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Oxygen-binding molecules are ancient proteins. They
probably evolved from enzymes that protected the organism
against the toxic oxygen molecule. As single-celled organisms
developed the capacity to use oxygen as an electron acceptor,
capturing oxygen and transferring it to the respiratory chain
became increasingly important. Intracellular protoheme or
myoglobin-type proteins, and perhaps copper proteins, that
could enhance oxygen diffusion and storage began to appear.
When multicellular organisms increased in size and
complexity, their surface to volume ratios diminished, and
simple diffusion of oxygen across the body wall was
inadequate to reach all of the cells. The development of
vascular and coelomic circulatory systems that could move
oxygen away from the inner body wall enhanced the oxygen
diffusion rate, but the low solubility of oxygen in body fluids
was still limiting. The evolution of simple oxygen-binding
proteins into multisubunit, circulating proteins, in combination
with the advent of circulatory systems, made possible the
transport of oxygen on a significant scale from the periphery
of the organism to metabolizing cells in its interior. This
discussion will focus on animal hemoglobins, hemerythrins
and hemocyanins that function as oxygen-transport molecules
and will refer only briefly to monomeric heme proteins, such
as myoglobins and myohemerythrins involved in intracellular
oxygen transfer and storage. Of course, effective oxygen
transport includes delivery, and thus transport molecules have
a transfer function as well. So that evolutionary patterns may
become more clear, I will describe the phylogenetic
distribution and general properties and will present specific
examples of how organisms use these proteins to obtain
appropriate amounts of oxygen.
Oxygen-transport proteins
There are two major kinds of oxygen-transport proteins,
those with iron as the prosthetic group, which reversibly binds
to oxygen, and those with copper. As illustrated in Fig. 1, the
circulating iron proteins include (1) cellular hemoglobins or
red blood cells, (2) giant extracellular hemoglobins and
chlorocruorins, and (3) cellular hemerythrins or pink blood
cells. In hemerythrins, oxygen binds to iron that is covalently
linked to the protein molecule; in hemoglobins and
chlorocruorins, the iron is coordinately bound to a
protoporphyrin IX or heme group. The heme group is attached
to the protein, a globin. Copper-based, blue oxygen-transport
proteins include (1) molluscan hemocyanins and (2)
arthropodan hemocyanins. While the two hemocyanin proteins
are dissimilar in quaternary structure and sequence, the active
sites in both hemocyanins are similar although not identical.
The active sites include six highly conserved histidines that
bind two copper atoms; both coppers together bind one oxygen
molecule, reversibly. Hemerythrins and hemocyanins are
1085
The Journal of Experimental Biology 201, 1085–1098 (1998)
Printed in Great Britain © The Company of Biologists Limited 1998
JEB1499
Oxygen-transport proteins are multisubunit, circulating
molecules that provide an efficient supply of oxygen to
metabolically active metazoans. Hemoglobins,
hemerythrins and hemocyanins have evolved in both
structural and functional diversity and exhibit functional
repertoires beyond that of simple, monomeric tissue
myoglobins. Their phylogenetic distribution is intriguing,
especially with respect to those organisms that express
more than one type of oxygen-transport protein. An animal
can modify the delivery of oxygen to its tissues by varying
the rate of synthesis of these proteins or by selective
expression of individual subunits and/or molecules.
Changes in levels of allosteric modifiers that affect the
protein’s oxygenation properties will also modify oxygen
delivery; some organisms have more ability than others to
control concentrations of modulators. Hemoglobins have
assumed functions in addition to oxygen transport, while
hemocyanins have diversified through multiple gene
duplications and functional specializations. Understanding
the mechanisms of regulation of expression, synthesis and
modulator levels is a key focus of current investigations.
Key words: hemoglobin, hemerythrin, hemocyanin, hypoxia, oxygen
transport.
Summary
Introduction
FUNCTIONAL ADAPTATIONS OF OXYGEN-TRANSPORT PROTEINS
NORA B. TERWILLIGER*
Oregon Institute of Marine Biology, University of Oregon, Charleston, OR 97420, USA and Department of Biology,
University of Oregon, Eugene, OR 97403, USA
*e-mail: nterwill@oimb.uoregon.edu
Accepted 15 January; published on WWW 24 March 1998
1086
colorless in the deoxy form. Almost all of the iron and copper
proteins involved in oxygen transport are multi-subunit
proteins. Consequently, they exhibit cooperative oxygen
binding and allosteric modulation of oxygen affinity,
properties that expand their functional repertoires beyond that
of a simple monomeric myoglobin. In the oxygen-transport
proteins, then, the evolution of functional diversity parallels
that of form. Tracing these patterns requires an overlay of
phylogeny with form and function.
Molecular size and structure
Hemoglobins in circulating red blood cells are composed of
polypeptide chains or subunits of approximately 17kDa, each
containing a heme group (for a review, see Royer, 1992). Each
subunit is a functional unit, or monodomain, that combines
with one oxygen molecule. The subunits in red blood cells
occur as monomers or aggregate to form dimers, tetramers or,
rarely, octomers. The exception to this rule is the hemoglobin
of Barbatia reeveana, a bivalve mollusc found in the Sea of
Cortez. B. reeveana red blood cells have a typical tetrameric
hemoglobin, plus another large polymeric hemoglobin, of
430 kDa, composed of 34kDa didomain subunits. Each subunit
of the polymeric hemoglobin, the largest intracellular
hemoglobin known, contains two oxygen-binding functional
units, covalently linked (Grinich and R. Terwilliger, 1980).
The DNA coding for this unique hemoglobin is
correspondingly long (Riggs et al. 1986; Riggs and Riggs,
1990). Since there is one example, there are probably other
dogma-confounding large multidomain intracellular
hemoglobins; they remain to be discovered.
Extracellular hemoglobins are extraordinarily diverse in
both quaternary structure and subunit size, although they share
the same myoglobin fold and heme moiety as the red blood
cell hemoglobins (for a review, see Terwilliger 1992). Annelid
extracellular hemoglobins and chlorocruorins use 17kDa
monodomain subunits, but assemble approximately 200 of
them, plus some non-heme-containing linker chains, into
beautiful hexagonal bilayers of approximately 3500kDa
(Vinogradov et al. 1986; Riggs, 1990, 1998). Arthropod
extracellular hemoglobins are composed of mono-, di- or
nonadomain subunits, with a range of matching quaternary
structures, depending on the species (Moens and Kondo, 1976;
Dangott and Terwilliger, 1981; Manning et al. 1990).
Molluscan extracellular hemoglobins are huge. Each subunit
consists of 17kDa functional units, 10–12 in planorbid snails,
18–20 in the bivalve families Carditidae and Astartidae, that
are covalently linked into long polypeptide chains. The
170kDa snail subunits assemble into 1700kDa ring-shaped
hemoglobins (Wood and Mosby, 1975; Terwilliger et al.
1976), while the 340kDa bivalve hemoglobin subunits form
8000–12000kDa rod-shaped assemblages (Waxman, 1975;
Terwilliger and Terwilliger, 1978).
Pink blood cell hemerythrin subunits are 13.5kDa
monodomain polypeptides. They are usually assembled into
108 kDa octomers, although trimers, tetramers and dimers have
also been described (for a review, see Kurtz, 1992). A 690kDa
hemerythrin aggregate has been reported for the vascular
hemerythrocyte of the polychaete annelid Magelona
papillicornis (Manwell and Baker, 1988); it probably has a
low-molecular-mass, monodomain subunit similar to the
14.7kDa subunit of a closely related species, Magelona alleni
(N. B. Terwilliger, unpublished observations).
Molluscan hemocyanins, like molluscan extracellular
hemoglobins, are composed of large multidomain subunits that
self-assemble into elegant symmetries (for a review, see van
Holde and Miller, 1995). Seven or eight oxygen-binding units
per 350–450kDa subunit are assembled into decameric
cylindrically shaped oligomers of 3500–4500kDa in chitons
and cephalopods. Gastropod and bivalve hemocyanins are
didecameric cylinders of 8000–10000kDa.
Arthropod hemocyanins are intermediate in size between
annelid extracellular hemoglobins and molluscan
hemocyanins. Six 75kDa subunits, each with one oxygen-
binding site, assemble into a 450kDa hexamer. The hexamers
assemble further into two-hexamers, four-hexamers, six-
hexamers or eight-hexamers depending on the class or species
(for a review, see Markl and Decker, 1992).
Most of these oxygen-transport proteins do not consist of a
single homogeneous array of subunits, but instead show
varying degrees of subunit heterogeneity. Arthropod
N. B. TERWILLIGER
Fig. 1. Oxygen-transport proteins. (A) Cellular hemoglobins or red
blood cells; (B) extracellular hemoglobins (annelid chlorocruorin and
hemoglobin, mollusc bivalve extracellular hemoglobin, arthropod
extracellular hemoglobin); (C) cellular hemerythrins or pink blood
cells; (D) molluscan hemocyanin (chiton or cephalopod, gastropod or
bivalve); (E) arthropod hemocyanin (one-, two-, six-, four- and
eight-hexamers). Models not drawn to scale.
1087Functional adaptations of oxygen-transport proteins
hemocyanins are particularly renowned for the number of
different kinds of polypeptides within a multisubunit molecule.
The heterogeneity expands the functional properties of the
respiratory protein, since different subunits may have different
oxygen affinities or responses to allosteric modifiers.
Phylogenetic distribution
The expression throughout the animal kingdom of these
oxygen-transport proteins and of the tissue protoheme proteins
is portrayed in Table 1. Phyla in which no respiratory protein
has been identified have not been listed. The extent to which
these proteins are expressed within a phylum varies greatly.
For example, almost all annelids contain hemoglobins
(myoglobin, red blood cells and/or extracellular hemoglobins),
while only certain families within two of the six classes of
echinoderms contain hemoglobin. Tissue myoglobins are most
widely distributed throughout the biosphere and are present in
prokaryotes, protists, plants and animals. Red blood cell
hemoglobins are represented in almost all protostomes and
deuterostomes, triploblasts with the requisite mesodermal
potential to form coeloms and vascular systems within which
the cells can circulate. A notable exception is the phylum
Arthropoda, with no examples of circulating red blood cells.
Hemerythrins are expressed in four phyla; however, they are
mostly small phyla, except for the Annelida. Here, hemerythrin
is known only in one family of worms, the Magelonidae.
Extracellular hemoglobins occur frequently in the protostomes
but are not expressed in the deuterostome line of evolution.
The two kinds of hemocyanins are each found only in a single
phylum, and thus at first glance are much more limited in
expression than are the heme proteins. The Mollusca and
Arthropoda are such huge, diverse phyla, however, that there
are a great many species utilizing these copper-based proteins
for oxygen transport (Fig. 2). At the same time, many molluscs
and some arthropods contain hemoglobins instead of, or in
addition to, hemocyanin. What are the selection factors that led
to the utilization of a particular oxygen-transport protein? Even
more intriguing, why do some organisms express more than
one kind, what are the regulatory signals for each protein, and
how is the expression of multiple oxygen-transport proteins
coordinated?
Adaptations of oxygen-transport molecules
With phylogeny of oxygen-transport proteins as a
framework, one can develop a broader understanding of
different ways in which organisms use these proteins to
facilitate the uptake and delivery of oxygen in a changing
environment. How are these molecules adapted to provide
adequate oxygen to the tissues under variable external and
internal conditions? Detailed information on both the structure
and function of oxygen-transport proteins are available in
chapters by individual authors in Mangum (1992), as well as
in reviews by van Holde and Miller (1995) and Mangum
(1997).
Regulation of oxygen-carrying capacity
Changing the rate of synthesis or catabolism of an oxygen-
transport protein will obviously affect how much oxygen the
blood can transport.
Environmental hypoxia
Environmental hypoxia is a key stimulus to increase the
production of an oxygen-transport protein and thereby to
increase the oxygen-carrying capacity of the blood. Oxygen
sensors and the regulation of hemoglobin expression are topics
of current research (Bunn et al. 1998; Hand, 1998; Hardison,
1998; Hochachka, 1998; Ratcliffe et al. 1998). Several
arthropods respond to environmental oxygen levels by
increasing or decreasing the rate of synthesis of an extracellular
hemoglobin, thus changing the oxygen-carrying capacity to
meet the need. The primitive crustaceans Artemia salina (Heip
Table 1. Phylogenetic distribution within the animal kingdom of oxygen-transport proteins and tissue protoheme proteins
Tissue Red blood cell Extracellular Extracellular Molluscan Arthropod
protoheme hemoglobin hemoglobin chlorocruorin hemocyanin hemocyanin Hemerythrin
Protista x
Platyhelminthes x
Nematoda x x
Phoronida x
Nemertea x x x
Priapulida x
Brachiopoda x
Sipunculida x
Annelida x x x x x
Vestimentifera x
Pogonophora x
Echiura x x
Mollusca x x x x
Arthropoda x x
Echinodermata x
Chordata x x
1088
et al. 1978b) and Daphnia magna (Kobayashi et al. 1988) both
respond to hypoxia with an increase in hemoglobin synthesis.
Kobayashi and Gonoi (1985) showed that the swimming
ability of Daphnia magna in hypoxic water was directly
proportional to the animal’s hemoglobin content. Red Daphnia
raised under conditions of low oxygen level swam faster and
for longer than pale Daphnia. Larvae of the phantom midge
Chironomus thummi thummi, an insect, also produce more
hemoglobin under hypoxic conditions (Weber et al. 1985). The
amphipod Cyamus scammoni, an obligate ectosymbiont on the
gray whale Eschrictius robustus, has an 1800kDa,
multidomain extracellular hemoglobin in its hemolymph
(Terwilliger, 1991). Cyamus scammoni, along with its host,
summers off the coast of Alaska and rides down to Baja,
Mexico, for the late winter whale calving season in Scammon’s
Lagoon. Does Cyamus scammoni synthesize more hemoglobin
in Baja than in the icy oxygen-rich waters of Alaska?
Hypoxia sensors responsible for the regulation of
hemocyanin synthesis are unknown, although oxygen-sensitive
receptors in the crustacean gills and central nervous system
play roles in the ventilatory and cardiovascular responses to
external hypoxia (Wilkens et al. 1989; Ishii et al. 1989;
Massabuau and Meyrand, 1996; Reiber, 1997). Several
laboratory studies have indicated that higher hemocyanin
levels in crustaceans are induced by hypoxia (Hagerman, 1986;
Baden et al. 1990). An increase in hemocyanin concentration
has been demonstrated in response to hyposalinity and hypoxia
in the blue crab Callinectes sapidus (Mason et al. 1983; deFur
et al. 1990) and will be discussed below in connection with
hypoxia-related changes in hemocyanin subunit composition.
Internal hypoxia or hyperoxia
Internal hypoxia or hyperoxia may be involved in regulating
the production of arthropod hemocyanin. Hemocyanin levels
are directly related to the nutritional state of a crab (see Dall,
1974; Dumler and Terwilliger, 1996), leading some to suggest
that, in times of plenty, hemocyanin serves not only as an
oxygen carrier but also as a storage protein. This may occur,
although storing excess nutrients as circulating oligomeric
proteins seems a bit expensive. An alternative hypothesis is
that, with increased nutrition, a potential physiological hypoxia
may develop even though external levels of oxygen remain
constant. As the well-fed crab’s activity levels, metabolic
demands and oxygen requirements increase, so might its
hemocyanin concentration.
Just as hypoxia may regulate hemocyanin synthesis,
incipient hyperoxia may also be involved. It has long been
observed that the concentration of hemocyanin plummets when
a crab molts (Drach, 1939). This is due both to increased water
uptake and to a change in hemocyanin biosynthesis (Mykles,
1980; Mangum et al. 1985; Spindler et al. 1992; Terwilliger
and Otoshi, 1994), but a physiological reason remains unclear.
It seems counterproductive that, at such a critical time, late
premolt, ecdysis and early postmolt, when the crab is resorbing
its old exoskeleton and synthesizing a new one, it would have
a lower oxygen-carrying capacity (Fig. 3). Perhaps we should
look at the problem from a different perspective. Could the
decrease in synthesis of hemocyanin at ecdysis be a defense
against too much oxygen related to a temporary increase in
oxygen permeability of the new exoskeleton? Might a lower
oxygen-carrying capacity help prevent premature cross-linking
of internal skeletal structures by slowing the activation cascade
of prophenoloxidase? Prophenoloxidase (tyrosinase) is a
copper enzyme involved in melanization and arthropod
exoskeleton cross-linking (Mason, 1965; Soderhall, 1982;
Ashida and Yamazaki, 1990; and see below). These
suggestions on the relationship between hypoxia and
hemocyanin are obviously speculative but testable.
Experimental design will need to take into account the
developmental and molt stage of the organism as well as
external factors such as oxygen levels, food availability and
salinity.
More generally, what are the oxygen response elements in
arthropods and other invertebrates and how do they compare
with those currently under investigation in bacteria, yeast and
mammals? Might oxygen-transport proteins participate in
oxygen sensing, as suggested for mitochondrial heme in yeast
and Artemia (Kwast et al. 1998; Hand, 1998)? How do the
immediate oxygen sensors translate external or internal
hypoxia into regulation of hemocyanin or hemoglobin
production? Is there an erythopoietin-like hormonal pathway
(Bunn et al. 1998; Ratcliffe et al. 1998) for regulation of the
cells that synthesize extracellular hemoglobin or hemocyanin?
Changes in oxygen affinity
Rather than regulating the rate of expression of a single
respiratory protein, many organisms synthesize multiple
N. B. TERWILLIGER
Fig. 2. Relative phylogenetic distribution of hemocyanin and
hemoglobin. The lower pie chart shows the relative distribution
among all animals except insects. Blue, hemocyanin; dark blue,
hemocyanin and/or hemoglobin; red, hemoglobin. Adapted from
frontispiece, Ruppert and Barnes (1994).
1089Functional adaptations of oxygen-transport proteins
oxygen carriers with different oxygen affinities. These carriers
are produced and function either simultaneously, to enhance
oxygen transport and transfer to different portions of the body,
or sequentially, to meet oxygen demand under changing
conditions.
Simultaneous expression
Among the Annelida, some contain red blood cells, while
others, especially the oligochaetes and hirudineans, have only
extracellular vascular hemoglobin. In contrast, terebellid and
opheliid polychaete annelids are outstanding examples of
organisms that use multiple proteins in separate body
compartments simultaneously to ensure an adequate oxygen
supply (Terwilliger, 1974; Mangum et al. 1975; Terwilliger et
al. 1980). Pista pacifica, a large terebellid that lives in a
vertical leathery tube extending several feet down into the
anoxic sediments of a mudflat, contains three different oxygen-
binding proteins (Terwilliger, 1974). First, it circulates a giant
(3400kDa) extracellular hemoglobin with a low oxygen
affinity (P50=22mmHg at 20°C; 1mmHg=0.1333kPa) in its
vascular system. Blood vessels are present in thin-walled gills
at the head of the worm; at high tide, these gills expand into
the oxygen-rich sea water above the mudflat. Second, the low-
affinity extracellular hemoglobin readily transfers its oxygen
to a moderate-affinity hemoglobin (P50=3.8 mmHg at 20°C) in
the red blood cells circulating in the coelomic fluid of the
worm. Third, the coelomic cell hemoglobin in turn tranfers
oxygen to a high-affinity body wall myoglobin.
Alvinellid worms, also in the order Terebellida, are endemic
to the deep-sea hydrothermal vents in the Pacific Ocean
(Desbruyeres and Laubier, 1986). In addition to an
extracellular hemoglobin in the vascular system (Terwilliger
and Terwilliger, 1984; Toulmond et al. 1990), alvinellids
contain a coelomic cell hemoglobin (Jouin-Toulmond et al.
1996). The coelomic cells are concentrated in an internal
periesophageal pouch surrounding a plexus of thin-walled
blood vessels, suggesting the presence of a complex respiratory
gas-transfer system between extracellular and intracellular
hemoglobins previously undescribed in polychaetes. The
physiological properties of these proteins are still unknown; the
authors suggest that the system may also participate in sulfide
detoxification.
A similar strategy of using multiple proteins for oxygen
transfer is seen in several sipunculids (Manwell, 1960;
Mangum and Burnett, 1987; N. B. Terwilliger, unpublished
results). Themiste sp. produces two different populations of
hemerythrin-containing blood cells. Pink blood cells with a
low-affinity hemerythrin are present in vessels that extend into
the introvert tentacles and back into the coelomic cavity. The
tentacles reach into the water column and are probably the
prime source of oxygen uptake. Coelomic pink blood cells
have a moderate-affinity hemerythrin, and a body wall
myohemerythrin has a high oxygen affinity, ensuring oxygen
transfer from regions of high to low concentration. The
tentacular and coelomic blood cells not only have
electrophoretically distinct hemerythrins with different
oxygenation properties but differ morphologically as well
(Terwilliger et al. 1985), reinforcing the conclusion that each
hemerythrin is a distinctively different molecule produced by
a unique cell population. Other thin-walled, stubby-tentacled
sipunculans seem to transfer oxygen in the opposite direction,
across the body wall, and there is little difference in oxygen
affinity between coelomic and tentacular hemerythrins
(Manwell, 1960).
Mollusca show many variations on a theme of multiple
oxygen-transporter molecules (Terwilliger and Terwilliger,
1985). Chitons and gastropods have a high-affinity radular
muscle myoglobin. This myoglobin ensures a supply of oxygen
for the scraping, grazing activities of the radula and
odontophore complex. Other body muscles do not contain
myoglobin, and their creamy white color is in stark contrast to
the red radular muscles. An exception to this is the red-footed
Fig. 3. A red king crab Paralithodes
camtschatica emerging from its old exoskeleton
during ecdysis. Carapace width approximately
10cm. Photograph by Jeff Goddard.
1090
chiton Lepidochiton rugatus (Fig. 4). This member of an
ancient suborder of chitons, mostly deep-water, has a striking
red coloration of its foot and soft tissues, including the mantle,
gills and radular muscle, due to a tissue hemoglobin (Eernisse
et al. 1988). This hemoglobin seems to have a high oxygen
affinity. Typical of all other chitons, L. rugatus has a
circulating hemocyanin. Unlike more advanced, intertidal
chitons, it has only a few gills and thus has relatively less
surface area for gas exchange. It lives in an oxygen-poor
shallow-water habitat. We hypothesize that the presence of the
tissue hemoglobin facilitates oxygen transfer from the
hemocyanin to the respiring tissues under hypoxic conditions.
A note of caution is in order when cataloguing molluscan
myoglobins and perhaps other molluscan heme proteins.
Suzuki et al. (1996) and Suzuki and Imai (1997) have recently
reported that the amino acid sequence and gene structure of the
radular muscle ‘myoglobin’ of the abalone Sulculus
diversicolor is more closely related to the enzyme 2,3-indole
dioxygenase than to myoglobins from other sources. These
data suggest that we take another look at radular muscle
proteins in chitons and gastropods. Perhaps there are others
with a didomain structure and sequence like that of Sulculus.
Another example of multiple hemoglobin expression in
molluscs is found in one group of pulmonate gastropods, the
planorbid snails. In addition to a radular muscle myoglobin,
they contain a unique high-molecular-mass extracellular
hemoglobin in their vascular system, but no hemocyanin. Most
other gastropods, including pulmonates, have a high-
molecular-mass hemocyanin in their vascular system, along
with the radular myoglobin. What evolutionary accident
encouraged the expression of hemoglobin in the planorbid
snails and hemocyanin in the others?
The expression of oxygen-transport proteins is most varied
in the bivalve molluscs. First, primitive bivalves, mostly active
foragers, contain a circulating hemocyanin; more advanced
bivalves do not (Morse et al. 1986; Mangum et al. 1987;
Terwilliger et al. 1988). Second, a tissue myoglobin is often
present in the ganglia and sometimes in the adductor, foot and
other muscles of bivalves (Kraus and Colacino, 1986). Third,
those primitive bivalves such as Solemya velum,S. reidi,
Myrtea spinifera and Lucina pectinata that incorporate
chemoautotrophic bacteria in their gills to utilize sulfide or
methane contain several tissue hemoglobins in their gills
(Read, 1965; Dando et al. 1985; Doeller et al. 1988). One or
more of these ‘branchioglobins’ is involved in oxygen uptake,
while the others participate in sulfide metabolism (Doeller et
al. 1988; Kraus, 1995; and see below). Fourth, circulating red
blood cells are present in the bivalve family Arcidae (for a
review, see Mangum, 1997). The hemoglobins are mostly
tetrameric and dimeric, except for the 430kDa oligomers in
Barbatia reeveana mentioned above. One bivalve hemoglobin,
the cooperative homodimer found in the red blood cells of
Scapharca inaequivalvis, has an interesting back-to-front
assembly of its subunits, the reverse of vertebrate hemoglobins,
such that the hemes are in direct contact (Royer et al. 1989).
Recent crystallography work by Royer et al. (1996) suggests
that the cooperativity between hemes in Scapharca is mediated
by ordered water molecules. Two deep-water heterodont
bivalves, Calyptogena magnifica (Terwilliger et al. 1983) and
C. soyoae (Suzuki et al. 1989), also contain red blood cell
hemoglobins. A fifth example of oxygen-transport proteins in
bivalves is the largest extracellular hemoglobin (12×103kDa)
known, found in only two families of bivalves, the Astartidae
and the Carditidae (see above). Despite their size and
polymeric nature, with approximately 20 functional domains
per subunit and many subunits per molecule, the giant
hemoglobins have neither the moderate cooperativity of
Scapharca homodimers nor the supercooperativity of annelid
extracellular hemoglobins. Somewhat disappointingly for such
dramatically designed oligomers, they show moderate to
relatively high oxygen affinities and little or no cooperativity
(Terwilliger and Terwilliger, 1978; Yager et al. 1982).
With this plethora of hemoglobin and hemocyanin
expression among the bivalves, it may soon be possible to
correlate the respiratory physiology and molecular phylogeny
of bivalve globins with the morphological phylogeny of the
bivalve species. This would allow us to determine whether
observed functional properties are more closely related to
genetics or to habitat and are therefore the result of homology
or convergent evolution.
Parasites offer an intriguing view of how two very different
respiratory proteins can be utilized for transport and transfer of
oxygen – keeping in mind that one protein is synthesized by
the host and the other by the parasite. The female
rhizocephalan barnacle Briarosaccus callosus invades its host,
a king crab, and proceeds to grow internally, mature, extrude
a brood pouch through the abdomen of the crab and then
produce gametes. After fertilization by a male barnacle, the
ensuing embryos develop in the barnacle’s brood pouch or
externa until they are ready to swim away as naupliar larvae.
During this bizarre process, the female barnacle synthesizes an
extracellular hemoglobin (Fox, 1953; Shirley et al. 1986;
Terwilliger et al. 1986). The hemoglobin circulates through the
barnacle’s externa and also through thin-walled tissues or
N. B. TERWILLIGER
Fig. 4. A red-footed chiton Lepidochiton rugatus; ventral view.
Length approximately 2cm. Photograph by Doug Ernisse.
1091Functional adaptations of oxygen-transport proteins
rootlets of the barnacle inside the crab, in close proximity to
the crab’s hemocyanin-filled hemolymph (N. B. Terwilliger,
unpublished observations). Presumably a low-affinity crab
hemocyanin and a higher-affinity barnacle hemoglobin provide
an oxygen-transfer system to the developing embryos of the
barnacle. The interplay between barnacle and crab hormones
and oxygen sensors in this system must be a fascinating story.
A similar parasitism occurs between the hermit crab Pagurus
samuelis and rhizocephalans Peltogaster paguri and
Peltogasterella gracilis. Hemocyanin from P. samuelis,
whether parasitized or not, has a low oxygen affinity (Torchin,
1994).
Sequential expression
Oxygen-transport capabilities can be varied by sequentially
expressing multiple oxygen carriers that have different
oxygen-binding properties. Developmentally linked changes in
hemogobin gene expression are well documented among
viviparous vertebrates. Fetal/maternal oxygen affinity
differences in red blood cells can be the result of sequential
expression of different hemoglobin chains, different
concentrations of allosteric effectors such as the organic
phosphates 2,3-bisphosphoglycerate or ATP, different
sensitivities of the hemoglobins to the effectors, or
combinations thereof (for a review, see Ingermann, 1992). The
higher affinity of human adult hemoglobin for 2,3-
bisphosphoglycerate, a modulator that lowers the oxygen
affinity, results in a net higher oxygen affinity in the fetal red
blood cell, even though fetal hemoglobin has a slightly lower
intrinsic oxygen affinity than adult hemoglobin.
Some arthropod extracellular hemoglobins, such as the two
multidomain chains aand bin the branchiopod crustacean
Artemia, are sequentially expressed as dimers aa, ab and bb
(referred to as Hbs I, II and III) from the naupliar stage onwards
(Heip et al. 1978a,b). They differ functionally, but the adaptive
response is not clear. The hemoglobin with the lowest oxygen
affinity, Hb I, is predominantly expressed in the adult. The
phenotype can be altered by hypoxia as well as ontogeny; the
highest-affinity Hb III is the most responsive. A more
advanced branchiopod, Daphnia (Kobayashi et al. 1988), and
the insect Chironomus have also been demonstrated to undergo
developmental shifts in hemoglobin synthesis. The chironomid
hemoglobins, expressed only in larval hemolymph, have
markedly different oxygen affinities and Bohr effects (Weber
et al. 1985). Because the synthesis of these hemoglobins is
under both ontogenetic and environmental control (see above),
they would be good candidates in which to identify an
arthropod oxygen sensor. I suspect that there are many other
examples of ontogenetic changes in hemoglobin expression,
especially in benthic annelids and molluscs that have
planktotrophic larvae, but they have not yet been investigated.
Arthropod hemocyanin undergoes a developmental shift in
expression that is tied in with the development of the renal
system. In the Dungeness crab Cancer magister, megalopa and
juvenile crab hemocyanin differs from adult hemocyanin in
both structure and function (Terwilliger and Terwilliger,
1982). The adult crab hemocyanin contains a subunit, C mag
6, that is absent in the young crabs until approximately the
sixth juvenile instar. This is the same time at which C mag 6
mRNA first appears in the hepatopancreas (Durstewitz and
Terwilliger, 1997a,b). Two other subunits change in relative
abundance during development, while levels of three
constitutive subunits are constant. The oxygen affinity of the
juvenile hemocyanin is lower by approximately 50% than that
of the adult (Terwilliger and Terwilliger, 1982; Terwilliger and
Brown, 1993). At first glance, this seemed to be an
environmentally adaptive difference: the megalopa, swimming
in well-oxygenated oceanic waters, gets by with a low-affinity
oxygen carrier, while the adult, scuttling about and burying
itself in the more hypoxic muddy sand floor of the nearshore
and estuary, needs a higher-affinity transport protein. In fact,
the oxygen affinities of juvenile and adult whole hemolymphs
are indistinguishable, in contrast to that of their purified
hemocyanins (Brown and Terwilliger, 1998). The growing
crab is able to maintain a relatively constant oxygen affinity
by coordinated developmental changes in both hemocyanin
and ionic regulation (Fig. 5). The juvenile has more
magnesium in its hemolymph than the adult (Brown and
Terwilliger, 1992). In addition, the juvenile hemocyanin is
more sensitive to magnesium, an allosteric effector that causes
an increase in hemocyanin oxygen-affinity, than is the adult
hemocyanin (Terwilliger and Brown, 1993). With this double
jeopardy, more magnesium and a greater hemocyanin
sensitivity to it, the juvenile crab would have difficulty
unloading oxygen at the tissues if it had an adult-type
hemocyanin. Fortunately, juvenile hemocyanin has a lower
intrinsic oxygen affinity. As the young crab’s ability to excrete
more magnesium develops and hemolymph levels of
magnesium drop, the synthesis of hemocyanin shifts from the
juvenile to the adult form. Changes in the concentration of an
internal cofactor rather than environmental oxygen levels seem
to be the stimulus for this ontogenetic change in hemocyanin
expression and function (Brown and Terwilliger, 1998). It is
an interesting case of using different hemocyanins to maintain
oxygen affinity while allosteric modulator concentrations vary.
Sometimes the sequential change in oxygen transporter
structure and function occurs on a cyclical basis rather than on
Fig. 5. Ontogeny of hemocyanin expression and ion regulation in the
Dungeness crab Cancer magister. The low-affinity hemocyanin in
the juvenile crab counterbalances high levels of magnesium in the
hemolymph (left). As hemolymph magnesium levels decrease during
development, the synthesis of hemocyanin shifts from the juvenile
form to the higher-affinity adult hemocyanin (right).
1092
a developmental time scale, and the cue is environmental. The
blue crab Callinectes sapidus migrates seasonally from oceanic
waters to dilute coastal marshes and is subjected to major
changes in both salinity and oxygen levels due to estuarine
stratification. Like Cancer magister, Callinectes sapidus
hemocyanin is composed of six heterogeneous polypeptides
that self-assemble into one-hexamers and two-hexamers. Three
of the polypeptides are reported to be invariant, the other three
are highly variable, from one crab to another and within one
adult crab, depending on conditions (Mangum and Rainer,
1988). Normoxic animals have all six hemocyanin chains and
low oxygen affinities; hypoxic animals have reduced levels of
the three variable chains and progressively higher oxygen
affinities. Purification of the oligomers by HPLC revealed that
the two-hexamers contain all six chains and have a lower
affinity and higher cooperativity, while the one-hexamers are
built entirely of the three invariant chains and have a higher
oxygen affinity (Mangum et al. 1991). Studies on crabs caught
in the field show a pattern consistent with the laboratory
studies. Crabs caught in the normoxic areas of the York River
estuary exhibited the hemocyanin subunit pattern associated
with a low oxygen affinity, normoxic conditions and high
proportions of two-hexamers, while crabs from hypoxic strata
displayed the three-subunit pattern of hemocyanin consistent
with high oxygen affinity, hypoxia and one-hexamers
(Mangum, 1994). Details at the molecular level have not yet
been investigated; it will be interesting to see the level of
regulation. Whether undergoing a permanent transition from
juvenile to adult or dealing with seasonal hypoxia, functional
adaptations in crustacean oxygen-transport proteins utilize
sequential changes in protein expression.
Changes in allosteric modifiers
Regulation of the function of oxygen-transport proteins by
allosteric modifiers in response to relatively short-term
environmental or metabolic changes varies widely depending
on the protein. Invertebrate red blood cells are perhaps the least
responsive, while annelid extracellular hemoglobins,
vertebrate red blood cells and arthropod hemocyanins show a
remarkable range of oxygen affinities and cooperativities that
are sensitive to pH and inorganic ions. The latter two groups
of proteins are modulated by organic metabolites as well (for
a review, see Mangum, 1997). Crustacean hemocyanin is
particularly responsive to different allosteric modifiers and
conditions. In many decapods, increases in pH, CO2
concentration, divalent cation levels and concentrations of
organic molecules such as lactate, urate and dopamine result
in an increase in hemocyanin oxygen-affinity, while an
increase in temperature causes a decrease in oxygen affinity.
Different combinations of these factors come into play, along
with ventilatory, cardiovascular and behavioral modifications,
to stabilize oxygen uptake during hypoxia or to increase
oxygen uptake during exercise (for reviews, see Burnett, 1992;
Truchot, 1992; Morris and Bridges, 1994; Morris and Airriess,
1998; Mangum, 1997). In subtidal or intertidal crabs,
environmental hypoxia often evokes a hyperventilation
response. This promotes CO2excretion and results in
hemolymph alkalosis and an adaptive increase in oxygen
affinity. Since the metabolic demand is not increased, the
increased affinity facilitates loading from a low-oxygen
environment without adversely affecting oxygen unloading at
the tissues. Moderate exercise, in contrast, causes both an
increase in oxygen demand and an increase in CO2production.
The ensuing hemolymph acidosis results in a decrease in
hemocyanin oxygen-affinity. External oxygen is not limited
during exercise, however. If the crab can keep hyperventilating
and perfusing its tissues, oxygen loading at the gills should not
be a problem, and the lower affinity will assist at the site of
unloading. Prolonged exercise is more of a problem. As a result
of lowered blood oxygen levels, urate, an intermediate of
purine metabolism, may accumulate (Dykens, 1991), and urate
increases hemocyanin oxygen-affinity (Morris et al. 1985;
Lallier et al. 1987). As the exercise continues, insufficient
oxygen will be delivered to the tissues and anaerobic
metabolism will begin, with the accompanying endproduct, L-
lactic acid. Decreased pH will cause a decreased oxygen
affinity, but the L-lactate will partially counterbalance the pH-
induced reduction in affinity and assist oxygen uptake at the
gill (Truchot, 1980). Superimposed on the patterns resulting
from hypoxia and exercise are the effects of neurohormones
and monoamines; while their primary functions appear to be
directed at mechanically tuning the cardiovascular system of
the crustacean, they also tend to cause an increase in
hemocyanin oxygen-affinity (for a review, see Morris and
Airreiss, 1998). Not all hemocyanins are equally responsive to
these allosteric effectors. Those from terrestrial crabs seem to
be especially insensitive to metabolic modulation. This may be
due to the high oxygen content of air versus water and the high
levels of hemocyanin oxygen saturation at the gas exchange
surfaces (Morris, 1991). The Christmas Island red crab
Gecarcoides natalis shows a decrease in hemocyanin oxygen-
affinity with increasing concentrations of lactate. This reverse
lactate effect, which may assist in unloading oxygen at the
tissues during increased oxygen demand, is unique among the
Crustacea (Adamczewska, 1997). Several hemolymph factors
have been noted (but not yet identified) that lower oxygen
affinity (see Bridges et al. 1997; Lallier and Truchot, 1997).
These unknown factors, like H+, act in marked contrast to most
of the other allosteric modifiers that cause an increase in
oxygen affinity. Collectively, the responses of oxygen-
transport proteins to modulators help the organism deal with
environmental changes and metabolic demands.
Other functions of oxygen-transport molecules
Besides transporting oxygen, several oxygen carriers have
taken on additional roles. One function reported for some
invertebrate hemoglobins is transport or detoxification of
hydrogen sulfide. Unlike vertebrate hemoglobins, which form
sulfhemoglobin in the presence of hydrogen sulfide, the
extracellular hemoglobin of the hydrothermal vent
vestimentiferan Riftia pachyptila binds sulfide reversibly at a
site on the molecule different from that binding oxygen (Arp
N. B. TERWILLIGER
1093Functional adaptations of oxygen-transport proteins
and Childress, 1983; Arp et al. 1987). The hemoglobin thus
transports both oxygen and sulfide to the sulfur-oxidizing
chemotrophic endosymbionts of Riftia. Recent studies by Zal
et al. (1997c) suggest that free cysteine residues on the heme-
containing globin chains, and also on the linker chains, of
Riftia hemoglobin are the principle sulfide-binding sites.
Several other extracellular hemoglobin sequences, including
those of the mudflat polychaete Arenicola marina and the vent
polychaete Alvinella pompejana from sulfide-rich
environments, also have free cysteine groups (Zal et al.
1997a,b). Sulfide binding may not be a universal property of
hemoglobins with free cysteines, however, as no sulfide-
binding activity was observed in the vascular blood from either
Alvinella pompejana or Alvinella caudata (Martineau et al.
1997).
Bivalves with chemoautotrophic bacteria in their gills
contain several hemoglobins in the gill tissue, as mentioned
above. These hemoglobins, like the vestimentiferan
hemoglobin, are involved in oxygen and/or sulfide uptake and
metabolism. Hemoglobin I from the gill of the mangrove
swamp clam Lucina pectinata is sulfide-reactive, while Hb II
and Hb III are oxygen-reactive. Hb I combines with oxygen
with high affinity, but at low oxygen concentrations, oxyHb I
readily reacts with sulfide to form ferric Hb I sulfide (Kraus
and Wittenberg, 1990). The heme-bound sulfide is stabilized
by three phenylalanine residues forming a ‘cage’ around the
sulfide (Rizzi et al. 1996). Sulfide is believed to be released to
the symbiotic bacteria upon the formation of the ferrous protein
by electron transfer from an unknown reductant. Thus, this
oxygen-binding protein, Hb I, is also implicated in sulfide
transport and electron transfer (Navarro et al. 1996).
Vertebrate red blood cells have recently been reported to
play a significant role in regulating blood pressure through the
ability of the oxyhemoglobin molecule to bind nitric oxide as
an S-nitrosothiol (SNO) (Jia et al. 1996). Nitric oxide had been
identified as an endothelial relaxing factor (Furchgott and
Zawadski, 1980). Its mechanism of action is unclear, because
the heme iron of vertebrate hemoglobin is a potent scavenger
of nitric oxide, binding it much more tightly than oxygen
(Lancaster, 1994). In the red blood cell, however, an
equilibrium has been described between nitric oxide bound to
the heme iron and to reactive thiol groups of the hemoglobin
chain, especially a conserved cysteine residue, CysB193,
versus nitric oxide bound to glutathione and other small thiols
in the erythrocyte. In the lungs, oxygenated hemoglobin is S-
nitrosylated. During the red blood cell’s transit through the
body, thiols can transfer nitric oxide from the red blood cell to
endothelial receptors as the hemoglobin becomes
deoxygenated. The result is vasodilation and a decrease in
blood pressure (Jia et al. 1996; Kagan et al. 1996).
Is this phenomenon restricted to vertebrate red blood cells
or does its evolutionary history include invertebrate red blood
cells or even extracellular hemoglobins and hemocyanins?
Perhaps the thiol groups on the linker chains of polychaete and
vestimentiferan extracellular hemoglobins (Zal et al.
1997a,b,c) can function as transfer agents of SNO from the
heme-containing chains of the giant molecules to receptors in
cells lining the heart-body or vessels of the worm. Does
hemocyanin also form S-nitrosothiols and transport them
through the hemolymph, and does nitric oxide affect blood
pressure in arthropods or molluscs? Our perception of
crustacean circulation from a loosely regulated ‘open’ system,
at least in part, to one that is precisely designed and controlled
has grown significantly (for reviews, see Airriess and
McMahon, 1994; Morris and Airriess, 1998). For many years,
it was believed that there was no vasoconstrictive musculature
in the blood vessel walls of decapod crustaceans. Recently,
striated muscle has been described in the posterior aorta of the
prawn Sicyonia ingentis (Martin et al. 1989) and in the lobster
Homarus americanus (Wilkens et al. 1997). Resistance to
blood flow in Homarus americanus is thought to be due to
cardioarterial valves and to a variety of neurotransmitters and
neurohormones (Wilkens, 1997). Perhaps nitric oxide is also
involved. Among the molluscs, it would be interesting to look
for a nitric oxide effect and an SNO–hemocyanin in the finely
tuned respiratory system of cephalopods.
Diversification of function among the hemocyanins seems
to have involved gene duplications that resulted not only in
multiple hemocyanin subunits with unique oxygen-binding
properties but also in new members of the hemocyanin gene
family. Phenoloxidases, or tyrosinases, are widespread in the
animal kingdom, as well as in plants, fungi and procaryotes;
they are able to catalyze the oxidation of monophenols to o-
diphenols and the oxidation of o-diphenols to the
corresponding o-quinones (Mason, 1965). Molluscan
hemocyanin has phenoloxidase activity (Salvato et al. 1983;
Nakahara et al. 1983), as does chelicerate hemocyanin (H.
Decker, personal communication). Crustacean hemocyanin
does not exhibit prophenoloxidase activity under normal
circumstances, but it catalyzes the reaction with low efficiency
when the hemocyanin molecule is partially unfolded (Zlateva
et al. 1996). Some chelicerates, crustaceans and insects have a
separate protein, a prophenoloxidase, that, when activated,
shows strong activity. This enzyme plays a key role in
melanization, cuticle hardening and other defense reactions
(Ashida and Yamazaki, 1990). It contains two copper-binding
sites whose amino acid sequences are nearly identical to those
of arthropod hemocyanin, and the entire protein sequence
shows some similarity to that of hemocyanin, although the
quaternary structures appear unrelated (Aspan et al. 1995; Hall
et al. 1995; Fujimoto et al. 1995; Kawabata et al. 1995). Thus,
it is likely that arthropod hemocyanins and prophenoloxidases
evolved from an ancestral arthropod copper protein. Molluscan
hemocyanins seem to be more closely related to plant, fungal
and vertebrate phenoloxidases, on the basis of sequence
comparisons of their copper-binding sites (Drexel et al. 1987;
Beintema et al. 1994; Kawabata et al. 1995; Durstewitz and
Terwilliger, 1997b). They share a similar copper A site that
differs from the amino acid sequence of the arthropod copper
A site. The copper B sites are similar among the molluscan and
arthropod hemocyanins and the phenoloxidases, suggesting
that there was a common ancestral copper protein that gave rise
1094
to today’s proteins through a series of gene duplications and
fusion events (see van Holde and Miller, 1995; Durstewitz and
Terwilliger, 1997b).
Crustaceans contain another hemolymph protein,
cryptocyanin, that is even more similar in sequence and
structure to crustacean hemocyanin than is prophenoloxidase
– except that it neither contains copper nor binds reversibly
with oxygen (Terwilliger and O’Brien, 1992; N. B. Terwilliger,
unpublished results). Levels of cryptocyanin increase
dramatically during premolt and decrease at ecdysis
(Terwilliger and Otoshi, 1994). Insect hemolymph storage
proteins, hexamerins, also resemble hemocyanin in quaternary
structure and sequence (Telfer and Kunkle, 1991). Like
cryptocyanin, they lack copper, and some show molt-cycle-
related patterns of biosynthesis and have potential roles in
cuticle formation. It is likely that gene duplications in the
arthropod hemocyanins allowed the loss of the copper-binding
capability in one of the gene products. This then gave rise first
to crustacean cryptocyanin and then to insect hexamerins.
These proteins have since taken on new functions related to the
molt cycle and exoskeleton formation. Thus, there are several
members of the arthropod hemocyanin gene family, including
crustacean and chelicerate hemocyanins, cryptocyanin,
prophenoloxidases and insect hexamerins. Rather than assume
additional functions, as some of the hemoglobins have done,
hemocyanin has diversified through multiple gene duplications
and specializations.
Conclusions
Oxygen-transport proteins are a colorful group of
multisubunit molecules with a common function. The observed
patterns of biosynthesis of crustacean hemocyanin and related
proteins such as cryptocyanin, investigated in individually
monitored crabs, indicate that the patterns are closely linked to
development and molting and therefore are under some level
of hormonal control. If similar monitoring studies were
performed on individual polychaetes or molluscs, for example,
rather than on pooled samples from many organisms, we would
gain further insights into the effects of factors such as
develomental stage, nutrition and reproductive cycle on
oxygen-transport protein expression.
Changes in oxygen-transport protein synthesis, whether up-
or down-regulation or expression of a particular combination
of gene products, are often considered to be a long-term
response to developmental or environmental change, while
allosteric modifiers are thought to be responsible for more
immediate, short-term perturbations. This is not always the
case, as shown by the rise and fall in levels of modulators such
as lactate and urate in the hemolymph. During forced exercise
or other stress in crabs, lactate levels rise rapidly within
minutes in the hemolymph, but the lactate lingers for hours
afterwards until blood levels finally return to normal. Urate is
slow to accumulate and is generally felt to be more of a
response to prolonged hypoxia. Conversely, oxygen-transport
protein synthesis may be much more of a short-term response
than previously believed. Studies by Hofmann and Somero
(1996) on the mussel Mytilus trossulus have shown that two
stress proteins were synthesized during the first 2h of recovery
from thermal stress. During the same period of recovery from
tidal emersion, damaged proteins were rapidly ubiquitinated
and degraded. These results indicate that rapid induction of
protein synthesis can occur on a tidal cycle basis. Monitoring
the in vivo expression of oxygen-transport proteins during a
6–8h exposure to hypoxia might reveal dynamic changes in
synthesis that have not been obvious in longer-duration studies.
Finally, as advances in molecular techniques allow us more
easily to obtain gene and protein sequence information, we
may discover, as we have for myoglobin and indole amine
oxidase, that some of the apparently homologous oxygen-
transport proteins are more likely to be the result of the
convergent evolution of different proteins. The continuing
debate on the relative significance of similarity of structure
versus amino acid sequence will help sort out molecular
phylogenies. Which is more significant, for example, the total
number of identical amino acids shared between two proteins
or the fact that they have identical active sites? Combining
molecular, functional and morphological data should
eventually clarify (and probably revise) organismal phylogeny
as well. New information may resolve our understanding of
some of the paradoxes about the evolution of oxygen-transport
proteins pointed out recently by Mangum (1998). Just as new
technologies have reintroduced the importance of cell lineage
studies to developmental biology, so investigations of
respiratory proteins at the level of gene expression and high-
resolution three-dimensional structure will provide significant
new information about the structure, function and adaptations
of oxygen-transport molecules.
This work was supported by the National Science
Foundation (IBN 9217530, IAB 9603521) and the
Environmental Protection Agency (CR824178-01-0).
References
ADAMCZEWSKA, A. M. (1997). Eco-physiology of the Christmas
Island red crab (Gecarcoidea natalis). PhD dissertation, University
of Sydney.
AIRRIESS, C. N. AND MCMAHON, B. R. (1994). Cardiovascular
adaptations enhance tolerance of environmental hypoxia in the crab
Cancer magister. J. exp. Biol. 190, 23–41.
ARP, A. E. AND CHILDRESS, J. J. (1983). Sulfide binding by the blood
of the hydrothermal vent tube worm Riftia pachyptila. Science 219,
295–297.
ARP, A. E., CHILDRESS, J. J. AND VETTER, R. D. (1987). The sulphide-
binding protein in the blood of the vestimentiferan tubeworm, Riftia
pachyptila, is the extracellular hemoglobin. J. exp. Biol. 128,
139–158.
ASHIDA, M. AND YAMAZAKI, Y. H. (1990). Biochemistry of the
phenoloxidase system in insects: with special reference to its
activation. In Molting and Metamorphosis (ed. E. Ohnishi and H.
Ishizaki), pp. 239–265. Berlin: Springer.
ASPAN, A., HUANG, T.-S., CERENIUS, L. AND SODERHALL, K. (1995).
N. B. TERWILLIGER
1095Functional adaptations of oxygen-transport proteins
cDNA cloning of prophenoloxidase from the fresh water crayfish
Pacifastacus leniusculus and its activation. Proc. natn. Acad. Sci.
U.S.A. 92, 939–943.
BADEN, S. P., PIHL, L. AND ROSENBERG, R. (1990). Effects of oxygen
depletion on the ecology, blood physiology and fishery of the
Norway lobster Nephrops norvegicus. Mar. Ecol. Prog. Ser. 67,
141–155.
BIENTEMA, J. J., STAM, W. T., HAZES, B. AND SMIDT, M. P. (1994).
Evolution of arthropod hemocyanins and insect storage proteins
(hexamerins). Molec. Biol. Evol. 11, 493–503.
BRIDGES, C. R., HUPPERTS, V., ESHKY, A. A. AND TAYLOR, A. C.
(1997). Haemocyanin oxygen transport in Ocypode spp.:
modification of oxygen affinity. J. mar. biol. Ass. U.K. 77,
145–158.
BROWN, A. C. AND TERWILLIGER, N. B. (1992). Developmental
changes in ionic and osmotic regulation in the Dungeness crab,
Cancer magister. Biol. Bull. mar. biol. Lab., Woods Hole 182,
270–277.
BROWN, A. C. AND TERWILLIGER, N. B. (1998). Ontogeny of
hemocyanin function in the Dungeness crab Cancer magister:
hemolymph modulation of hemocyanin oxygen-binding. J. exp.
Biol. 201 (in press).
BUNN, H. F. GU, J., HUANG, L. E., PARK, J.-W. AND ZHU, H. (1998).
Erythropoietin: a model system for studying oxygen-dependent
gene regulation. J. exp. Biol. 201, 1197–1201.
BURNETT, L. E. (1992). Integrated function of the respiratory pigment
hemocyanin in crabs. Am. Zool. 32, 438–446.
DALL, W. (1974). Indices of nutritional state in the western rock
lobster Panulirus longipes (Milne-Edwards). I. Blood and tissue
constituents and water content. J. exp. mar. Biol. Ecol. 16,
167–180.
DANDO, P. R., SOUTHWARD, A. J., SOUTHWARD, E. C., TERWILLIGER,
N. B. AND TERWILLIGER, R. C. (1985). Sulphur-oxidising bacteria
and haemoglobin in gills in the bivalve mollusc Myrtea spinifera.
Mar. Ecol. Prog. Ser. 23, 85–98.
DANGOTT, L. AND TERWILLIGER, R. C. (1981). Arthropod extracellular
hemoglobins: structural and functional properties. Comp. Biochem.
Physiol. 70B, 549–557.
DEFUR, P. L., MANGUM, C. P. AND REESE, J. E. (1990). Respiratory
responses of the blue crab Calllinectes sapidus to long-term
hypoxia. Biol. Bull. mar. biol. Lab., Woods Hole 178, 46–54.
DESBRUYERES, D. AND LAUBIER, L. (1986). Les Alvinellidae, une
famille nouvelle d’annelides infeodées aux sources hydrothermales
sous-marines: systematique, biologie et ecologie. Can. J. Zool. 64,
2227–2245.
DOELLER, J. E., KRAUS, D. W., COLACINO, J. M. AND WITTENBERG, J.
B. (1988). Gill hemoglobin may deliver sulfide to bacterial
symbionts of Solemya velum (Bivalvia, Mollusca). Biol. Bull. mar.
biol. Lab., Woods Hole 175, 388–396.
DRACH, P. (1939). Mue et cycle d’intermue chez les crustaces
decapodes. Annls Inst. Oceanogr. Monaco 19, 103–329.
DREXEL, R., SIEGMUND, S., SCHNEIDER, H. J., LINZEN, B., GIELENS, C.,
LONTIE, R., KELLERMANN, J. AND LOTTSPEICH, F. (1987). Complete
sequence of a functional unit from a molluscan hemocyanin (Helix
pomatia). Biol. Chem. Hoppe Seyler 368, 617–635.
DUMLER, K. AND TERWILLIGER, N. B. (1996). Effects of food levels
and temperature on growth and hemocyanin ontogeny in juvenile
Cancer magister. Am. Zool. 36, 14A.
DURSTEWITZ, G. AND TERWILLIGER, N. B. (1997a). Developmental
changes in hemocyanin expression in the Dungeness crab, Cancer
magister. J. biol. Chem. 272, 4347–4350.
DURSTEWITZ, G. AND TERWILLIGER, N. B. (1997b). cDNA cloning of
a developmentally regulated hemocyanin subunit in the crustacean
Cancer magister and phylogenetic analysis of the hemocyanin gene
family. Molec. Biol. Evol. 14, 266–276.
DYKENS, J. (1991). Purinolytic capacity and origin of hemolymph
urate in Carcinus maenas during hypoxia. Comp. Biochem. Physiol.
98B, 579–582.
EERNISSE, D. J., TERWILLIGER, N. B. AND TERWILLIGER, R. C. (1988).
The red foot of a lepidopleurid chiton: evidence for tissue
hemoglobins. Veliger 30, 244–247.
FOX, H. M. (1953). Hemoglobin and biliverdin in parasitic cirripede
Crustacea. Nature 171, 162–163.
FUJIMOTO, K., OKINO, N., KAWABATA, S., IWANAGA, S. AND OHNISHI,
E. (1995). Nucleotide sequence of the cDNA encoding the
proenzyme of phenol oxidase A1of Drosophila melanogaster.
Proc. natn. Acad. Sci. U.S.A. 92, 7769–7773.
FURCHGOTT, R. F. AND ZAWADSKI, J. V. (1980). The obligatory role
of endothelial cells in the relaxation of arterial smooth muscle by
acetylcholine. Nature 288, 373–376.
GRINICH, N. AND TERWILLIGER, R. (1980). The quaternary structure of
an unusually high molecular weight intracellular hemoglobin from
the bivalve mollusc Barbatia reeveana. Biochem. J. 189, 1–8.
HAGERMAN, L. (1986). Haemocyanin concentration in the shrimp
Crangon crangon (L.) after exposure to moderate hypoxia. Comp.
Biochem. Physiol. 85A, 721–724.
HALL, M., SCOTT, T., SUGUMARAN, M., SODERHALL, K. AND LAW, J.
H. (1995). Proenzyme of Manduca sexta phenol oxidase:
Purification, activation, substrate specificity of the active enzyme
and molecular cloning. Proc. natn. Acad. Sci. U.S.A. 92,
7764–7768.
HAND, S. C. (1998). Quiescence in Artemia franciscana embryos:
reversible arrest of metabolism and gene expression at low oxygen
levels. J. exp. Biol. 201, 1233–1232.
HARDISON, R. (1998). Hemoglobins from bacteria to man: evolution
of different patterns of gene expression. J. exp. Biol. 201,
1099–1117.
HEIP, P., MOENS, L., JONIAU, M. AND KONDO, M. (1978a).
Ontogenetical studies on extracellular hemoglobin of Artemia
salina. Dev. Biol. 64, 73–81.
HEIP, P., MOENS, L. AND KONDO, M. (1978b). Effect of concentrations
of salt and oxygen on the synthesis of extracellular hemoglobins
during development of Artemia salina. Dev. Biol. 63, 247–251.
HOCHACHKA, P. W. (1998). Mechanism and evolution of hypoxia-
tolerance in humans. J. exp. Biol. 201, 1243–1254.
HOFMANN, G. E. AND SOMERO, G. N. (1996). Protein ubiquitination
and stress protein synthesis in Mytilus trossulus occurs during
recovery from tidal emersion. Moec. mar. Biol. Biotech. 5,
175–184.
INGERMANN, R. (1992). Structure–function relationships of the
ectothermic vertebrate hemoglobins. In Advances in Comparative
and Environmental Physiology, vol. 13, Blood and Tissue Oxygen
Carriers (ed. C. P. Mangum), pp. 411–431. Heidelberg: Springer
Verlag.
ISHII, K., MASSABUAU, J. C. AND DEJOURS, P. (1989). Oxygen sensitive
chemoreceptors in the branchiocardiac veins of the crayfish,
Astacus leptodactylus. Respir. Physiol. 78, 73–81.
JIA, L., BONAVENTURA, C., BONAVENTURA, J. AND STAMLER, J. S.
(1996). S-nitrosohaemoglobin: a dynamic activity of blood
involved in vascular control. Nature 380, 221–226.
JOUIN-TOULMOND, C., AUGUSTIN, D., DESBRUYERES, D. AND
TOULMOND, A. (1996). The gas transfer system in alvinellids
1096
(Annelida Polychaeta, Terebellida). Anatomy and ultrastructure of
the anterior circulatory system and characterisation of a coelomic,
intracellular, haemoglobin. Cah. Biol. Mar. 37, 135–151.
KAGAN, V. E., DAY, B. W., ELSAYED, N. M. AND GORBUNOV, N. V.
(1996). Dynamics of haemoglobin. Nature 383, 30–31.
KAWABATA, T., YUSAHARA, Y., OCHIAI, M., MATSUURA, S. AND
MASAAKI, A. (1995). Molecular cloning of insect pro-phenol
oxidase: a copper-containing protein homologous to arthropod
hemocyanin. Proc. natn. Acad. Sci. U.S.A. 92, 7774–7778.
KOBAYASHI, M., FUJIKI, M. AND SUZUKI, T. (1988). Variation in and
oxygen binding properties of Daphnia magna hemoglobin. Physiol.
Zool. 61, 415–419.
KOBAYASHI, M. AND GONOI, H. (1985). Horizontal movement of pale
and red Daphnia magna in low oxygen concentration. Physiol.
Zool. 58, 190–196.
KRAUS, D. W. (1995). Heme proteins in sulfide-oxidizing
bacteria/mollusc symbioses. Am. Zool. 35, 112–120.
KRAUS, D. W. AND COLACINO, J. M. (1986). Extended oxygen delivery
from the nerve hemoglobin of Tellina alternata (Bivalvia). Science
232, 90–92.
KRAUS, D. W. AND WITTENBERG, J. B. (1990). Hemoglobins of the
Lucina pectinata/bacterial symbiosis. I. Molecular properties.
Kinetics and equilibria of reactions with ligands. J. biol. Chem. 265,
16043–16053.
KURTZ, D. M., JR(1992). Molecular structure/function relationships
of hemerythrins. In Advances in Comparative and Environmental
Physiology, vol. 13, Blood and Tissue Oxygen Carriers (ed. C. P.
Mangum), pp. 151–171. Heidelberg: Springer Verlag.
KWAST, K. E., BURKE, P. V. AND POYTON, R. O. (1998). Oxygen
sensing and the transcriptional regulation of oxygen-responsive
genes in yeast. J. exp. Biol. 201, 1177–1195.
LALLIER, F., BOITEL, F. AND TRUCHOT, J.-P. (1987). The effect of
ambient oxygen and temperature on the haemolymph L-lactate and
urate concentrations in the shore crab Carcinus maenas. Comp.
Biochem.Physiol. 86A, 255–260.
LALLIER, F. H. AND TRUCHOT, J. P. (1997). Hemocyanin oxygen
binding properties of a deep-sea hydrothermal vent shrimp –
Evidence for a novel cofactor. J. exp. Zool. 277, 357–364.
LANCASTER, J. R. (1994). Simulation of the diffusion and reaction of
endogenously produced nitric oxide. Proc. natn. Acad. Sci. U.S.A.
91, 8137–8141.
MANGUM, C. P. (1992). (ed.) Advances in Comparative and
Environmental Physiology, vol. 13, Blood and Tissue Oxygen
Carriers. Heidelberg: Springer Verlag.
MANGUM, C. P. (1994). Subunit composition of hemocyanins from
Callinectes sapidus: Phenotypes from naturally hypoxic waters and
isolated oligomers. Comp. Biochem. Physiol. 108B, 337–341.
MANGUM, C. P. (1997). Invertebrate blood oxygen carriers. In
Handbook of Physiology, section 13, Comparative Physiology (ed.
W. H. Dantzler), pp. 1097–1131. New York: Oxford University
Press.
MANGUM, C. P. (1998). Major events in the evolution of the oxygen
carriers. Am. Zool. (in press).
MANGUM, C. P. AND BURNETT, L. E. (1987). Response of sipunculid
hemerythrins to inorganic ions and CO2. J. exp. Zool. 244, 59–65.
MANGUM, C. P., GREAVES, J. AND RAINER, J. S. (1991). Oligomer
composition and oxygen binding of the hemocyanin of the blue
crab Callinectes sapidus. Biol. Bull. mar. biol. Lab., Woods Hole
181, 453–458.
MANGUM, C. P., MCMAHON, B. R., DEFUR, P. L. AND WHEATLY, M.
G. (1985). Gas-exchange, acid–base balance and the oxygen-supply
to the tissues during a molt of the blue crab Callinectes sapidus. J.
crust. Res. 5, 207–215.
MANGUM, C. P., MILLER, K. I., SCOTT, S. L., VAN HOLDE, K. E. AND
MORSE, M. P. (1987). Bivalve hemocyanins – structural, functional
and phylogenetic relationships. Biol. Bull. mar. biol. Lab., Woods
Hole 173, 205–221.
MANGUM, C. P. AND RAINER, J. S. (1988). The relationship between
subunit composition and oxygen binding of blue crab hemocyanin.
Biol. Bull. mar. biol. Lab., Woods Hole 174, 77–82.
MANGUM, C. P., WOODIN, B. L., BONAVENTURA, C., SULLIVAN, B. AND
BONAVENTURA, J. (1975). The role of coelomic and vascular
hemoglobins in the annelid family Terebellidae. Comp. Biochem.
Physiol. 51A, 281–294.
MANNING, A., TROTMAN, C. AND TATE, W. (1990). Evolution of a
polymeric globin in the brine shrimp Artemia. Nature 348,
653–656.
MANWELL, C. (1960). Histological specificity of respiratory pigments.
II. Oxygen transfer systems involving hemerythrins in sipunculid
worms of different ecologies. Comp. Biochem. Physiol. 1, 277–285.
MANWELL, C. AND BAKER, C. M. A. (1988). Magelona papillicornis
haemerythrin: tissue specificity, molecular weights and oxygen
equilibria. Comp. Biochem. Physiol. 89B, 453–463.
MARKL, J. AND DECKER, H. (1992). Molecular structure of the
arthropod hemocyanins. In Advances in Comparative and
Environmental Physiology, vol. 13, Blood and Tissue Oxygen
Carriers (ed. C. P. Mangum), pp. 325–376. Heidelberg: Springer
Verlag.
MARTIN, G. G., HOSE, J. E. AND CORZINE, C. J. (1989). Morphological
comparison of major arteries in the ridgeback prawn, Sicyonia
ingentis. J. Morph. 200, 175–183.
MARTINEAU, P., JUNIPER, S. K., FISHER, C. R. AND MASSOTH, G. J.
(1997). Sulfide binding in the body fluids of hydrothermal vent
alvinellid polychaetes. Physiol. Zool. 70, 578–588.
MASON, H. S. (1965). Oxidases. A. Rev. Biochem. 34, 595–634.
MASON, R. P., MANGUM, C. P. AND GODETTE, G. (1983). The influence
of inorganic ions and acclimation salinity on hemocyanin oxygen
binding in the blue crab Callinectes sapidus. Biol. Bull. mar. biol.
Lab., Woods Hole 164, 104–123.
MASSABUAU, J. C. AND MEYRAND, P. (1996). Modulation of a neural
network by physiological levels of oxygen in lobster stomatogastric
ganglion. J. Neurosci. 16, 3950–3959.
MOENS, L. AND KONDO, M. (1976). The structure of Artemia salina
hemoglobins: a comparative characterisation of four naupliar and
adult hemoglobins. Eur. J. Biochem. 67, 397–402.
MORRIS, S. (1991). Respiratory gas exchange and transport in
crustaceans: Ecological determinants. Mem. Queensland Mus. 31,
241–261.
MORRIS, S. AND AIRRIESS, C. N. (1998). Integration of physiological
responses of crustaceans to environmental challenge. South Afr. J.
Zool. (in press).
MORRIS, S. AND BRIDGES, C. (1994). Properties of respiratory
pigments in bimodal breathing animals: air and water breathing by
fish and crustaceans. Am. Zool. 34, 216–288.
MORRIS, S., BRIDGES, C. AND GRIESHABER, M. (1985). A new role for
uric acid: Modulator of haemocyanin oxygen affinity in
crustaceans. J. crust. Biol. 235, 135–139.
MORSE, M. P., MEYERHOFF, E., OTTO, J. J. AND KUZURIAN, A. M.
(1986). Hemocyanin respiratory protein in bivalve mollusks.
Science 231, 1302–1304.
MYKLES, D. (1980). The mechanism of fluid absorption at ecdysis in
N. B. TERWILLIGER
1097Functional adaptations of oxygen-transport proteins
the American lobster Homarus americanus. J. exp. Biol. 84,
89–101.
NAKAHARA, A., SUZUKI, S. AND KINO, J. (1983). Tyrosinase activity
of squid hemocyanin. Life Chem. Reports Suppl. I, 319–322.
NAVARRO, A. M., MALDONADO, M., GONZALEZ-LAGOA, J., LOPEZ-
MEJIA LOPEZ-GARRIGA, J. AND COLON, J. L. (1996). Control of
carbon monoxide binding states and dynamics in hemoglobin I of
Lucina pectinata by nearby aromatic residues. Inorg. chim. Acta
243, 161–166.
RATCLIFFE, P. J. O’ROURKE, J. F., MAXWELL, P. H. AND PUGH, C. W.
(1998). Oxygen sensing, hypoxia-inducible factor-1 and the
regulation of mammalian gene expression. J. exp. Biol. 201,
1153–1162.
READ, K. R. H. (1965). The characterization of the hemoglobins of
the bivalve mollusc Phacoides pectinatus (Gmelin). Comp.
Biochem. Physiol. 15, 137–158.
REIBER, C. L. (1997). Oxygen sensitivity in the crayfish Procambarus
clarkii: peripheral O2receptors and their effect on cardiorespiratory
functions. J. crust. Biol. 17, 197–206.
RIGGS, A. (1990). Studies of invertebrate hemoglobins: structure,
function and evolution. In Invertebrate Dioxygen Carriers (ed. G.
Preaux), pp. 9–16. Leuven: Leuven University Press.
RIGGS, A. (1998). Self-association, cooperativity and
supercooperativity of oxygen binding by hemoglobins. J. exp. Biol.
201, 1073–1084.
RIGGS, A. F., RIGGS, C. K., LIN, R. I. AND DOMDEY, H. (1986). Cloning
of the cDNA for the globin from the clam, Barbatia reeveana. In
Invertebrate Oxygen Carriers (ed. B. Linzen), pp. 473–476.
Heidelberg, Berlin, New York: Springer Verlag.
RIGGS, C. AND RIGGS, A. (1990). cDNA-derived amino acid sequences
of single and two-domain globins from the clam Barbatia
reeveana. In Invertebrate Dioxygen Carriers (ed. G. Preaux), pp.
57–60. Leuven: Leuven University Press.
RIZZI, M., WITTENBERG, J. B., CODA, A. AND ASCENZI, P. (1996).
Structural bases for sulfide recognition in Lucina pectinata
hemoglobin I. J. molec. Biol. 258, 1–5.
ROYER, W. E., JR(1992). Structures of red blood cell hemoglobins.
In Advances in Comparative and Environmental Physiology, vol.
13, Blood and Tissue Oxygen Carriers (ed. C. P. Mangum), pp.
87–116. Heidelberg: Springer Verlag.
ROYER, W. E., JR, HENDRICKSON, W. A. AND CHIANCONE, E. (1989).
The 2.4Å crystal structure of Scapharca dimeric hemoglobin:
cooperativity based on directly communicating hemes at a novel
subunit interface. J. biol. Chem. 264, 21052–21061.
ROYER, W. E., PARDANANI, A., GIBSON, Q. H., PETERSON, E. S. AND
FRIEDMAN, J. M. (1996). Ordered water molecules as key allosteric
mediators in a cooperative dimeric hemoglobin. Proc. natn. Acad.
Sci. U.S.A. 93, 14526–14531.
RUPPERT, E. E. AND BARNES, R. D. (1994). Invertebrate Zoology, sixth
edition. Fortworth: Saunders College Publishers.
SALVATO, B., JORI, G., PIAZZESE, A., GHIRETTI, F., BELTRAMINI, M.
AND LERCH, K. (1983). Enzymic activities of type-3 copper pair in
Octopus vulgaris haemocyanin. Life Chem. Rep. Suppl. 1, 313–317.
SHIRLEY, S. M., SHIRLEY, T. M. AND MEYERS, T. R. (1986).
Hemolymph responses of Alaskan king crabs to rhizocephalan
parasitism. Can. J. Zool. 64, 1774–1781.
SODERHALL, K. (1982). Prophenoloxidase activating system and
melanization – a recognition mechanism of arthropods? A review.
Dev. comp. Immunol. 6, 601––611.
SPINDLER, K. D., HENNECKE, R. AND GELLISSON, G. (1992). Protein
production and the molting cycle in the crayfish Astacus
leptodactylus (Nordmann, 1842). II. Hemocyanin and protein
synthesis in the midgut gland. Gen. comp. Endocr. 85, 248–253.
SUZUKI, T. AND IMAI, K. (1997). Comparative studies of the
indoleamine dioxygenase-like myoglobin from the abalone
Sulculus diversicolor. Comp. Biochem. Physiol. 117B, 599–604.
SUZUKI, T., TAKAGI, T. AND OHTA, S. (1989). Amino acid sequence
of the dimeric hemoglobin (HbI) from the deep-sea cold-seep clam
Calyptogena soyoae and the phylogenetic relationship with other
molluscan globins. Biochim. biophys. Acta 993, 254–259.
SUZUKI, T., YUASA, H. AND IMAI, K. (1996). Convergent evolution:
The gene structure of Sulculus 41kDa myoglobin is homologous
with that of human indoleamine dioxygenase. Biochim. biophys.
Acta 1308, 41–48.
TELFER, W. H. AND KUNKEL, J. G. (1991). The function and evolution
of insect storage hexamers. A. Rev. Ent. 36, 205–228.
TERWILLIGER, N. B. (1991). Arthropod (Cyamus scammoni,
Amphipoda) hemoglobin structure and function. In Structure and
Function of Invertebrate Oxygen Carriers (ed. S. Vinogradov and
O. Kapp), pp. 59–63. Berlin, Heidelberg, New York: Springer
Verlag.
TERWILLIGER, N. B. (1992). Molecular structure of the extracellular
heme proteins. In Advances in Comparative and Environmental
Physiology, vol. 13, Blood and Tissue Oxygen Carriers (ed. C. P.
Mangum), pp. 193–229. Heidelberg: Springer Verlag.
TERWILLIGER, N. B. AND BROWN, A. C. (1993). Ontogeny of
hemocyanin function in the Dungeness crab Cancer magister: the
interactive effects of developmental stage and divalent cations on
hemocyanin oxygenation properties. J. exp. Biol. 183, 1–13.
TERWILLIGER, N. B. AND O’BRIEN, K. (1992). Cancer magister ‘non-
respiratory protein’ is a molt-related hemocyanin. Am. Zool. 32,
44A.
TERWILLIGER, N. B. AND OTOSHI, C. (1994). Cryptocyanin and
hemocyanin: fluctuations and functions of crab hemolymph
proteins during molting. Physiologist 37, A67.
TERWILLIGER, N. B. AND TERWILLIGER, R. C. (1978). Oxygen binding
domains of a clam (Cardita borealis) extracellular hemoglobin.
Biochim. biophys. Acta 537, 77–85.
TERWILLIGER, N. B. AND TERWILLIGER, R. C. (1982). Changes in the
subunit structure of Cancer magister hemocyanin during larval
development. J. Exp. Zool. 221, 181–191.
TERWILLIGER, N. B. AND TERWILLIGER, R. C. (1984). Hemoglobin
from the ‘Pompeii worm’, Alvinella pompejana, an annelid from a
deep sea hot hydrothermal vent environment. Mar. biol. Lett. 5,
191–201.
TERWILLIGER, N. B., TERWILLIGER, R. C., MEYERHOFF, E. AND MORSE,
M. P. (1988). Bivalve hemocyanins – a comparison with other
molluscan hemocyanins. Comp. Biochem. Physiol. 89B, 189–195.
TERWILLIGER, N. B., TERWILLIGER, R. C. AND SCHABTACH, E. (1976).
The quaternary structure of a molluscan (Helisoma trivolvis)
extracellular hemoglobin. Biochim. biophys. Acta 453, 101–110.
TERWILLIGER, N. B., TERWILLIGER, R. C. AND SCHABTACH, E. (1985).
Intracellular respiratory proteins of Sipuncula, Echiura and
Annelida. In Blood Cells of Marine Invertebrates (ed. W. Cohen),
pp. 193–225. New York: Alan R. Liss
TERWILLIGER, R. C. (1974). Oxygen equilibria of the vascular and
coelomic hemoglobins of the terebellid polychaete, Pista pacifica.
Evidence for an oxygen transfer system. Comp. Biochem.Physiol.
48A, 745–755.
TERWILLIGER, R. C., GARLICK, R. L. AND TERWILLIGER, N. B. (1980).
Characterization of the hemoglobins and myoglobin of Travisia
foetida. Comp. Biochem. Physiol. 66B, 261–266.
1098
TERWILLIGER, R. C. AND TERWILLIGER, N. B. (1985). Molluscan
hemoglobins. Comp. Biochem. Physiol. 81B, 255–261.
TERWILLIGER, R. C., TERWILLIGER, N. B. AND ARP, A. (1983). Thermal
vent clam (Calyptogena magnifica) hemoglobin. Science 219,
981–983.
TERWILLIGER, R. C., TERWILLIGER, N. B. AND SCHABTACH, E. (1986).
Hemoglobin from the parasitic barnacle, Briarosaccus callosus. In
Invertebrate Oxygen Carriers (ed. B. Linzen), pp. 125–127. Berlin,
Heidelberg: Springer Verlag.
TORCHIN, M. (1994). The effects of parasitism on the hemocyanin of
an intertidal hermit crab, Pagurus samuelis. MS thesis, University
of Oregon.
TOULMOND, A., ELIDRISSI SLITINE, F., DE FRESCHEVILLE, J. AND JOUIN,
C. (1990). Extracellular hemoglobins of hydrothermal vent
annelids: structural and functional characteristics in three alvinellid
species. Biol. Bull. mar. biol. Lab., Woods Hole 179, 366–373.
TRUCHOT, J. P. (1980). Lactate increases the oxygen affinity of crab
hemocyanin. J. exp. Zool. 214, 205–208.
TRUCHOT, J. P. (1992). Respiratory function of arthropod
hemocyanins. In Advances in Comparative and Environmental
Physiology, vol. 13, Blood and Tissue Oxygen Carriers (ed. C. P.
Mangum), pp. 377–410. Heidelberg: Springer Verlag.
VAN HOLDE, K. V. H. AND MILLER, K. I. (1995). Hemocyanins. In
Advances in Protein Chemistry, vol. 47, pp. 1–81. New York:
Academic Press.
VINOGRADOV, S. N., LUGO, S., MAINWARING, M., KAPP, O. AND
CREWE, A. (1986). Bracelet protein: a quaternary structure
proposed for the giant extracellular hemoglobin of Lumbricus
terrestris. Proc. natn. Acad. Sci. U.S.A. 83, 8034–8038.
WAXMAN, L. (1975). The structure of annelid and mollusc
hemoglobins. J. biol. Chem. 250, 3790–3795.
WEBER, R. E., BRAUNITZER, G. AND KLEINSCHMIDT, T. (1985).
Functional multiplicity and structural correlations in the
hemoglobin system of larvae of Chironomus thummi thummi
(Insecta, Diptera): Hb components CTT I, CTT II, CTT III, CTT
IV, CTT VI, CTT VIIB, CTT IX and CTT X. Comp. Biochem.
Physiol. 80B, 747–753.
WILKENS, J. L. (1997). Possible mechanisms of control of vascular
resistance in the lobster Homarus americanus. J. exp. Biol. 200,
487–493.
WILKENS, J. L., DAVIDSON, G. W. AND CAVEY, M. J. (1997). Vascular
peripheral resistance and compliance in the lobster Homarus
americanus. J. exp. Biol. 200, 477–485.
WILKENS, J. L., YOUNG, R. E. AND DICAPRIO, R. A. (1989). Responses
of the isolated crab ventilatory central pattern generators to
variations in oxygen tension. J. comp. Physiol. B 159, 29–36.
WOOD, E. AND MOSBY, L. (1975). Physicochemical properties of
Planorbis corneus erythrocruorin. Biochem. J. 149, 437–445.
YAGER, T., TERWILLIGER, N. B., TERWILLIGER, R. C., SCHABTACH, E.
AND VAN HOLDE, K. (1982). Organization and physical properties
of the giant extracellular hemoglobin of the clam Astarte castenea.
Biochim. biophys. Acta 709, 194–203.
ZAL, F., GREEN, B. N., LALLIER, F. H. AND TOULMOND, A. (1997a).
Investigation by electrospray ionization mass spectrometry of the
extracellular hemoglobin from the polychaete annelid Alvinella
pompejana: an unusual hexagonal bilayer hemoglobin.
Biochemistry 36, 11777–11786.
ZAL, F., GREEN, B. N., LALLIER, F. H., VINOGRADOV, S. N. AND
TOULMOND, A. (1997b). Maximum entropy analysis of electrospray
ionisation mass spectra of the heteromultimeric hemoglobin of
Arenicola marina. Eur. J. Biochem. 243, 85–92.
ZAL, F., SUZUKI, T., KAWASAKI, Y., CHILDRESS, J. J., LALLIER, F. H.
AND TOULMOND, A. (1997c). Primary structure of the common
polypeptide chain bfrom the multi-hemoglobin system of the
hydrothermal vent tube worm Riftia pachyptila: An insight on the
sulphide binding site. Proteins (in press).
ZLATEVA, T., DIMURO, P., SALVATO, B. AND BELTRAMINI, M. (1996).
The o-diphenol oxidase activity of arthropod hemocyanin. FEBS
Lett. 384, 251–254.
Note added in proof
The Pogonophora have been reduced from phylum to the
rank of family (Siboglinidae) within the Polychaeta [Rouse, G.
W. and Fauchald, K. (1997). Cladistics and polychaetes.
Zoologica Scripta 26, 139–204], a classification consistent
with the structures of the extracellular hemoglobins of
pogonophores, vestimentiferans and annelids (Terwilliger,
1992).
N. B. TERWILLIGER
