass Spectrometry
A more accurate technique, electrospray ionization-mass spectrometry (ESI-MS), was successfully applied to some HBL Hbs,
allowing the determination of their complete polypeptide chain
composition. The EHbs examined were from Macrobdella decora
(Weber et al., 1995), Tylorrynchus heterochaetus (Green et al.,
1995), Lumbricus terrestris (Martin et al., 1996b), Riftia pachyptila
(Zal et al., 1996a), and Arenicola marina (Zal et al., 1997a, 1997b).
The models of these molecules, obtained by ESI-MS, were
compared to those obtained with HPLC in Table 16.3. The
model obtained for Arenicola marina EHb HBL, using ESIMS data, is shown in Figure 16.9. The one-twelfth protomer
would be constituted by the following assembly [(3a1)(3a2)2T],
where T corresponds to either T3, T4, or T5. T1 and T2 were
probably located at the molecule center, as noted in previous
studies.
(a)
(b)
T3
T5
T4
T
T
T4
T5
T3
Figure 16.8 Cleavage possibility of the extracellular hemoglobin from
Lumbricus terrestris, according to the bracelet model proposed by
Vinogradov et al. (1986). This scheme explains the heterogeneity of SDS–
PAGE results. (a) Face view (T ¼ trimer; M ¼ monomer; D1 (V) and D2
(VI) ¼ linker chains; (b) Profile view. V and VI correspond with the first
nomenclature used to name these chains. Courtesy of S. Vinogradov.
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a2 a2
a1 a1
a1 T
a2
a2 a2
a2
Figure 16.9 Model of Arenicola marina HBL Hb, adapted from Waxman
(1971). (a) Drawing of typical HBL Hb, top view. Each one-twelfth is
constituted by one trimer (T2, T3 and T4), as noted by Waxman (1971),
with nine monomeric chains. The two trimers (T), for example, T1 and T1
or T1 and T2 at the center of the HBL structure, are represented as viewed
on the TEM image; (b) Detail of a one-twelfth, showing the arrangement of
monomeric chains (Zal et al., 1997a).
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16 Extracellular Hemoglobins from Annelids, and their Potential Use in Biotechnology
16.5
Biotechnology Applications
16.6
Organ Preservation
As noted briefly above, many research groups worldwide have
investigated the annelid EHb, demonstrating the great interest
in this molecule among the international scientific community. Among these groups, the more active in the field in
France was led by Prof. Andre Toulmond and was located
initially in Paris on the University Paris VI campus (Pierre-etMarie-Curie University). In 1993, this laboratory moved to the
Marine Biological Station Roscoff (Finistere, France), and
Prof. Toulmond became the Director of this very old and
famous center (founded in 1872 by Prof. Henri de LacazeDuthiers, at the Sorbonne University), between 1993 and
2003. Franck Zal, a PhD student of Andre Toulmond, began
to work on Arenicola marina in 1993, and described in detail
the structure of several EHbs. Zal discovered that the Hb
molecule of Arenicola marina (HbAm) had all the characteristics of a “universal oxygen carrier” that medics had been
seeking for several decades for therapeutic applications.
HbAm has exceptional properties:
In 2005, almost 93 000 organ transplants were conducted
worldwide, and the number of transplants is growing each
year by 5%. For example, 25 000 transplants were conducted
in the US alone in 2003. In France, the annual number of
transplants is constantly growing, having risen from 3115 in
1998 to 4946 in 2011. During the latter year about 300 people
died in France due to a lack of transplants, whilst in 2012 some
10 400 patients were awaiting transplant. The success of organ
transplantation has resulted in a rapid increase in the discrepancy between the number of patients waiting to be transplanted
and the number of transplants available. The available solutions
include splitting an organ between several receivers (e.g., liver),
xenotransplantation (using organs from animals, mostly cardiac
valves or islets of Langerhans from pigs), or the culture of
organs from stem cells. Unfortunately, as these solutions are
only at the exploratory stages, a much better solution might be to
extend the duration of organ preservation after collection from a
cadaver, by using hypothermic continuous reperfusion so as to
minimize organ loss.
The main objective of organ preservation is to extend the
period of ex vivo viability of an organ, allowing for its transfer
from the removal center to the transplant center. Under classical
static organ conservation conditions, the conservation times
possible before ischemia sets in differ widely and depend on
the organ concerned. For example, the kidney, heart, liver, lungs
and pancreas are very sensitive to cold ischemia.
The limited life span of transplants is due to deleterious
effects induced by interruption of the blood circulation (ischemia) which, at 37 C, causes a rapid cellular necrosis of the
removed organ. Ischemia can be retarded by refrigerating the
organ, as a decrease in temperature causes a decline in cellular
energy requirements (the O2 requirement at 5 C is only 5% of
that at 37 C). Ischemia in hypothermia (cold ischemia) starts as
soon as the washed organ is refrigerated, and continues until it
is reperfused in the recipient. Cooling does not completely stop
cellular metabolism, but does slow down the speed of enzymatic
reactions and delays cellular death. However, the use of cold
ischemia is time-limited, as the absence of a sufficient supply of
O2 causes a metabolic shift towards anaerobic glycolysis. This
leads to the generation of lactic acid with cellular acidification
from the rupture of lysosomal membranes and cellular destruction by the liberated proteolytic enzymes. The results is an
accumulation of various toxins, including cytotoxic free radicals,
that are passed to the receiver during organ reperfusion.
It is naturally extracellular and polymerized.
Its molecular weight is 50-fold that of human Hb.
It has functional O2-binding and -liberating properties similar
to those of human Hb (HbA inside the red blood cell).
Each HbAm molecule can bind 156 molecules of oxygen,
compared to four in the case of human HbA
It has naturally antioxidative properties.
Historically, and before this discovery, two approaches had
been investigated to develop universal oxygen carriers: (i) a
chemical method using perfluorocarbons; and (ii) biological
methods using human or bovine Hb (Ketcham and Cairns,
1999; Gulati, Barve, and Sen, 1999; Winslow, 2000. In March
2007, Dr Franck Zal and Dr Morgane Rousselot created the
French biotechnology company, HEMARINA, in order to
develop and promote the molecule HbAm, which was seen
to possess all of the characteristics necessary to become the
leading third-generation blood substitute. In addition to the
data outlined above, the principal characteristics of HbAm are
that:
The functional properties are totally independent of
secondary molecules, such as 2,3-DPG.
The molecule functions without any chemical modification,
and with no additional treatment.
The molecule has a functional capability at temperatures
between 4 C and >30 C, which is a major and essential
advantage.
The absence of any vasoconstrictor effects is in contrast to all
other products developed to date and referred to as hemoglobin oxygen carriers (HBOCs) (Tsai et al., 2012).
Although several major applications for HbAm have already
been identified, attention here will be focused on only two,
namely organ preservation pending transplantation and the
development of an innovative oxygen carrier.
16.6.1
Preservation Solutions
The deleterious effects of cold ischemia can be attenuated
through the use of preservation solutions, which allow the
reduction of cellular edema by maintaining the extracellular
osmotic pressure, thus preventing intracellular acidosis and
reducing reperfusion injuries due to free radical accumulation.
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16.7 Anemia
Some years ago, the ANSM (Agence National de Securite du
Medicament) questioned the efficiency of organ preservation
solutions.
16.6.2
Hypothermic Continuous Reperfusion
In general, following its removal a transplant can be conserved by
simple immersion in a preservation solution at 4 C. This static
technique is simple and inexpensive; however, due to the urgency
of the situation related to lack of organs and to the degree of
ischemia of organs currently being transplanted, a hypothermic
continuous reperfusion technique has been developed. This
dynamic technique, which is presently being applied to kidney
transplants, requires sophisticated and expensive equipment.
Currently, two main dynamic systems have been developed,
the performances of which are similar. Unfortunately, the use
of perfusion equipment is limited to a small number of centers in
Europe, and the preliminary results do not indicate a significantly
better performance than static methods.
Because it is functional in the natural environment at low
temperature, HbAm was manufactured by HEMARINA as the
product HEMO2life1 which, when added to several clinical
storage media, insured oxygenation of the organs. The recovery
of organ grafts maintained in media with HEMO2life1 and then
transplanted into pig models was shown to be excellent (Figure 16.10). HEMO2life1 is currently at the final stage of
registration, with efficiency having already been demonstrated
for kidney, lung, and heart. HEMO2life1 also possesses naturally an intrinsic superoxide dismutase (SOD) activity on
HbAm, which allows it to protect the cells against oxidative
damage caused by an accumulation of free radicals liberated
during cold ischemia. HEMO2life1 could also increase the
efficiency of dynamic systems for the conservation of kidneys,
and probably also for other organs with shorter preservation
times (e.g., heart, liver, pancreas, lung).
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16.7
Anemia
According to a World Health Organization reports, one in ten
people admitted to hospital requires a blood transfusion. The
majority of these cases are anemia, due to a decrease in Hb
levels following the loss or reduced production of red blood cells
(RBCs). A loss of RBCs may be due to hemorrhage during a
trauma (accidents, injuries, burns) or during surgery (coronary
artery bypass, organ transplants, hip replacement, etc.). Anemia
can also be caused by hemolytic anemia (e.g., drepanocytosis). A
reduced RBC production may occur in cases of global medullary
insufficiency and bone marrow cancer, as well as being a side
effect of radiotherapy or chemotherapy. The aim of transfusing
RBC concentrates is to maintain or to restore a sufficient level of
Hb. Typically, 50% of all RBCs are transfused to patients aged
>65 years, and demographics show that, in developed countries,
this age category will double over the next 30 years. Certain types
of surgery that are becoming increasingly common require an
essential number of RBCs (e.g., up to 100 RBC units for one
liver transplantation). Moreover, these trends are also observed
in emerging countries (India, China, Brazil), and it is agreed
that worldwide RBC demands to treat anemia will continue to
increase.
As an example, in 2009 the number of RBCs transfused was
about 93 million units, which represents an increase of 33%
compared to 2003. Moreover, this situation is not expected to
improve with demands continuing to increase, especially when
associated with a stagnation or even regression in donations.
Today, the exclusion criteria for donors are expanding due to
emergent risks relating to transfusions (Nile virus, severe acute
respiratory syndrome, prions, chikungunya virus, etc.), and this
is causing a regression in the number of regular donors. Experts
in the field maintain that the shortage in blood products worldwide is 100 million liters per year, and these requirements are
constantly increasing.
Figure 16.10 Kidney function following reperfusion. Porcine kidneys were cold-flushed and preserved for 24 h with UW or HTK supplemented with M101
at a concentration of 0 g l1 (UW and HTK groups) or 5 g l1 (UW þ M101 and HTK þ M101 groups). Follow-up was performed on the day before
transplantation (D-1) and after transplantation (at 1 h ¼ R60; from day 1 to day 14 ¼ D1 to D14; and 1 month ¼ M1). Values shown are mean SEM.,
p <0.05 versus UW; y, p <0.05 versus HTK; n ¼ 5. UW ¼ University of Wisconsin organ preservation solution, patented by the university (also called
VIASPAN); HTK ¼ Histidine-tryptophan-ketoglutarate organ preservation solution (also called Custodiol); M101 ¼ Molecule 101, the development name
given by HEMARINA to the hemoglobin of Arenicola marina.
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16 Extracellular Hemoglobins from Annelids, and their Potential Use in Biotechnology
16.7.1
Hemoglobin Oxygen Carriers
Initially, HBOCs were developed in response to this blood
shortage. The short half-life of HBOCs (ranging from several
hours to 2–3 days) compared to RBCs (120 days) means that
they are only suitable for use in acute anemia (programmed
surgery, traumatology), though these represent 60% of current
prescriptions for RBCs. Today, experts estimate that oxygen
transporters will not replace RBCs from donors for the treatment of anemia. In the case of HBOCs, this is due to the very
large amounts of active material that must be produced, the
limited sources of raw materials, and the costs related to
chemical modifications. Perfluorinated compounds (PFCs)
are limited in their therapeutic applications, as only small
quantities can be used because of their high retention, and the
need for concomitant use of an oxygen mask. Nevertheless,
HBOCs can represent an additional therapy for certain applications, notably normovolemic hemodilution and emergencies relating to hemorrhagic shocks.
16.7.2
Normovolemic Hemodilution
Normovolemic hemodilution has been recommended for
decades. The principle is simple: blood is taken from the
patient who will undergo surgery and replaced by filling
liquids (colloids and crystalloids) in order to maintain the
intravascular volume. After surgery, the blood that had been
removed is re-transfused to the patient. Dilution of the circulating blood reduces the number of RBCs lost by hemorrhage
during the intervention, which in turn reduces the need for
homologous transfusions postoperatively. This has a double
advantage, in that homologous RBC stocks are saved, and
there is an avoidance of exposure to homologous blood, which
carries a sizeable residual risk. In France today, three accidents
occur for every 1000 homologous transfusions. These problems occur mainly due to blood typing errors (donor or
receiver) or to sanitary issues (bacteria or viruses below
detection levels during tests). Available data on normovolemic
hemodilution show, however, that the objective targeted by
this strategy (a reduction in homologous blood use) is rarely
attained under most surgical conditions. The mathematical
modeling of normovolemic hemodilution indicates that the
chances of reaching this goal are much higher if the volume of
blood removed is high before preoperative blood losses. However, in practice this theoretically optimal hemodilution is
rarely achieved because of issues relating to individual tolerance levels of patients.
16.7.2.1 HEMOXYCarrier®
HEMARINA is currently developing a product called
HEMOXYCarrier1, which contains HbAm, for such applications. The coadministration of HEMOXYCarrier1 with filling
liquids would allow a greater degree of hemodilution in order
to obtain a higher efficiency of this practice. HEMOXYCarrier1, which is highly soluble and stable when diluted, should
temporarily mediate the lack of oxygen inherent in the simple
use of a filling solution.
Anemia can be caused by intense and brutal bleeding
after a trauma, typically accidents, gunshot wounds or
rupture of the vascular system (aneurysm). The tolerance
of certain vital organs (brain, heart) to a massive reduction
in O2 supply is weak. Trauma situations generally occur
outside hospitals and are managed by mobile emergency
units, and often the blood group of the victim is not known.
The stocks of O blood (universal donor) carried by emergency vehicles are insufficient to compensate for important
losses and, in the worst cases, only filling solutions are
used. In this situation, emergency units require a product
that is compatible with all blood groups, and easy to store
and use. This type of product would be of special interest
for the treatment of victims of natural catastrophes and of
war casualties. The intravenous administration of HEMOXYCarrier1 permitted a rapid, efficient and simple restoration of the oxygenation capacity affected during massive
blood loss in an animal model, without any side effects
(Rousselot et al., 2006; Tsai et al., 2012). This molecule is
stable at ambient temperature and can also be lyophilized.
Moreover, its properties would allow mobile emergency
units to dispose of sufficient products to meet important
and unpredictable demands.
Preclinical studies performed on rodents have shown that the
systemic injection of this molecule does not generate immunogenicity and allergic reactions, and does not provoke any vasoconstrictive effects. It has been possible to directly visualize the
efficiency of the molecule on hypoxic tissues immediately after
systemic perfusion (Rousselot et al., 2006; Tsai et al., 2012; T. Le
Gall et al., unpublished results).
16.8
Conclusion
To summarize, as opposed to the development of first- and
second-generation HBOCs that modify intracellular hemoglobin (human or bovine) in order to function extracellularly, a
natural extracellular hemoglobin from the invertebrate Arenicola marina (HbAm) was used. This circulatory pigment,
which is present in the blood vessels of the invertebrate,
has evolved over millions of years and is able to function
extracellularly. It was shown, using vertebrate animal models,
that this molecule does not induce vasoconstriction, which
was the major secondary effect of the last generation of
HBOCs. As a consequence, HbAm can be considered a
lead molecule for the future development of an oxygen carrier
for ischemia or anemia applications. In this respect, two
products are currently under development by the HEMARINA
company, namely HEMO2life1 for organ preservation, and
HEMOXYCarrier1 as a universal oxygen carrier.
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References
Acknowledgments
Dr Franck Zal is very grateful to Prof. A. Toulmond, who was his
advisor during his PhD studies and introduced him to the
hemoglobin research field. The authors would also like to thank
j 373
Nelly Rolland for its help to get on time all journal authorisations for this review. Conflict of interest: It should be noted
that the authors are the founders of HEMARINA and hold
company stock.
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About the Authors
Franck Zal is the cofounder of HEMARINA and recognized
internationally for his studies in the field of the respiratory
pigments of invertebrates. He received his PhD degree with
honors in oceanography in 1996 from the University of Paris
VI (Pierre-et-Marie-Curie), where his doctoral research focused
on the structure–function relationships of the annelid extracellular hemoglobin. He then spent three years abroad as part of
a post-doctoral program, two years at the University of Santa
Barbara in California (USA), and one year at the University of
Antwerp (Belgium). For his work, he received in 2001 the
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16 Extracellular Hemoglobins from Annelids, and their Potential Use in Biotechnology
bronze medal of CNRS (French National Research Center).
Dr Zal is the author of over 70 publications in international
peer-reviewed scientific journals, the author of ten patents, and
has made presentations at over 100 international scientific
meetings. He was also the head of an academic research group
working on marine ecophysiology at a marine station in
Brittany, CNRS-UPMC. In 2007, he decided to create HEMARINA, a marine biotechnology start-up located in Morlaix
(Finistere, France). The Capital Magazine identified this company in France as the most promising biotechnology company.
HEMARINA won almost all prizes for innovative enterprise
in France, and especially the great prize of the Senat in France
in 2007. Franck is also the cofounder of the Enterprise club
Bretagne Biosciences and was vice-president of France Biotech.
Morgane Rousselot PhD is cofounder of HEMARINA, and in
charge of the operational aspects of Hemarina research and
development programs. In 2001, she was employed for a year in
the Research and Development Analytical Chemistry Department of GlaxoSmithKline in the UK. In 2002, she obtained a
degree in Chemical Engineering (Dipl^
ome d’Ingenieur) at
ENSCMu (Ecole Nationale Superieure de Chimie de Mulhouse)
with qualifications in Chemistry-Biology-Healthcare. During the
same year she obtained her master’s degree in Interface Chemistry-Biology and molecular modeling. She received her PhD
with honors in biochemistry in 2006 from the University of Paris
VI (performed at the Station Biologique of Roscoff), where her
doctoral research focused on the properties of Arenicola marina
hemoglobin (HbAm) and its potential use as a blood substitute.
In 2006, she obtained a grant “Jeune Createur d’Entreprise”
delivered by Bretagne Innovation in order to create the Biotechnology society Hemarina. She possesses strong expertise in
analytical chemistry and structural biology). Dr Rousselot is the
author of eight publications in international peer-reviewed scientific journals, the author of four patents, and has made
presentations at over 10 international meetings.
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