AMER. ZOOL., 26:235-248 (1986)
The Molecular Ecology of Fundulus heteroclitus
Hemoglobin-Oxygen Affinity1
DENNIS A. POWERS, PAULA M. DALESSIO,2
EDWARD LEE, AND LEONARD DIMICHELE
Department of Biology and The Chesapeake Bay Institute,
The Johns Hopkins University, Baltimore, Maryland 21218
SYNOPSIS. Animals faced with environmental perturbations must adapt or face extinction.
The respiratory complex, specifically hemoglobins, is perhaps the best system to study
such adaptation because it exists at the organism—environment interface. Fish are particularly useful models because they respond directly to such environmental variables as
temperature, oxygen, pH, carbon dioxide, and salinity. Our experiments have addressed
the molecular, cellular, and physiological mechanisms employed by fish to maintain respiratory homeostasis in the wake of changing temperature and oxygen. Immediate, intermediate, and long-term adaptation can only be understood when the hemoglobin's ligand
binding properties and the cellular and hormonal regulation of various ligands are considered simultaneously. We describe a detailed thermodynamic model for the binding of
oxygen, protons, and organic phosphates to hemoglobin; discuss the role of multiple
hemoglobins; and present evidence for physiological and genetic regulation of hemoglobin's major allosteric modifiers in response to environmental stress in the mummichog,
Fundulus heteroclitus.
logical function depends on the hemoglobin's
ability to form a reversible complex
Comparative biochemists and physiologists have found that the blood-oxygen between oxygen and the ferrous iron in the
affinities of various fish species are com- hematoporphyrin. Under constant physipatible with the physical and chemical cal conditions and in the absence of modparameters of their environments. For ifying molecules, the affinity of the hemoexample, fish that live in low oxygen envi- globin for oxygen depends primarily on
ronments have high oxygen affinities while the hydrophobic nature of the heme pocket
those that live in high oxygen environ- and the roles of specific amino acid resiments have lower oxygen affinities. More- dues which may directly or indirectly affect
over, fish that live in environments where hemoglobin-oxygen (Hb-O 2 ) affinity.
physical parameters periodically change Since these amino acids are coded for by
have the necessary molecular machinery the globin genes, this intrinsic Hb-O 2
required for adaptation. This machinery affinity is genetically determined. While
includes species-specific hemoglobins and/ intrinsic H b - O 2 affinity is genetically
or the regulation of various modifier determined, physiological plasticity is proligands. The oxygen transport system of vided by regulating intracellular concenmodifiers, such as
the fish Fundulus heteroclitus is an ideal trations of allosteric
+
HCO
",
Cl~,
H
,
and
organic
phosphates.
3
model in which to study these adaptative
The mathematical concept that describes
mechanisms.
the interactions between hemoglobin and
The primary function of hemoglobin is its various ligands was formalized by
to carry oxygen from the "organism-envi- Wyman (1948, 1964).
ronment interface" (e.g., the gills, lungs,
etc.) to the respiring tissues. This physioINTRODUCTION
1
From the Symposium on The Biology of Fundulus
heteroclitus presented at the Annual Meeting of the
American Society of Zoologists, 27-30 December
1983, at Philadelphia, Pennsylvania.
8
Paula M. Dalessio's home institution is: Department of Biology, The George Washington University,
Washington, D.C.
THE LINKAGE BETWEEN HEMOGLOBIN,
OXYGEN, PROTONS, AND
ORGANIC PHOSPHATE
When hemoglobin (M) binds four oxygen molecules (X) and one molecule of an
organic phosphate (D) (Scheme 1), the
simultaneous equilibria can be depicted by
the following linkage scheme:
235
236
D. A. POWERS ET AL.
»K
D
«k
"k
M
|
» MX
D
K
MD-
|
IVQ
»k
MX,
D
K,
'MXD
XU
4
>*k
|
°K IX
MX,DSXI.
IVp
(1 +
MX,
|
(Scheme 1)
H
H
K D [H] + * H K D [ H ] ' + . . . * H K D [H]')
K[H] +
2H
K[Hf +
SH
K[H] 5 + . . .
mH
K[H]™)
MX,
°K 5x
*MX,D-
3x1.
KQ
K(1 +
j
'MX.D
4x1.
Kp
°K 4X
(eq. 1)
and the equation for oxyhemoglobin is:
"Kg*
_ °K 4 x (l +
H
(1 +
H
K 4 x D [H] + ' " K 4 x D [ H ] ' + . . .
K 4 x [H] +
2H
!
rH
K 4xD [H]Q
H
K [ H ] + . . . " K 4x [H]")
(eq. 2)
The derivation for these equations has been
published by Hobish and Powers (submitwhere the k's are the microconstants for ted).
individual oxygenation steps, and the K's
The various DKaPP values can be deterare macroconstants. The presuperscripts on mined for oxy- and deoxyhemoglobin at
all constants indicate the number and type defined pH values by the method of Powers
of ligands being bound; while the subscripts et al. (1981). These data cannot be fitted
indicate the number and type of ligands to eqs. 1 and 2 easily because there are an
already bound to the hemoglobin.
infinite number of possible constants. Since
If temperature, pH, etc. are constant, we know only a few protons are involved
and the hemoglobin does not dissociate or (Greaney et al., 1980a), the data can be
polymerize, Scheme 1 can be described by fitted by a non-linear least squares analysis
9 of the 13 equilibrium constants. How- to simplified versions of these equations.
ever, when proton concentration is allowed
These (eqs. 3 and 4) are functionally simto vary, it affects oxygen binding as well as ilar to a Hill equation.
the binding of organic phosphate. At rel_ D K(1 + Z H K D [H] Z )
atively high pH (e.g., pH 10) there is little
(eq- 3)
or no Bohr or organic phosphate effects
(1 + M H K[H] m )
on Hb-O 2 affinity; conversely, at low pH
there can be a reversal or inhibition of both
-K4X[H]")
the Bohr and organic phosphate effects on
Hb-O 2 affinity. Thus, the Bohr and organic Examination of Figure 1 indicates several
phosphate effects are most pronounced possibilities for Hb-O 2 affinity regulation.
over the intermediate, physiologically For example, it can be differentially
important pH range. Assuming that the affected by changing intracellular concenprotons that affect the binding of organic trations of ligands, modifying one or more
phosphate are between zero and z for of the ligand-protein binding constants, a
deoxyhemoglobin and between zero and r combination of these, or other strategies.
for oxyhemoglobin, the wiacro-interaction Moreover, the dissociation of all of the
between hemoglobin, oxygen, organic complexes (Fig. 1) is increased by increasphosphate, and protons can be described ing temperature; providing an additional
by Figure 1.
mode of physiological adjustment to enviWhen the hemoglobin is at the extremes ronmental temperature.
of oxygenation (Fig. 1), the apparent bindMULTIPLE FISH HEMOGLOBINS
ing of organic phosphate to oxy- or deoxPossessing a number of hemoglobins,
yhemoglobin can be measured directly
(Powers et al., 1981). The macroconstants some of which have unique functional
for the other faces of Figure 1 are obtain- characteristics, is one strategy for adapting
able from oxygen binding measurements to a changing environment. While the
carried out as a function of pH and organic presence of multiple hemoglobins in lower
phosphate concentrations.
vertebrates is a well-established phenomFor deoxyhemoglobin (M) the apparent enon, there are discrepancies regarding the
binding constant for organic phosphate actual number, proportion, and structural
and functional properties of species-spe(DKggxy) is defined by:
MOLECULAR ECOLOGY OF FUNDULUS HEMOGLOBIN
-±-
MDHz*
• MX«DH,-*-
FIG. 1. A generalized schematic of the linkage equilibria relating the binding of protons (H), organic
phosphate (D), and oxygen (X) to hemoglobin tetramers (M). The macrobinding constants (K) are those
described in the text with the presuperscripts indicating the number and kind of ligands being bound
and the postsubscripts, the numbers and kind of ligands
bound to the macromolecules (M).
cific isohemoglobins (reviewed by Riggs,
1970; Fyhn and Sullivan, 1975; Powers,
1980). Such differences can arise from
thermoacclimatory variation, developmental changes, polymerization, dissociation, and susceptibility to autooxidation.
One of the most interesting sources of
electrophoretic variation arises from
genetically unique globin chains that combine to give a series of unique hemoglobin
tetramers. An analysis of the subunit structure of multiple fish hemoglobins has only
rarely been attempted. In those instances,
two adaptive strategies have become evident. First, there are multiple hemoglobins
that are structurally different but functionally equivalent. Second, there are
structurally different hemoglobins, some
of which (but not all) are functionally
unique (see Powers, 1980 and references
therein).
STRUCTURAL ASPECTS OF FUNDULUS
HEMOGLOBINS
One of the better analyzed hemoglobin
systems is that of Fundulus heteroclitus (Mied
and Powers, 1978). Fundulus has four isohemoglobins, each a tetramer of about
64,000 daltons (Fig. 2, Table 1). They have
different isoelectric points (Table 1); all are
237
between pH 5-9. These fish hemoglobins
show very little tetramer-dimer dissociation in low salt (~10~8 M heme) and only
slightly more in 1.0 M NaCl (~10"7 M
heme). Moreover, the presence of up to 4
M urea has little effect on the dissociation.
Each hemoglobin is composed of two a
and two /3 subunits. There are four different globin chains: a3, ab, /3a and j3b. The
Hbl is a homotetramer composed of two
ab and two /3b chains; HblV is a homotetramer consisting of two a* and two fit* subunits. Isohemoglobins II and III are heterotetramers consisting of all four chains.
A model of these combinations can be seen
in Figure 2. While the amino acid compositions of the chains are significantly different the end-groups of homologous
chains (i.e., a*:ah and /3a:/3b) are identical
(Mied and Powers, 1978). The NH2-termini of the a chains are blocked with an
acetyl group, while the /3 chains have free
NH2-terminal Val. The COOH-termini are
-Tyr-Arg and -Tyr-His for the a and /3
chains, respectively.
FUNCTION OF PURIFIED FUNDULUS
HEMOGLOBINS
The effect of pH on the P50 of the unfractionated hemoglobin and of the isolated
components I, II, III and IV is illustrated
in Figure 3. All of the components are similarly affected by pH. The data in Figure
3 suggest that components II and IV have
slightly higher oxygen affinity at pH values
greater than 7.5. Although subtle differences may exist among the components,
these differences in general appear to lie
within the range of experimental variation. Finally, oxygen equilibria studies done
at high pH as a function of temperature
indicate significant thermal sensitivity of
the hemoglobin components. The enthalpy
calculated from van't Hoff plot is AH =
-15.5 ± 0.5 kcal/mole.
THE ORGANIC PHOSPHATE EFFECT
The major organic phosphate in fish
erythrocytes is either adenosine triphosphate (ATP) or guanosine triphosphate
(GTP), while in mammalian red blood cells
it is 2,3-diphosphoglycerate (2,3 DPG). The
effects of ATP and GTP on the oxygen
binding properties offish hemoglobins are
238
D. A. POWERS ET AL.
f2
a2
PI
HbET
a
a
N O
"CD
° p?
az
Hb HI
b
b
Ra
oa
O (VI
a , P,
i
I
I
b • o b•
•
1
I
1
i
i
i
Q
ob
-8,b
b
O CVI
ijSJSaJ !
'1
•2
«2
Pi
»2b
Hbl
Hb E
FIG. 2. A model for the subunit compositions of F. heteroclitus hemoglobins. The four different dimers that
can be formed, a"/3", a*/9b, ab0*, and a'/S1", have been entered at the left as a,(3, dimers and above each column
as a,f}2 dimers. For association into tetramers, the oy?2 dimers have been written |82a2 to more accurately
depict the actual spatial relationship to the 0,18, dimer that exists in the tetrameric hemoglobin molecule and
to emphasize the a,/32 and a ^ , contacts between dimers. To aid in visualizing the association-dissociation
process, the integrity of the dimers has been maintained within each tetramer. The upper portion of the
matrix, which contained tetramers identical with those in the lower half, has been omitted for the sake of
simplicity. The four tetramers shown with dotted lines between the subunits contain double a-b type contacts
(a,|82 and Qfj/9, contacts) and/or four different polypeptide chains, and therefore are assumed to be unstable
(see "Discussion"). The six remaining stable hemoglobins constitute the four electrophoretically distinguishable components, I, II, III, and IV, of F. heteroclitus hemoglobin.
similar to those of 2,3 DPG on mammalian
hemoglobins. Furthermore, the influence
of ATP on both the Bohr effect and on
the pH dependence of subunit cooperativity indicates that the reactions of many fish
hemoglobins with oxygen, organic phosphates, and protons are functionally linked.
In addition to its allosteric effect on oxygen
affinity, organic phosphate has an important influence on the Donnan distribution
of protons across the erythrocyte membrane (Wood andjohansen, 1972). Several
workers have demonstrated that adaptive
changes in fish blood hemoglobin-oxygen
239
MOLECULAR ECOLOGY OF FUNDULUS HEMOGLOBIN
TABLE 1. Selected physiochemical properties of the hemoglobins from Fundulus heteroclitus.
Electrophoretic component
Parameter
Hb I
Hb II
H b III
Hb IV
11.5 ± 0.9
40.5 ± 1.0
10.2 ± 0.7
37.8 ± 1.1
8.20 ±0.15
7.52 ± 0.16
5.82 ± 0.09
6.48 ± 0.16
4.4
4.4
4.4
4.4
S»ow b
—
—
K.i'lmMheme)
<0.12
<0.04
K 4 , d (mMheme)
6.04
2.41
2.38
3.85
64,000
64,000
64,000
64,000
M r '(0.1 M NaCl)
* Determined by gel scanning of 16 gels.
b
At several protein concentrations between 1 mg/ml and 10 mg/ml in 0.1 M phosphate, 0.1 M NaCl and
pH 7.0.
c
Tetramer-dimer dissociation constants in mM heme determined in 0.1 M phosphate buffer, pH 7.0, 0.1
Af NaCl and 1 mM EDTA.
d
Same as c but performed in 1.0 M NaCl at three different protein concentrations and three rotor speeds.
e
Determined by gel filtration (see: Mied and Powers, 1978).
Proportions (%)*
Isoelectric point
affinity during acclimation to low oxygen
or increased temperature are inversely
related to changes in red blood cell organic
phosphate concentrations (Wood and
Johansen, 1972; Wood et al., 1975; Greaney and Powers, 1978).
ATP is the major allosteric modifier of
Fundulus hemoglobins. ATP binds to F.
heteroclitus deoxyhemoglobin with a stoichiometry of one major binding site per
hemoglobin tetramer. The affinity of the
deoxyhemoglobins for ATP is elevated as
temperature decreases. At 11.5°C, its dissociation constant is 4.0 ± 1.2 x 10~6 M.
However, at 22.5°C, it increases to 1.18 ±
0.8 x 10"5 M. The van't Hoff enthalpy
(AH), calculated from the equilibrium association constants, is approximately —15.0
kcal/mole of tetramer. As illustrated in
Figure 3, the presence of ATP dramatically decreases the hemoglobin-oxygen
affinity of Fundulus hemoglobins and the
effect is amplified at low pH.
A number of studies have shown that the
NH2-termini of the 0 chains are involved
in ionic interactions with the organic phosphate. Organic phosphate binds to certain
basic residues in the central cavity of the
human deoxytetramer (Arnone, 1972).
Presumably, salt bridges are formed with
the NH?-termini of the 0 chains (Val, 01),
and the imidazole side chains (His: 02, 0143)
of both 0 chains, as well as the e-amino
group of Lys, /382, from one of the 0 chains.
The homologous residues in Fundulus and
most other fish hemoglobins are: Val (01),
Glu (/32), Lys (082), and Arg (0143)
(reviewed by Powers, 1980). Assuming that
the same amino acid residues are involved
in the binding of ATP by Fundulus hemoglobins, the major residues that would be
titrated over a physiological pH range
would be the amino termini of the 0 chains.
ADAPTATIONS TO TEMPERATURE CHANGES
As temperature increases, the availability of oxygen in water decreases because
the solubility of oxygen decreases with
increasing temperature and elevated biological activity may reduce dissolved oxygen to below saturation. Consequently, at
higher temperatures fish require more
oxygen, but less is available. Fish respond
to this dilemma by increasing ventilation
volume, heart rate, and oxygen carrying
capacity of the blood.
The effect of temperature on the oxygen
equilibria of fish hemoglobin has been
reviewed (Riggs, 1970; Johansen and Lenfant, 1972; Johansen and Weber, 1976).
Although the number of species examined
is limited, the functional properties of
teleost hemoglobins can be divided into
three major categories as suggested by
Weber etal. (1976). Class I contains species
with one or more hemoglobins all of which
are sensitive to both temperature and pH
(Gillen and Riggs, 1971; Gillen and Riggs,
1972; Bonaventura et al, 1974; Weber,
1975; Mied and Powers, 1978). Class II has
species with multiple components, some of
which are functionally similar to the Class
240
D. A. POWERS ET AL.
affinity in fish experiencing large fluctuations, as suggested by Johansen and Lenfant (1972), then it must be primarily associated with the regulation of intracellular
pH, the levels and types of organic phosphates, and other ligand-linked phenomena rather than selection of hemoglobins
per se. Thus, blood-oxygen affinity adaptation to different thermal regimes must
be primarily at the erythrocyte level rather
than in the intrinsic enthalpy of hemoglobin oxygen binding.
Greaney and Powers (1977, 1978) demonstrated that Fundulus heteroclitus adapt
to thermal changes by changing ATP concentrations within erythrocytes. This
FIG. 3. A plot of variation of log P50 with pH for F. prompted us to ask if this response was
heteroclitus hemoglobins. Data were obtained using the
Aminco Hem-O-Scan oxygen dissociation analyzer. triggered by reduced oxygen (due to the
Open symbols represent stripped Hb; solid symbols rep-increased temperature), increased temperresent Hb in the presence of 10 ATP/Hb. O and • , ature, or both of these variables. Thereunfractionated Hb; V, HB I; 0 . Hb II; O, HB III; A, fore, fish were acclimated to various temHb IV.
peratures (10°C, 22°C, and 30°C), but with
oxygen concentration maintained constant
I hemoglobins, while other components are (about 7 ppm) at each temperature. These
not strongly affected by temperature and animals also decreased their erythrocyte
pH (Hashimoto et al., 1963; Binotti et al., ATP with increased temperature. More1971; Powers and Edmundson, 1972a; over, fish acclimated to 10°C but in air satPowers, 1972, 1974; Wyman et al., 1977). urated water (12.5 ppm) showed the same
Class III fishes have hemoglobins that are red blood cell ATP levels as those mainpH sensitive, but temperature insensitive tained at 10°C but with 7 ppm oxygen.
(Rossi-Fanelli and Antonini, 1961; Ander- These data demonstrate that the ATP
response was elicited by increased temperson et al., 1973).
ature
alone and was independent of disThe overall enthalpy of bood oxygen
solved
oxygen levels in the range 7—12.5
equilibria represents both the intrinsic heat
of oxygen binding to hemoglobin and con- ppm. However, studies on other fishes sugtributions due to other ligand-linked pro- gest the strategy employed by Fundulus is
cesses (see earlier discussion). One such not universal (Powers, unpublished).
process is associated with proton equilibria
STRATEGIES OF ADAPTING TO HYPOXIA
of amino acid side chains (i.e., the Root and
Bohr effects). At alkaline pH (i.e., pH 9There are numerous strategies by which
10) the Root and Bohr effects are essen- fish are able to maintain respiratory
tially inoperative. Thus, data collected on homeostasis when environmental oxygen
"stripped" hemoglobins at alkaline pH, is reduced. Perhaps the most common is
over a range of temperatures, provide to seek out a more favorable environment.
information primarily on the intrinsic Species that remain in oxygen poor envithermodynamic parameters of the hemo- ronments have: (1) immediate, (2) interglobins per se. Under such conditions, mediate, and (3) long-term adaptation
enthalpies are very similar for a number of strategies.
fish hemoglobins from a variety of thermal
environments (Powers et al., 19796).
Immediate response to hypoxia
Powers et al. (1979a) have clearly shown
The immediate response to an oxygen
that if evolution has favored a decrease in poor environment is an increase in heart
temperature sensitivity of blood-oxygen rate and ventilation volume (Prosser, 1973).
MOLECULAR ECOLOGY OF FUNDULUS HEMOGLOBIN
241
20
3
E
15
o
10
120
I0"
1
,-7
10
!
IO"
FIG. 5. The effect of epinephrine on the PM values
of washed erythrocytes obtained from Fundulus hetFIG. 4. The effect of varying concentrations of epi- eroclitus. The solid circles (•) represent P50 values of
nephrine on the P50 values of washed erythrocytes untreated erythrocytes. The open triangles (A) repfrom Fundulus heteroclitus. Bars indicate ± standard resent P50 values of epinephrine treated erythrocytes.
error of the mean (SEM).
Epinephrine (M)
In addition, fish will often "gulp" air and/
or utilize the water at the air-interface
which has the greatest oxygen content.
Furthermore, fish are able to rapidly
increase or decrease blood oxygen affinity
by employing hormonal and/or other factors in the serum.
Circulating catecholamine concentrations increase markedly in fish subjected to
stress such as hypoxia, surgery, physical
disturbance, and confinement (Nakano and
Tomlinson, 1967; Butlers al., 1978,1979;
Wahlqvist and Nilsson, 1980; LeBras,
1982). Nikinmaa (1982, 1983) assessed the
role of epinephrine in controlling the
blood-oxygen affinity in rainbow trout.
Both in vivo and in vitro, epinephrine
increased blood-oxygen affinity and erythrocyte volume, decreased mean erythrocyte hemoglobin and ATP concentrations,
and decreased the extracellular to intracellular proton gradient. All of which are
similar responses seen in hypoxic trout.
Fundulus heteroclitus whole blood has a
much higher oxygen affinity than washed
Fundulus erythrocytes—a phenomenon
also observed in trout and flounder (Soivio
et al, 1980; Nikinmaa, 1983; Dalessio et
al., 1984,1985 manuscript in preparation).
Since removal of catecholamines from the
medium of washed erythrocytes results in
a general instability of these cells (Bourne
and Cossins, 1982), then perhaps the
decreased oxygen affinity of washed fish
erythrocytes is also due to the absence of
catecholamines.
To test this hypothesis, epinephrine was
added to washed Fundulus erythrocytes and
the oxygen affinity of red cells was measured (Fig. 4). The P50 of washed cells
decreased in a dose-dependent fashion in
concentrations ranging from 10~7 M to 10"6
M epinephrine. Further decreases in P50
did not occur with epinephrine in concentrations exceeding 10~6 M. The P50 of
washed cells decreased within 10 min following addition of epinephrine to the
medium and remained constant for at least
110 min (Fig. 5). Although the P50 of
washed cells decreased from 18.0 mm Hg
to 7.5 mm Hg following treatment with
\0~6 M epinephrine, the P50 value of whole
blood, 4.5 mm Hg, was never attained.
In vivo, epinephrine is generally degraded
very rapidly. It is not known if washed
erythrocytes maintained in an artificial
medium are capable of degrading epinephrine and hence, if the decreased P50
seen over 2 hr is due to the continued presence of epinephrine. Washed cells incubated for 5 min in medium containing epi-
242
TABLE 2.
ities.
D. A. POWERS ET AL.
In vitro and in vivo erythrocyte-oxygen affinTreatment
P,o (mm Hg)
In vitro (washed erythrocytes)"
Control
13.4 ± 0.1
Epinephrine treated
7.3 ± 0.1
13.5 ± 0.1
Control
Epinephrine treated
(washed)
8.5 ± 0.1
12.7 ± 0.1
Control
Epinephrine treated
(washed, 4 hr later)
7.7 ± 0.1
In vivo (whole blood)6
Non-injected control
4.9 ± 0.2
Bull saline injected
control
6.4 ± 0.3
Epinephrine 10"6M
8.2 ± 0.4
Epinephrine 10"sM
7.6 ± 0.4
Epinephrine 10~*Af
7.2 ± 0.3
• Following P50 measurements of washed cells in
presence of epinephrine, cells were washed with and
resuspended in epinephrine free medium, and the P50
followed for 4 hr. Errors are reported as ±SEM.
b
Each fish was injected with 0.01 ml/g body wt of
one of the above substances. Each group consisted of
10 fish. Errors are reported as ±SEM.
nephrine and then washed with and
resuspended in fresh medium without epinephrine have a decreased P50 that was
maintained over a 4 hr period (Table 2).
Stadel etal. (1983) have shown that washed
frog erythrocytes incubated for 3 hr in
medium containing the /?-adrenergic agonist, isoproterenol, results in desensitization of the /3-adrenergic receptor-coupled
adenylate cyclase in the plasma membrane.
Furthermore, they show that prolonged
stimulation of adenylate cyclase by isoproterenol results in a 50-60% decrease in
receptor sites. Perhaps epinephrine causes
a shift in erythrocyte metabolism resulting
in a new steady-state that is temporarily
independent of the presence of the hormone. At low concentrations (10~9 M and
10~8 M) epinephrine has little effect on the
rate of oxygen consumption of washed
erythrocytes. However, 5 min after the
addition of 10~6 M epinephrine, oxygen
consumption increases markedly and
remains elevated for 5-10 min (Fig. 6).
Oxygen consumption decreases slightly
below basal levels for a short time, after
which it returns to basal levels for an indef-
inite period. However, it is not known
whether this shift in metabolism lasts for
5 min after which it returns to a pre-epinephrine state or if it reaches a new steadystate.
In Fundulus injected with various concentrations of epinephrine, P50 values of
blood increase slightly; this response does
not appear to be dose-dependent (Table
2). The effects of epinephrine on blood in
vivo appear to be opposite from those of
washed erythrocytes. Epinephrine added
to whole blood in vitro yielded P50 values
similar to those obtained in epinephrine
injected fish.
Intermediate response to hypoxia
Intermediate responses are generally
activated after several hours of hypoxic
conditions and last many days or until longterm compensation is achieved. These
responses include increasing hematocrit by
retaining serum in muscle tissues (Cameron, 1970), or releasing stored erythrocytes from the spleen, reducing intraerythrocyte organophosphate concentrations
(Wood and Johansen, 1972; Greaney and
Powers, 1977, 1978), changing pH, and
changing the ionic micro-environments of
erythrocytes (Houston and Mearow, 1979).
Such attempts to maintain oxygen delivery
to respiring tissues is usually accompanied
by large fluctuations in various enzyme
activities during metabolic readjustment
(Greaney etal, 19806).
During the intermediate responses
described above, fish synthesize new erythrocytes so that the total oxygen carrying
capacity of the blood is increased. Eventually, a new steady-state between new cells,
enzyme levels, organophosphates and
hemoglobin function is achieved. This level
and the balance is different for each fish
species. When exposed to low oxygen environments for several days, both mammals
and fish (Krogh and Leitch, 1919; Johansen, 1970) increase the oxygen carrying
capacity of their blood. This is accomplished by a number of strategies. The
common mechanisms between mammals
and fish are: increased hematocrit,
increased hemoglobin content, and
increased blood buffering capacity. On the
MOLECULAR ECOLOGY OF FUNDULUS HEMOGLOBIN
120 -
•
Control
A
Epinephrine (IO" 6 M)
243
100
80
il2 E
60
-20
60
30
90
MINUTES
FIG. 6. The effect of epinephrine on the oxygen consumption (Vo2) of washed erythrocytes obtained from
Fundulus heteroclitus. The solid circles (•) represent Vo2 values of untreated erythrocytes. The open triangles
(A) represent Vo2 values of treated erythrocytes. The arrow indicates the time at which epinephrine was
added to erythrocytes.
other hand, mammals and fish differ considerably in other aspects of their response
to low oxygen. Mammals decrease the
affinity of their hemoglobin for oxygen.
Hypoxic fish, in contrast, increase hemoglobin-oxygen affinity as characterized by
a decrease in the P50 of their corresponding
oxygen-saturation curves (Wood and
Johansen, 1972). For example, mammals
acclimated to high altitudes increase levels
of 2,3 DPG in their erythrocytes (Lenfant
et al., 1968). Since mammals typically live
in an oxygen-rich environment, it has been
suggested that this response might be best
suited to the more commonly encountered
forms of low altitude hypoxia, such as
chronic hypoxemia (Gerlach and Duhm,
1972; Eaton, 1974). The response of waterbreathing vertebrates to chronic hypoxia
is quite different (Powers, 1974). Eels have
been shown to decrease erythrocyte ATP
and increase Hb-O 2 affinity when acclimated to low environmental oxygen (Wood
and Johansen, 1972). In addition, Fundulus
heteroclitus acclimated to hypoxic conditions lower red cell ATP by as much as
40% and increase the percent hematocrit
which presumably increases the oxygen
carrying capacity of the blood (Fig. 7)
(Greaney and Powers, 1978). Moreover,
there were concomitant increases in serum
lactate and a decrease in blood pH.
We predicted that if the control mechanism was directed at the erythrocyte level,
then fish red blood cells should decrease
ATP, in vitro, under anoxic conditions.
Consistent with our in vivo observation, we
found that anaerobic F. heteroclitus red cells
significantly lowered their ATP levels in
vitro (Greaney and Powers, 1977). Since
244
D. A. POWERS ET AL.
PHYSIOLOGICAL CORRELATION BETWEEN
LACTATE DEHYDROGENASE GENOTYPE AND
HEMOGLOBIN FUNCTION IN FUNDULUS
During our hypoxic studies, we observed
significant variability in red cell ATP concentrations among individual fish. We
questioned whether this variability could
be the result of a genetic component. Consequently, we analyzed F. heteroclitus for
red cell ATP, concomitantly screening for
a series of enzyme variants. Not only were
ATP levels under genetic control, but they
were also highly correlated with lactate
dehydrogenase (LDH) phenotype (Powers
etal., 1979c).
There are three major electrophoretically distinguishable phenotypes of the
heart-type LDH (the LDH-B locus) in F.
heteroclitus (Place and Powers, 1978). The
polymorphism is due to two co-dominant
alleles which exhibit a dramatic north-south
cline in gene frequency along the Atlantic
coast of the U.S. (Powers and Powers, 1975;
acclimation time (days)
Powers and Place, 1978). The phenotypes
are
LDH-BaBa, LDH-BaBb and LDH-BbBb.
FIG. 7. Fundulus heteroclitus hypoxia studies: A. Time
course of % blood hematocrit of hypoxic (•) and nor- The B locus is the only LDH expressed in
moxic (•) fish. B. Time course of acclimation of F.
the red blood cells of this fish. When intraheteroclitus to hypoxic (•) and normoxic (O) conditions erythrocyte ATP levels were compared
at 22°C. Dissolved oxygen values were 0.2—2.0 parts
for individuals of different LDH-B pheper million (ppm) for hypoxic and 8.5-9.0 ppm for
notypes, there was a significant association.
control fish. Red cell ATP for this experiment and
all others described in this paper were determined
As the intraerythrocyte ATP/Hb ratio is
using the firefly luciferase assay. All points represent
different for each of the LDH-B phenoaverages of 6-7 fish. Bars indicate ±standard error
types, we expected to find differences in
of the mean (SEM) (from Greaney and Powers, 1978).
hemoglobin-oxygen affinity. In agreement with this expectation, LDH-BaBa
homozygotes with the lowest A T P / H b
the highest oxygen affinity and
the nucleated erythrocytes of fish possess ratio had
b b
LDH-B
B
homozygotes with the highest
mitochondria, we reasoned that this
A
T
P
/
H
b
value
have the lowest oxygen
response may be mediated by way of a
decrease in oxidative phosphorylation. This affinity (Powers et al., 1979c). This was
hypothesis was supported when aerated found to be an important factor in differcells were incubated in the presence of low ential developmental rates and swimming
concentrations of cyanide. This inhibitor abilities (DiMichele and Powers, 1982a, b).
of aerobic respiration reduced intracellu- Since the differential developmental rates
lar ATP to levels similar to those found in of LDH-B genotypes are being addressed
the anoxic cells (Greaney and Powers, 1977, elsewhere in this volume (DiMichele et al.,
1978). These data have been confirmed by 1986 [this vol.]), we shall restrict our comred cell oxygen consumption studies (Pow- ments to swimming performance.
ers, 1983). Thus it seems that one reason
Our analysis of purified LDH-B allelic
fish have retained a functional oxygen-con- isozymes (Place and Powers, 1979, 1984a,
suming electron transport system in the b), indicated that the greatest catalytic difmitochondria of their red blood cells is to ferences between LDH-BaBa and LDH-BbBb
control the levels of hemoglobin allosteric existed at low temperature (10°C) while no
effector.
significant difference exists at 25°C. Thus,
MOLECULAR ECOLOGY OF FUNDULUS HEMOGLOBIN
if the LDH-B enzyme has a direct influence
on erythrocyte ATP concentration, differences in ATP and blood-oxygen affinity
should only exist at acclimation temperatures below 25°C. Furthermore, since
organic phosphate amplifies the Bohr effect
of Fundulus heteroclitus hemoglobins (see
Fig. 3), these phenomena should be exaggerated at low blood pH values, like those
produced during swimming performance
experiments. DiMichele and Powers
(19826) have reported that swimming performance is highly correlated with genetic
variation at the LDH-B locus for Fundulus
acclimated to 10°C while no such differences exist for 25°C acclimated fish.
After an acclimation period, fish of each
of the two homozygous LDH-B phenotypes were swum to exhaustion in a closed
water tunnel. The exhausted fish were sacrificed immediately and appropriate biochemical and physiological parameters
determined. Among resting fish acclimated to 10°C, hematocrit, blood pH,
blood-oxygen affinity, serum lactate, liver
lactate, and muscle lactate were not significantly different between the two LDH-B
homozygous phenotypes (DiMichele and
Powers, 19826). Exercising fish, acclimated to 10°C, to the point of fatigue
caused a significant change in all of these
parameters. The LDH-BbBb phenotype was
able to sustain a swimming speed 20%
higher than that of LDH-BaBa phenotype.
Blood-oxygen affinity, serum lactate and
muscle lactate also differed between the
phenotypes.
In an analysis of the binding of ATP to
carp deoxyhemoglobin, Greaney et al.
(1980a) have shown that the organophosphate-hemoglobin affinity constants
change by two orders of magnitude
between pH 8 and pH 7. The same general
phenomenon appears to be true for Fundulus heteroclitus hemoglobins. In resting
Fundulus at 10°C, the blood pH was about
7.9. At this pH, the difference in erythrocyte ATP between LDH-B phenotypes
(ATP/Hb were 1.65 ± 0.12 and 2.11 ±
0.22 for LDH-BaBa and LDH-BbBb respectively) is not reflected as a significant difference in blood-oxygen affinity. However, as blood pH fell with increasing
exercise, the organophosphate—hemoglo-
245
100
FIG. 8. Oxygen equilibrium curves for whole blood
of Fundulus heteroclitus acclimated to 10°C, as determined with an oxygen dissociation analyzer (Aminco).
The ordinate is the percentage saturation of hemoglobin by oxygen, and the abscissa is the partial pressure of oxygen (pO2). Oxygen equilibrium curves of
blood from (a) resting fish of both LDH-B phenotypes, (b) LDH-B'B" swum to exhaustion, and (c) LDHBbBb swum to exhaustion. The intraerythrocyte ratio
of ATP to hemoglobin (ATP/Hb) resting fish is 1.65
±0.12 for LDH-B>B* and 2.11 ± 0.22 for LDH-B"Bb
(from DiMichele and Powers, 19826).
bin affinity constant increased, and differences in oxygen affinity between homozygous LDH-B genotypes became apparent
(Fig. 8). As blood pH is lowered, ATP
amplifies the dissociation of oxygen from
Fundulus heteroclitus hemoglobin (i.e., P50
increases as pH decreases; see Fig. 3); the
more ATP, the greater the effect. This
difference is translated into a differential
ability to deliver oxygen to muscle tissue
which in turn affects swimming performance.
This phenomenon is particularly important because it illustrates how an enzyme
that is not involved in respiration can indirectly affect the availability of oxygen and
thus the ability to perform work.
ACKNOWLEDGMENTS
We thank the National Fishery Center,
Kearneysville, WV for providing us with
246
D. A. POWERS ET AL.
rainbow trout. We are also grateful for the
flounder blood supplied by Jeffrey Price.
This work was supported by NSF Grants
DEB-79-12216, DEB-82-07706, DCM-8402432 and NIH Grant 51201-HL-28893
to DAP. LDM was also supported by an
NIH Fellowship (F32-GM-078898). This is
contribution No. 1297 from the Department of Biology and the McCollum-Pratt
Institute of The Johns Hopkins University.
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