/. exp. Biol. 149, 425-437 (1990)
Printed in Great Britain © The Company of Biologists Limited 1990
425
INFLUENCE OF ORGANIC PHOSPHATES ON THE ROOT
EFFECT OF MULTIPLE FISH HAEMOGLOBINS
BY BERND PELSTER 1 AND ROY E. WEBER
Zoophysiology Laboratory, Aarhus University, Denmark and 1Institut fur
Physiologie, Ruhr-Universitat Bochum, FRG
Accepted 15 November 1989
Summary
The influence of organic phosphates on the reduction in oxygen-carrying
capacity at low pH (Root effect) in multiple fish haemoglobins has been analysed
spectrophotometrically. In stripped haemolysates of carp, trout and eel, the Root
effect in the presence of ATP was manifested below pH7.0. In the absence of
phosphates, it was only found in trout haemolysate.
In the pH range between 8.5 and 6.1 no Root effect could be induced in the
cathodic component (Hbl) of either trout or eel haemoglobin, even in the
presence of very high concentrations of ATP or GTP. This was also true for
component II (Hbll) of trout. The anodic component (HblV) of both species,
however, exhibited a strong Root effect potentiated by NTP. At the same
NTP/Hb 4 concentration ratio, GTP was much more effective than ATP in both
species.
The involvement of different haemoglobin components in the generation of
high oxygen tensions in the fish swimbladder is discussed by comparing in vivo
Root effect data obtained with an eel swimbladder preparation with in vitro data
measured in eel blood and haemoglobin.
Introduction
Fish haemoglobins commonly exhibit a Root effect, i.e. a reduction of the
oxygen-carrying capacity at low pH (Root, 1931; Bridges etal. 1983; Brittain,
1987), which appears to be involved in the secretion of oxygen into the
swimbladder (Fange, 1983) and in supplying oxygen to the eye (Ingerman and
Terwilliger, 1982). It has been shown that the major cofactors in fish erythrocytes
are nucleotide triphosphates (NTP), which strongly reduce the oxygen affinity of
the pigment by allosteric interaction (Weber et al. 1975; Weber and Jensen, 1988),
and that the in vitro Root effect observed in red cell haemolysates may be
increased in the presence of NTP (Weber and DeWilde, 1975; Vaccaro Torracca
etal. 1977).
Fish haemoglobins display a striking multiplicity of molecular structure and
function. Intraspecifically they are often differentiated into (electrophoretically)
cathodic components exhibiting high oxygen affinities and small Bohr effects, and
words: haemoglobin, Root effect, organic phosphates.
426
B. PELSTER AND R . E .
WEBER
anodic ones with lower affinities and larger sensitivities to pH (Gillen and Riggs,
1973; Weber et al. 1916a,b), suggesting a division of labour with the former
increasing in importance under conditions of environmental hypoxia and internal
acidosis. It has been shown that in the absence of phosphates there may be
differences in the magnitude of the Root effect between these two components
(Itada et al. 1970; Binotti et al. 1971), but the influence of varied concentrations of
NTP on the expression of the Root effect in cathodic and anodic haemoglobins
remains unknown. The relative effects of ATP and GTP, which differ in the
number of hydrogen bonds they form with the haemoglobin molecule (Gronenborn et al. 1984), are also of interest. These considerations call for a systematic
study of the effects of allosteric ligands on the composite and isolated haemoglobins of teleosts.
Materials and methods
Animals and collection of blood
Specimens of carp (Cyprinus carpio), trout (Salmo gairdneri) and freshwater eel
(Anguilla anguilla) were obtained from local suppliers and kept in the aquaria at
19°C. Heparinized blood samples were drawn by caudal vein puncture.
Preparation of haemoglobin solutions
The red cells were washed twice in ice-cold ISOmmoll" 1 NaCl, and lysed in
three times their volume of ice-cold distilled water, with a few drops of l m o l l " 1
Tris buffer, pH7.6, to stabilize the pH. The cell debris was removed by
centrifugation at 14 000 revs min" 1 in a Sigma centrifuge (Sigma 3MK, Osterode,
FRG) at 4°C. A sample of the supernatant was stripped on a Sephadex G-25
column using SOmmolT 1 Tris buffer, pH7.6, with lOOmmoll"1 NaCl for the
elution. The stripped haemoglobin solution was dialysed for 12 h against three
changes of lOmmoll" 1 Tris buffer, pH7.6.
Prior to separation of the anodic and cathodic components of eel and trout
haemoglobin, the unstripped haemolysate was dialysed against 20mmoll~ 1 Tris
buffer, pH8.4. The dialysed sample was then applied to a Sephacel DEAE ionexchange column (2cmx20cm) and eluted with ZOmmoll"1 Tris buffer, pH8.4,
and a linear, increasing (0-0.3moll" 1 ) NaCl gradient. After absorbance measurements, fractions containing the same haemoglobin components were pooled and
dialysed for 12 h against 2-3 changes of lOmmoll" 1 Tris buffer at pH7.6.
The fractional size of the different haemoglobin components was calculated
from the area of the peaks obtained by plotting optical density against fraction
number (see Fig. 5).
Experimental protocol and analytical procedures
The oxygen saturation of the pigment was measured spectrophotometrically at
very high P O2 levels. SOmmolP 1 Tris buffers (pH range 8.6-7.5) and BisTris
buffers (pH range 6.1-7.6), through which pure oxygen had been bubbled for
Root effect in multiple fish haemoglobins
427
least 40min, were mixed with haemoglobin solutions in lml cuvettes to give an
optical density of 0.7-0.8 at a wavelength of 540 nm. The pH of each cuvette was
measured immediately with a micro pH electrode (G 299, Radiometer, Copenhagen, Denmark) coupled to a PHM 73 monitor (Radiometer). Pure oxygen was
again bubbled through the cuvette for about 10 s before sealing it with Paranhn.
This procedure resulted in PQ2 values higher than 60kPa, as checked with an
oxygen electrode (E 5047, Radiometer). The optical density of the samples was
recorded at different wavelengths (540nm; 548/549nm; 558nm; 576/577nm;
585/586 nm) using a spectrophotometer (Ultrospec II, LKB, Cambridge, England). The 1 nm variation in the wavelength of absorbance measurements related
to small differences in the absorption maxima and minima of the different
components. To analyse the influence of organic phosphates 5 or 10 (A of
appropriate solutions was added to the cuvettes and the optical densities recorded
again. Finally, absorption of the deoxygenated samples was measured after
addition of a pinch of dithionite. The influence of chloride or lactate was
determined by replacing 100 or 200 [A of the buffer with 1 mol I" 1 NaCl solution or
l m o l P 1 sodium lactate, respectively.
The concentration of ATP and GTP in the stock solutions was measured
enzymatically (Sigma enzymes and reagents). The chloride concentration was
measured using a chloride titrator (CMT10, Radiometer).
The solutions were prepared at 4°C whereas absorbance measurements were
performed at 20°C.
Evaluation of the Root effect
The fractional oxygen saturation (y) was calculated as:
_ ["AOD576 (oxy _ deoxy)] / [AOD 576 ( oxy _ deoxy )]
V
L OD 576 (deoxy) J ' L OD 576 (deoxy) J max
where AOD is the change in optical density. The maximum values were taken at
pH values above 7.8, where no Root effect is present.
The slight dilution caused by addition of organic phosphates resulted in slight
shifts in the spectra which could be corrected by multiplying by the factor
(F=OD o x y /OD d e o x y ) at the isosbestic points (A=548/549nm and 585/586nm),
where absorption is independent of oxygen saturation and should be constant.
Evaluation of the Root effect from the measurements at 540 and 558 nm gave
slightly different percentages of deoxygenation, which may be due to slight
changes in absorbance of the haemoglobins when the Root effect is manifested.
Results
In stripped haemolysates of all three species, haemoglobin oxygen-saturation
was independent of pH between pH8.6 and 7.4, even in the presence of high
concentrations of organic phosphates (Fig. 1). In carp and eel a further increase in
p>roton concentration down to pH6.1 induced only a slight Root effect, while in
428
B. PELSTER AND R. E. WEBER
100
-ATP + ATP ATP/Hb4
50
on
Carp
o
Eel
o
Trout
0L
6.0
6.5
7.0
7.5
8.0
•
80
•
10
A
8
8.5
pH
Fig. 1. Fractional oxygenation of stripped haemolysates of carp, eel, and trout at high
POl (above 60 kPa) in relation to blood pH and in the presence (solid symbols) and
absence (open symbols) of organic phosphates.
trout haemolysate the oxygen saturation decreased markedly below pH7.0, falling
to about 62 % at pH 6.2. In the presence of saturating ATP concentrations, oxygen
saturation decreased at acidic pH values, carp haemoglobin showing the smallest
effect (Fig. 1).
The effect of ATP at different pH values on the oxygen saturation of eel
haemoglobin reaches a maximum when available phosphate-binding sites are
saturated. Below pH 6.5 even an eightfold increase in the phosphate concentration
could not reduce oxygen saturation (Fig. 2).
Physiological concentrations of small anions, e.g. chloride, decreased oxygen
saturation of eel haemolysate only slightly at very low pH (Fig. 3). Similarly,
pH7.05
pH6.57
pH6.16
ATP/Hb 4
24
lOO
48
92
184
:• • • ~ " \ ]
50
oo
Fig. 2. Fractional oxygenation of stripped eel haemolysate at different pH values and
ATP/Hb4 ratios.
Root effect in multiple fish haemoglobins
429
NaCl
3 50
0L
lOOmmoll"1
200mmoir'
6.0
6.5
7.0
7.5
PH
8.0
8.5
Fig. 3. Fractional oxygenation of stripped eel haemolysate at different NaG concentrations.
100 mmol I" 1 lactate did not enhance the Root effect in the anodic component of
trout haemoglobin (data not shown).
The elution profiles of anion-exchange chromatography (Fig. 4) reveal a clear
separation of positively charged and anodic components in both eel and trout. In
eel the cathodic component comprised 37 % and the anodic component 63 %. In
trout the cathodic component comprised only 22% (peak I), while 66% was
anodic (peak IV) and a small intermediate fraction (peak II) formed 12 %.
The dependence of oxygen saturation upon pH and organic phosphate
concentration showed clear differences between the two components. The oxygen
saturation of the cathodic components of eel and trout and also of the second
component of trout haemoglobin (peak II) remained independent of proton or
phosphate concentrations in the tested range (Fig. 5). The anodic components of
both species, in contrast, exhibited a strong Root effect that increased with
phosphate/Hb 4 ratio (Figs 6, 7). The maximum reduction of oxygen saturation
attained was almost 65 % for eel Hb IV. At pH values of about 6.5 or below in the
presence of phosphates the absorbances at 576 and 540 nm of the hyperoxic anodic
trout haemoglobin (peak IV) dropped almost to the level of deoxygenated
haemoglobin immediately after addition of phosphates.
The sensitivity of oxygen saturation at pH 6.8 to phosphate/Hb 4 ratio is shown
in Fig. 8 for the anodic components of both eel and trout haemoglobin. In both
species GTP exerted a greater effect than ATP on oxygen saturation.
Discussion
As high POl values (above 60 kPa) were used throughout the study to measure
oxygen saturation of the haemoglobin, the deoxygenation at specific proton and
NTP concentrations is assumed to be due to the Root effect. The original
observation of Root (1931) and especially the study of Scholander and van Dam
PP-954) using oxygen tensions of several atmospheres show that at low pH the
430
B. PELSTER AND R. E. WEBER
A
Eel
I
IV
¥
I
100
50
E
2
a
0
•8
8
I V.
B
Trout
IV
/
¥'1 •
<§• 6
ft
20
u
1
I
0
100
50
\
40
\. .1
60
80 100 120
Fraction number
140
160
Fig. 4. Separation of cathodic and anodic haemoglobin components of (A) eel and (B)
trout by anion-exchange chromatography. • , optical density; x, Cl~ concentration.
oxygen saturation of fish haemoglobins exhibiting a Root effect asymptotically
approaches the maximal value observed at high pH and thus complete saturation is
never achieved. It is generally assumed that, when the Root effect is manifested,
the haemoglobin molecule is fixed in the deoxygenated state and the cooperativity
is depressed (Riggs, 1988).
Interspecific differences in the magnitude of the Root effect have been observed
in the stripped haemolysate of several species and are also obvious in our study.
Itada et al. (1970) reported a reduction in the oxygen-carrying capacity of eel
haemoglobin of about 20 % at a pH of 6.5 in the absence of NTP, whereas our data
show no significant effect under these conditions in the stripped haemolysate. The
difference may be attributed to the fact that their measurements were performed
in the presence of phosphate buffer, which has an effect similar to that of organic
phosphates on the oxygen-binding properties of the pigment.
The importance of phosphates for the expression of the Root effect is clearly
demonstrated in the present study. Organic phosphates enhance the Root effect in
the haemolysate in all three species, and in carp and eel NTP is essential for the
induction of the Root effect in the pH range studied. The results on stripped ^
Root effect in multiple fish haemoglobins
Eel
Cathodic
1001
o
IUU1--
431
•-
oGTP/Hb 4 =2.7
• ATP/Hb4=450
80L
Trout
100 r Cathodic — A O
I
A-
A ATP/Hb4=7
AATP/Hb4=70
Trout
100 - Component II A-=J
AATP/Hb4=100
80
0<C i_
6.0
7.0
6.5
7.5
8.0
8.5
pH
Fig. 5. Fractional oxygenation of eel and trout cathodic haemoglobin components
(component I in Fig. 4) and of trout component II in relation to pH and increasing
NTP/Hb4 (mol/mol) ratios.
100
X
o
o
50
o
V
a
•S
ATP/Hb 4
0.2
0.4
0.8
1.6
75
0L
100]
GTP/Hb 4
0.12
0.21
0.43
2.25
50
6.0
6.5
7.0
7.5
8.0
8.5
pH
Fig. 6. Fractional oxygenation of eel anodic haemoglobin (component IV in Fig. 4A)
in relation to pH and increasing NTP/Hb4 (mol/mol) ratios.
432
B. PELSTER AND R. E. WEBER
Trout
100
ATP/Hb4
X
50
o
A
o
o
0.4
0.8
1.5
13
1 o
cx»<
100
a?
GTP/Hb4
50
23
6.0
6.5
7.0
8.0
7.5
8.5
PH
Fig. 7. Fractional oxygenation of trout anodic haemoglobin (component IV in
Fig. 4B) in relation to pH and NTP/Hb4 (mol/mol) ratio. Asterisks indicate the
presence of met-haemoglobin.
•
—
•
—
•
—
_
_
_
_
o
a so
ATP GTP
3?
Eel
Trout
1.0
NTP/Hb4
•
o
A
A
2.0
Fig. 8. Fractional oxygenation of eel and trout haemoglobin in relation to NTP/Hb4
(mol/mol) ratio at pH6.8.
haemolysate in the presence of added NTP show good correspondence to the
oxygen capacity vs pHi plot, obtained for whole blood using gasometric methods
(Pelster et al. 1989). This suggests that in eel no unidentified factors are involved in
the expression of the Root effect, as has been proposed by Vaccaro Torracca et al.
(1977) for goldfish.
Root effect in multiple fish haemoglobins
433
The Root effect in multiple haemoglobins
The component profile obtained by ion-exchange chromatography was similar
to that reported by Binotti et al. (1971) for trout haemoglobin and by Weber et al.
(1976a,b) for trout and eel haemoglobin, although the isoelectric focusing
technique applied by the latter authors resulted in greater resolution of anodic
components.
Previous studies on eel and trout cathodic components in the absence of NTP
(Itada et al. 1970; Binotti et al. 1971) failed to show a Root effect at low pH. This is
supported by our results. Our data show, moreover, that the Root effect cannot be
induced even at extremely high concentrations of ATP or GTP (see Fig. 5); this
also applies to trout component II. This result was surprising for the cathodic
component of eel haemoglobin, where oxygen affinity, in contrast to trout
haemoglobin, shows strong sensitivity to ATP and GTP (Weber et al. 1976a,b).
Thus, although the cathodic eel haemoglobin has a binding site for NTP,
phosphate binding evidently does not hinder the transition from the T to the R
state at low pH.
A strong Root effect could be induced by increasing NTP/Hb 4 ratios in the
anodic components, but interspecific differences again exist. In eel, where only
slight deoxygenation is seen in the absence of phosphate, the maximum reduction
in oxygen saturation in the presence of NTP was 65 %. In trout, in contrast, an
acidification to pH6.2 was sufficient for approximately 60% deoxygenation and
the stripped anodic haemoglobin became almost completely deoxygenated in the
presence of high phosphate concentrations.
The saturation levels of the stripped haemolysate may be interpreted in terms of
those of their components. With 40% saturation measured at pH6.2 for trout
HblV, which contributes 65% to the total haemoglobin, and with 35% of the
haemoglobin being completely saturated (Hbl and II), a saturation of 61 % may
be expected for the haemolysate, which is close to the measured value of 62%
(Fig. 1). It even fits with the saturation level reported in single erythrocytes
(Brunori et al. 1974), but this comparison is difficult as the NTP content of the cells
has not been reported. If deoxygenation of haem groups of the tetrameric
haemoglobin molecule were successive, changes in saturation should occur in steps
of 25 %. The lowest oxygen saturation obtained in the eel anodic component was
35 %. This suggests inhomogeneity of the eel anodic haemoglobin, which probably
includes three different components with very similar oxygen affinities (Weber
etal. 1976a).
At low NTP/Hb 4 ratios, GTP exerts a much greater effect than does ATP on
oxygen saturation of the anodic components. This correlates with the greater
effect of GTP on oxygen affinity and with modelling studies which indicate that in
carp haemoglobin GTP is bound by six bonds, compared with five in the case of
ATP (Gronenborn etal. 1984; Weber and Jensen, 1988). In trout anodic
component (Hb IV) the pi glutamate, which is involved in ATP and GTP binding
in carp, is replaced by aspartate, and Gronenborn et al. (1984) conclude that GTP
(and ATP should exhibit the same effect on this component. Weber et al. (19766),
434
B . PELSTER AND R. E.
WEBER
however, observed a much greater effect of GTP than ATP on the oxygen affinity
of trout haemolysate, corresponding with the present results on the Root effect.
These results indicate that trout Hb IV similarly forms an additional bond to GTP
compared with ATP.
Molecular mechanisms
The Root effect is considered to arise from a large reduction in oxygen affinity
and in cooperativity rather than by any absolute inability of certain haems to bind
oxygen, and to be associated with suppression of the 'add Bohr effect' [which
increases affinity in non-Root-effect haemoglobins (Brittain, 1987)] below pH6.5.
In terms of the two-state theory of allosteric transition (Monod etal. 1965), the
loss of cooperativity is accounted for by an inhibition of the T-R allosteric
transition (from the low-affinity Tense to the high-affinity Relaxed state of the
molecule).
The pH dependence of the Root effect suggests involvement of proton-binding
histidine residues (Riggs, 1988). Parkhurst etal. (1983) observed that removal of
the C-terminal histidines of the /S chains, /3147 His, inhibited the Root effect and
halved the Bohr factor (AlogPso/ApH) of carp haemoglobin. Perutz and Brunori
(1982) proposed that a single replacement (y393 cysteine —> serine) allows formation of hydrogen bonds with the histidyl COO~ of /3147 His and with its peptide NH, which stabilizes the T structure and thus lowers oxygen affinity, and that these
hydrogen bonds are the prime cause of the Root effect. This effect is compounded
by the fact that the salt bridge between this /S-terminal histidine and /J144 lysine,
which stabilizes the high-affinity state in mammalian haemoglobin, cannot form in
fish Root-effect haemoglobins where the lysine is replaced by non-bonding
glutamine. At low pH, the hydrogen bonds with /7147 His may, moreover, allow
the formation of a salt bridge with /?94 glutamic acid in the R structure (Perutz and
Brunori, 1982), which would lower not only the oxygen affinity of the T state but
also that of the R state, as indeed is observed in carp and tench haemoglobins
(Chien and Mayo, 1980; Weber etal. 1987).
The y393 serine hypothesis, however, does not seem to be the final explanation
since haemoglobins of some species, including the South American lungfish
Lepidosiren, have /S93 Ser (Rodewald et al. 1984) and do not show a Root effect.
Furthermore, introduction of /J93 Ser into human haemoglobin did not induce the
Root effect (Nagai et al. 1985). Nevertheless, the presence of ^393 Ser seems to be
one of the requirements for expression of the Root effect. In Squalus acanthias
haemoglobin, for example, which has a /^-terminal histidine residue, but lacks
serine at /S93 (Aschauer etal. 1985), no Root effect is found (Ingermann and
Terwilliger, 1982).
In carp haemoglobin and the anodic component (HblV) of trout haemoglobin
the occurrence of the Root effect correlates with persistence of /S-terminal
histidines and serine residues at /393 (Grujic-Injac etal. 1980; Petruzzelli etal.
1984); these residues are also preserved in goldfish haemoglobin (Rodewald and
Braunitzer, 1984), which similarly exhibits a marked Root effect (Vaccarq
Root effect in multiple fish haemoglobins
435
Torracca et al. 1977). The absence of a Root effect in the cathodic trout Hb
(component I) correlates with the replacement of /S147 histidine by phenylalanine
and of yS93 serine by alanine (Barra et al. 1983).
Determination of the primary structures of the anodic and cathodic components
of Anguilla anguilla haemoglobin would be valuable in assessing the validity of
these correlations, and in determining the molecular basis for the smaller Root
effect found in eel than in trout anodic haemoglobins. In the closely related species
Anguilla japonica, hydrazinolytic analyses (Amano et al. 1972) indicate that
C-terminal histidines of the two anodic components are replaced by arginine in
two cathodic haemoglobins.
Physiological implications
The Root effect plays a pivotal role in the generation of high oxygen partial
pressures and the secretion of oxygen into the swimbladder. In eel swimbladder
blood vessels, pH values of 6.6-6.8 have been reported (Steen, 1963; H.
Kobayashi, B. Pelster and P. Scheid, in preparation), giving an intra-erythrocytic
pH (which determines haemoglobin function) of 6.5-6.65. Taking additional
account of the erythrocytic NTP/Hb 4 ratios found in vivo (Weber et al. 1976a,b;
Bridges et al. 1983), we can thus predict near maximal expression of the Root
effect in the eel swimbladder.
Our data also reveal that at these pH values a variation of the NTP/Hb 4 ratio
between 1 and 2, which is found in vivo, has very little influence on the Root effect
(see Fig. 8). This implies there is limited scope for changing the magnitude of the
Root effect via phosphates. Thus, the main means of initiating deoxygenation in
vivo is by an increase in proton concentration.
Another important point arising from our results is the functional significance of
multiple haemoglobins. While the oxygen capacity of the anodic component will
be substantially reduced in swimbladder vessels, the cathodic component will be
completely saturated, and, at least in trout, this non-Root-effect component is
present in all erythrocytes (Brunori et al. 1974). Breepoel et al. (1980) suggest that
it may serve in transporting oxygen to other tissues. H. Kobayashi, B. Pelster and
P. Scheid (in preparation) have shown that the blood leaving the swimbladder
tissue is alkalinized to pH 7.3-7.4 because of acid movements in the rete mirabile.
The Root effect of the anodic component of the haemoglobin therefore is switched
off and, with a POl of about 6.7kPa, which is close to the arterial level, this
component would be able to deliver oxygen to other tissues, where, because of its
lower oxygen affinity, it will unload oxygen before the cathodic component does.
Powers (1972) links the occurrence of the cathodic components in some catfish
species with their occupation of fast-flowing water habitats, suggesting that it
provides an emergency oxygen transport system during periods of activity when
blood acidosis blocks oxygen loading by the pH-sensitive anodic component. This
interpretation is supported by the lack of cathodic haemoglobins in inactive fish
}such as carp and benthic flatfish (Weber and DeWilde, 1976).
436
B . PELSTER AND R. E .
WEBER
Financial support by the Deutsche Forschungsgemeinschaft to BP is gratefully
acknowledged (DFG - Pe 389/1-1).
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