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 pH 7.0. 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/Hb4 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.

Fish haemoglobins commonly exhibit a Root effect, i.e. a reduction of the oxygen-carrying capacity at low pH (Root, 1931; Bridges et al. 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 et al. A9T1).

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 anodic ones with lower affinities and larger sensitivities to pH (Gillen and Riggs, 1973; Weber et al. 1976a, 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.

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 150 mmol l−1 NaCl, and lysed in three times their volume of ice-cold distilled water, with a few drops of 1 mol l−1 Tris buffer, pH 7.6, 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 50 mmol l−1 Tris buffer, pH 7.6, with 100 mmol l−1 NaCl for the elution. The stripped haemoglobin solution was dialysed for 12 h against three changes of 10 mmol l−1 Tris buffer, pH 7.6.

Prior to separation of the anodic and cathodic components of eel and trout haemoglobin, the unstripped haemolysate was dialysed against 20 mmol l−1 Tris buffer, pH 8.4. The dialysed sample was then applied to a Sephacel DEAE ionexchange column (2cm×20cm) and eluted with 20 mmol l−1 Tris buffer, pH8.4, and a linear, increasing (0–0.3 mol l−1) NaCl gradient. After absorbance measurements, fractions containing the same haemoglobin components were pooled and dialysed for 12 h against 2–3 changes of 10 mmol l−1 Tris buffer at pH 7.6.

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 levels. 50 mmol l−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 atleast 40 min, were mixed with haemoglobin solutions in 1ml 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 10s before sealing it with Parafilm. This procedure resulted in values higher than 60 kPa, 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 μl 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 μl of the buffer with 1 mol l−1 NaCl solution or 1mol l−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:
formula
where ΔOD 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=ODoxy/ODdeoxy) at the isosbestic points (λ=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.

In stripped haemolysates of all three species, haemoglobin oxygen-saturation was independent of pH between pH 8.6 and 7.4, even in the presence of high concentrations of organic phosphates (Fig. 1). In carp and eel a further increase in proton concentration down to pH 6.1 induced only a slight Root effect, while in trout haemolysate the oxygen saturation decreased markedly below pH 7.0, 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).

Fig. 1.

Fractional oxygenation of stripped haemolysates of carp, eel, and trout at high PO2 (above 60 kPa) in relation to blood pH and in the presence (solid symbols) and absence (open symbols) of organic phosphates.

Fig. 1.

Fractional oxygenation of stripped haemolysates of carp, eel, and trout at high PO2 (above 60 kPa) in relation to blood pH and in the presence (solid symbols) and absence (open symbols) of organic phosphates.

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).

Fig. 2.

Fractional oxygenation of stripped eel haemolysate at different pH values and ATP/Hb4 ratios.

Fig. 2.

Fractional oxygenation of stripped eel haemolysate at different pH values and ATP/Hb4 ratios.

Physiological concentrations of small anions, e.g. chloride, decreased oxygen saturation of eel haemolysate only slightly at very low pH (Fig. 3). Similarly, 100 mmol l−1 lactate did not enhance the Root effect in the anodic component of trout haemoglobin (data not shown).

Fig. 3.

Fractional oxygenation of stripped eel haemolysate at different NaCl concentrations.

Fig. 3.

Fractional oxygenation of stripped eel haemolysate at different NaCl concentrations.

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 %.

Fig. 4.

Separation of cathodic and anodic haemoglobin components of (A) eel and (B) trout by anion-exchange chromatography. •, optical density; ×, Cl concentration.

Fig. 4.

Separation of cathodic and anodic haemoglobin components of (A) eel and (B) trout by anion-exchange chromatography. •, optical density; ×, Cl concentration.

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/Hb4 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.

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.

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.

Fig. 6.

Fractional oxygenation of eel anodic haemoglobin (component IV in Fig. 4A) in relation to pH and increasing NTP/Hb4 (mol/mol) ratios.

Fig. 6.

Fractional oxygenation of eel anodic haemoglobin (component IV in Fig. 4A) in relation to pH and increasing NTP/Hb4 (mol/mol) ratios.

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.

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.

The sensitivity of oxygen saturation at pH 6.8 to phosphate/Hb4 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.

Fig. 8.

Fractional oxygenation of eel and trout haemoglobin in relation to NTP/Hb4 (mol/mol) ratio at pH 6.8.

Fig. 8.

Fractional oxygenation of eel and trout haemoglobin in relation to NTP/Hb4 (mol/mol) ratio at pH 6.8.

As high 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 1954) using oxygen tensions of several atmospheres show that at low pH the 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 eel 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.

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 NIP, 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/Hb4 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 pH 6.2 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 pH 6.2 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 et al. 1976a).

At low NTP/Hb4 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 et al. 1984; Weber and Jensen, 1988). In trout anodic component (HblV) the β2 glutamate, which is involved in ATP and GTP binding in carp, is replaced by aspartate, and Gronenborn et al. (1984) conclude that GTP land ATP should exhibit the same effect on this component. Weber et al. (1916b), 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 HbIV 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 ‘acid Bohr effect’ [which increases affinity in non-Root-effect haemoglobins (Brittain, 1987)] below pH 6.5. In terms of the two-state theory of allosteric transition (Monod et al. 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 et al. (1983) observed that removal of the C-terminal histidines of the β chains, β147 His, inhibited the Root effect and halved the Bohr factor (ΔlogP50/ΔpH) of carp haemoglobin. Perutz and Brunori (1982) proposed that a single replacement (β93 cysteine→ serine) allows formation of hydrogen bonds with the histidyl COO of β147 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 β-terminal histidine and β144 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 β147 His may, moreover, allow the formation of a salt bridge with /β94 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 et al. 1987).

The β93 serine hypothesis, however, does not seem to be the final explanation since haemoglobins of some species, including the South American lungfish Lepidosiren, have β93 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 β93 Ser seems to be one of the requirements for expression of the Root effect. In Squalus acanthias haemoglobin, for example, which has a β-terminal histidine residue, but lacks serine at β93 (Aschauer et al. 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 β-terminal histidines and serine residues at β93 (Grujic-Injac et al. 1980; Petruzzelli et al. 1984); these residues are also preserved in goldfish haemoglobin (Rodewald and Braunitzer, 1984), which similarly exhibits a marked Root effect (Vaccaro Torracca et al. 1977). The absence of a Root effect in the cathodic trout Hb (component I) correlates with the replacement of β147 histidine by phenylalanine and of β93 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/Hb4 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/Hb4 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 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).

Financial support by the Deutsche Forschungsgemeinschaft to BP is gratefully acknowledged (DFG - Pe 389/1–1).

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