 Reference Base  Oxygen binding properties of hemoglobin from the white rh... |
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| Subject: |
Physiology |
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White Rhino |
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At the present time, the small mammalian order Perissodactyla includes three families: the Equidae, which are the largest group, the Tapiridae and the Rhinocerotidae. Hemoglobin sequences and functional studies have only been obtained for hemoglobins of the Equidae group (cf. Mazur and Braunitzer, 1982; Matsuda et al., 1980). All species of this group have glutamine at position 2, one of the binding sites for 2,3-diphosphoglycerate (2,3-DPG), which in human hemoglobin is occupied by histidine (Arnone, 1972). However, experiments with horse hemoglobin have shown that the substitution is apparently of little consequence for the effect of organic phosphates (Bunn and Kitchen, 1973; Braunitzer et al., 1978).
In contrast to the above results, the recently published primary structure of the hemoglobin from the white rhinoceros ( Ceratotherium simum ) shows a glutamic acid residue at position 2 (Mazur et al., 1982). Glutamic acid at position 2 has not been demonstrated in any other mammalian hemoglobin, but is present in some fish hemoglobins, notably the -chains of carp, goldfish and trout hernoglobin type I (Braunitzer and Rodewald, 1980; Grujic-Injac et al., 1980; Barra et al., 1983). In view of the unusual character of the substitution found in rhinoceros hemoglobin, we have investigated the functional properties. The results show that the substitution has a profound effect on the interaction of rhinoceros hemoglobin with most allosteric effectors. In addition, data are presented for tapir hemoglobin function.
Materials and methods
Preparation of hemoglobin solutions. Samples of rhinoceros and tapir hernoglobin were shipped on ice. Any methemoglobin present in the samples was converted to hemoglobin following the method described by Bauer and Pacyna (1975), The hemoglobin solutions were equilibrated with 0.1 M NaCl on a 1.5 - 90 em column of Sephadex G-25 fine. Stock solutions of hemoglobin with a concentration of 120 g/l were stored in liquid nitrogen until use.
Oxygen equilibrium survey of hemoglobin solutions (40 g/l) were determined spectrophotome-trically (Niesel and Thews, 1961; Sick and Gersonde, 1969). For the determination of the Bohr effect, the pH was varied using 0.05 M Bis-Tris or Tris buffers with a total concentration of 0.1 M /Cl , unless otherwise indicated.
For experiments done in the presence of CO2, bicarbonate buffers were used and the total concentration of bicarbonate and chloride adjusted to 0.1 M. The pH was measured with ph-meter 64 (Radiometer), and ATP and 2,3-diphosphoglycerate were purchased from Bochringer Co. Mannheim. The oxygen half-saturation pressure P50 and n-value between 20 and 80% saturation were determined from the Hill plot. The hemoglobin pattern was determined by isoelectric focussing (Drysdale et al., 1971) using ampholine pH 6A (LKB, Bromma) and the isoelectric points of the separate fractions were determined as described elsewhere (Petschow et al., 1977). Quantitative analysis of the gels was carried out by densi- tometry on a Gelman AC D 15 densitometer.
Photomicrographs of oxyhemoglobin crystals from rhinoceros and tapir hemoglobin were taken by phase contrast on a Leitz Stereoplan microscope.
Results
Hemoglobin pattern and solubility.
Tapir blood has 4 hemoglobin components, each accounting for 20- 30% of the total hemoglobin, while rhinoceros blood has a major component (80%) and one minor fraction of greater electrophoretic mobility (fig.1 and table I). Note that the estimated isoelectric points for all hemoglobin fractions are up to 0,3 pH units lower than for human hemoglobin A.
Table 1.
Hemoglobin pattern of tapir and rhinoceros blood. pI estimated at 20?C; corresponding value for major fraction of human adult hemoglobin, pI = 7.33
Fraction Tapir Hb Rhinoceros Hb
number Estimated pI % of total Hb Estimated pI % of total Hb
1 7.295 21 7.214 81
2 7,187 19.7 7.142 19
3 7,114 27.4
4 7,009 31.9
During the preparation of the hemoglobin solutions we found that oxyhemoglobin solutions from tapir and rhinoceros crystallize spontaneously at room temperature, physiological ionic strength and hemoglobin concentrations well below those present in the red cell. Thus, for rhinoceros hemoglobin one finds a concentration of 7.03 g/dl in the soluble phase at pH 6.97 and 22?C. Figure 2 is a photomicrograph of crystalline precipitate removed from an oxygenated solution of rhinoceros hemoglobin with 0.1 M per Cl and pH 7 at 22?C.
Oxygen affinity and Bohr Effect
At pH 7.2 and 37 ?C the oxygen half-saturation pressures (P50) of purified hemoglobin from tapir and rhinoceros are almost identical with 17.1 torr (2.28 kPa) and 17.4 torr (2.32 kPa). These values are higher than the P50 for human Hb under the same condition, which is 12 torr (1.6 kPa). The Bohr effect is -0.62 for rhinoceros hemoglobin and -0.58 for tapir hemoglobin in the physiological pH range (figs. 3 and 4, table 2).
Influence of organic phosphates and CO2 on oxygen affinity
The effect of 2,3-DPG and ATP on rhinoceros hemoglobin is small. With 10 mol 2,3-DPG/mol Hb, the P50 increases from 17.1 torr (2.28 kPa) to 19.5 torr (2.6 kPa) and to 25.3 torr (3.38 kPa) with a 50-fold excess of 2,3-DPG; the same P50 value is obtained with 50 root ATP/mol Hb4. However, ATP is more effective than 2,3-DPG at intermediate concentrations (fig. 5). With 10 mol ATP/mol Hb4, the P50 increases to 21.9 torr (2.9 kPa). In the presence of 40 torr (5.33 kPa) CO2 one observes a decrease of the oxygen affinity only at pH > 7.2 (fig. 3 and table 2).
Compared to rhinoceros Hb, the response of tapir Hb to both CO2 and 2,3-DPG is considerably increased, since 10 mol 2,3-DPG/mol Hb4 raises the P50 by 8,4 torr (1.12 kPa) at pH 7.2 and 40 torr (5.33 kPa) CO2 increases the P50 by 4.5 torr (0.6 kPa) at the same pH.
Effect of chloride on rhinoceros hemoglobin.
Because of the small specific effects of 2,3-DPG and CO2 on rhinoceros hemoglobin we determined the influence of chloride (fig. 3). At pH 7.2 the P50 increases from 7.6 torr (1.01 kPa) to 17.1 torr (2.28 kPa), when the chloride concentration is raised from 0.01 M to 0.1 M, and the Bohr effect decreases to -0.33 with only 0.01 M/ Cl. The n-values are dependent on pH but not the chloride concentration (fig. 6). Between pH 6.5 and 8.1 the n-value decreases from 2.9 to 2.1.
Table 2. P50 of rhinoceros and tapir hemoglobin solutions under various experimental conditions; values in brackets give P50 in torr; 37?C; [Hb4]=40 g/L
Condition PH 7.2 pH 7.5
Rhino Hb P50 Tapir Hb P50 Rhino Hb P50 Tapir Hb P50
kPA torr kPa torr kPa torr kPa torr
0.0.1 M per Cl 1.01 (7.58) n.d. 0.82 (6.16) n.d.
0.1 M per Cl 2.28 (17.1) 2.32 (17.4) 1.48 (11.1) 1.55 (11.61)
+5.33 kPa CO2 2.32 (17.4) 2.92 (21.9) 1.97 (14.79) 1.98 (14.78)
+2 mol 2,3-DPG/mol Hb 2.32 (17.4) 2.72 (20.4) n.d. n.d.
Discussion
Mammalian hemoglobins are customarily classified into two groups (Bunn, 1980; Perutz and Imai, 1980): (i) hemoglobins witli high intrinsic oxygen affinity, which depend on 2,3-DPG for the regulation of their oxygen affinity, (ii) hemoglobins with low intrinsic oxygen affinity, like ruminant and felidae hemoglobins, which are independent of organic phosphates. This classification does not cover hemoglobins of those mammalian species that have a high oxygen affinity in vivo, i.e. diving mammals, species habitually exposed to hypoxia, or excessively large animals. A common finding for all these hemoglobins is that their intrinsic oxygen affinity is relatively high, i.e. comparable to that of human hemoglobin, while the interaction with 2,3-DPG is considerably reduced. This may be the result of a substitution at the organic phosphate binding site, as in elephant and rhinoceros hemoglobin (Braunitzer et al., 1982; Bauer et al., 1980; Mazur et al., 1982) or due to altered tertiary/quaternary structures of the binding site that result from substitutions elsewhere in the molecule, as for instance in the hemogtobin of the mole (Jelkmann et al., 1981). Since these species belong to very different orders of mammals, the loss or reduction of organic phosphate binding is a striking example of convergent evolution.
The substitution of 2-HIS by glutamic acid has drastically reduced the affinity of rhinoceros hemoglobin for 2,3-DPG. A tenfold excess of 2,3-DPG over Hb increases the P50 only by 0.4 kPa at pH 7.2. Although the concentration of 2,3-DPG in the red cells of the rhinoceros is unknown, it is reasonable to assume that even if present in concentrations that represent the upper limit for mammalian red cells (10-12 mol 2,3-DPG/L RBC), it would not exert an important direct effect on oxygen affinity, except for the lowering of the intracellular pH. In this respect it is noteworthy that the estimated pI of rhinoceros hemoglobin is considerably lower than that of human hemoglobin, so that a normal intracellular pH (e.g. around pH 7.2 at pHe 7.4) could be maintained even in the absence of 2,3-DPG. That ATP is more effective than 2,3-DPG in reducing the oxygen affinity of rhinoceros hemoglobin is in accord with the recently proposed structure of the ATP-binding site in fish hemoglobins, where GLU 2 is thought to interact with the adenine (Perutz and Brunori, 1982). In absolute terms, the effect of ATP is still small, since even with a tenfold excess of ATP over hemoglobin the P50 is raised by less than 5 torr.
An effect of C02 on the oxygen affinity of rhinoceros hemoglobin could not he demonstrated at pH <7.2. The primary structure of the globin chains shows that all N-termini are free (Mazur et al., 1982). One obvious explanation for the reduced effect of CO2 would be an increased ionization of the N-terminal amino group of the P-chain (which is responsible for most of the oxygen-linked CO, binding of mammalian hemoglobin), which could be caused through interaction of the amino group with the gamma-carboxyl group of GLU P2.
The effect of chloride on the oxygen affinity of rhinoceros hemoglobin is of the same magnitude as that observed in human hemoglobin. At the -chains chloride is bound to LYS 82 (Bonaventura et al., 1976), but apparently the presence of GLU 2 does not interfere. Bonaventura et al. (1980) have suggested that a reduction of the positive charge density in the central cavity should decrease the intrinsic oxygen affinity. However, our data show that the intrinsic oxygen affinity of rhinoceros hemoglobin as indicated by the P50 measured at low (10 mM) chloride concentration is not much lower (7.5 torr at pH 7.2) than that of human hemoglobin (5.3 torr) under the same conditions (Baumann, 1980). This result supports the conclusion of Perutz and Imai (1980) that mammalian hemoglobins with a low intrinsic oxygen affinity are characterized by a hydrophobic residue at position 2, rather than a negatively charged one. From the data obtained in hemoglobin solutions it can be extrapolated that the P50 of rhinoceros blood under standard conditions should be around 20 torr, which is in keeping with results for other large mammals like the elephant or hippopotamus (Bartels et al., 1963; Leivestad et al., 1973). Although we did not separate the two hemoglobin fractions, large functional differences are not to be expected since the rhinoceros hemoglobins differ only at two positions of the -chain ( 62 and 116; Mazur et al., 1982). Less is known about the structure of the four tapir hemoglobins, which have different alpha and beta-chains. At position 2 one finds GLU as well as GLN (Mazur et al., unpublished observation), which explains the larger eftect of both CO2 and 2,3-DPG on the unfractionated hemoglobin solution.
Conclusion:
Rhinoceros hemoglobin represents an interesting example of a mammalian hemoglobin whose functional control is almost exclusively dependent on inorganic anions, i,e. chloride. The fact that the oxygen-linked binding of chloride is not changed through the presence of glutamic acid at position 2, underlines the highly specific nature of substitutions at strategic sites in the hemoglobin molecule and is a further indication for the high degree ol' independence existing between organic and inorganic anion binding sites.
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