Abstract. In order to understand the mechanism responsible for the high oxygen affinity of mole blood, we investigated in the mole, Talpa europaea, red cell parameters that determine hemoglobin function.
We have found that the oxygen half saturation pressure (Ps0) of mole blood is 2.85 kPa (21.4 Torr) at pCO 2 4.7 kPa, pH 7.4 and 37°C. The concentration of 2,3-diphosphoglycerate (2,3-DPG) averaged 5.3 mmol/ l in red cells. In addition, we have determined Ps0 in hemoglobin solutions at various
concentrat ions of 2,3-DPG at an assumed intraerythrocytic pH of 7.2 and 37°C. These data were used to calculate the association constants of 2,3-DPG to mole hemoglobin. Ps0 was 1.89 kPa (14.2 Torr) in hemoglobin solutions without 2,3-DPG. The response to 2,3-DPG was relatively low. Noteworthy, CO2
did not affect the oxygen affinity at constant pH in the presence of 2,3-DPG. Our results suggest that the high blood oxygen affinity of the mole can be attributed to a weak
interaction of its hemoglobin with 2,3-DPG.
Erythrocytes Phosphoglyceric acids
Hemoglobin Talpa europaea
Oxygen affinity Fossorial mammals
Blood O~-affinities have been shown to vary greatly among mammalian species (cf. Bunn, 1980). Schmidt-Nielsen and Larimer (1958) have demonstrated a direct relationship between blood O2-affinity and body size. In fact, small mammals have usually blood with a lower O2-affinity than large mammals. It may be that in small animals, with their relatively high rate of O~-consumption, the low O~-affinity favors the unloading of O2 from the blood to the tissues (Schmidt-Nielsen and Larimer, 1958).
More recent studies show that the general relationship between blood O~- affinity and body size does not apply to mammals which are adapted to a hypoxic
environment. Hypoxia adapted mammals tend to have increased blood O2-affinities. Namely, relatively high O2-affinities have been reported in fossorial (</i Nevo, 1979) and high altitude mammals (O< Scott et al., 1977; Bunn, 1980) and in divers (Dhindsa et al., 1974). The increased O2-affinity is thought to be of adaptive value to these animals, because it facilitates the oxygenation of the blood at lower arterial 02 pressures (Eaton, 1974).
Fossorial mammals must adapt to their subterranean habitation in sealed burrows and tunnels in which they may encounter severe hypoxia and hypercapnia due to the restricted gas permeability of the soil (Ar et al., 1977). An extreme example among fossorials is the mole, Talpa europaea, an insectivore, which spends almost its entire life in an underground tunnel system. In fact, studies on mole b4ood indicate that its O2-affinity is much higher than has been found in any non- fossorial terrestrial small mammal living at low altitude (Bartels et al., 1969; Quilliam et al., 1971).
The present study was undertaken to explore the mechanism responsible for the high O2-affinity of mole blood. Basically, a high O2-affinity of red cells can be attained by way of any of the following mechanisms: (a) a high intrinsic O2-affinity of hemoglobin; (b) a low level of 2,3-diphosphoglycerate (2,3-DPG) in red cells; and (c) a reduced interaction of hemoglobin with 2,3-DPG. We have examined these possibilities and our results indicate that the intrinsic O2-affinity of hemoglobin and the level of 2,3-DPG are not distinctly altered in mole blood. However, the effect of 2,3-DPG on hemoglobin function is relatively low.
Methods
BLOOD SAMPLING AND DETERMINATION OF RED CELL PHOSPHATES
Moles (Ta/pa europaea, 48-92 g body weight) of either sex were obtained from local meadows in autumn by digging them out with a spade. Heparinized blood was taken from the abdominal aorta under ether anesthesia on the day of capture. For the determination of red cell phosphates, neutralized perchloric acid extracts of blood were prepared. 2,3-diphosphoglycerate (2,3-DPG) in red cell extracts was assayed enzymatically according to Ericson and de Verdier (! 972). Nucleoside-triphosphates were determined as described by Jaworek et al. (1974). Enzymes and substrates were obtained from Boehringer, Mannheim.
OXYGEN EQUILIBRIUM CURVES OF BLOOD
O2-equilibrium curves of whole blood were determined by equilibrating 0.2 ml samples at 37°C for 15 min with water-vapor saturated gas mixtures containing varying concentrations of O~. CO, and N, (WOsthoff Gas-Mixing Pumps, BochumL
H E M O G L O B I N O X Y G E N A F F I N I T Y IN MOLE BLOOD 9
The Oz-content of samples was measured in a saturation range between 20 and 80% using a Lex-Oz-Con apparatus (Lexington, Mass.). Blood Oz-capacity was determined by equilibration with pure O z. The Oz-pressure at half-saturation of hemoglobin (Ps0) was calculated from the Hill equation (4-5 points per curve). The Bohr effect (Alog Ps0/ApH) was determined by changing the partial pressure of CO2.
O X Y G E N E Q U I L I B R I U M CURVES OF H E M O G L O B I N SOLUTIONS
For the preparation of hemoglobin solutions, red cells were washed in saline and lysed by adding distilled water. After dialysing the samples against distilled water, phosphates were removed from the hemolysate by chromatography on a mixed-bed ion-exchange column (AG 501 X8 [D], Bio-Rad, Mtinchen) according to Jelkmann and Bauer (1976). Oz-equilibrium curves of hemoglobin solutions (0.3 mmol Hb4/1 ) were measured spectrophotometrically at 37 °C as described previously (Jelkmann and Bauer, 1978). Measurements were done in the absence of CO 2 unless other- wise noted. For the determination of the fixed-acid Bohr effect, O~-binding curves were assayed in a pH-range between 6.0 and 8.0 with 100 mmol C1-/I and 100 mmol bis-Tris/1 at pH < 7.5 and 100 mmol Tris/1 at pH >7.5, respectively.
The effect of 2,3-DPG on Oz-binding properties of hemoglobin was investigated with commercial 2,3-DPG (Boehringer, Mannheim) that had been freed previously of cyclohexylammonium cation with Dowex-50 WX 8 (Serva, Heidelberg) and subsequently neutralized with NaOH. For the determination of Oz-equilibrium curves, mixtures of hemoglobin with 2,3-DPG were studied at pH 7.2 (assumed intraerythrocytic pH), 100 ml CI-/I and 100 mmol bis-Tris/1.2,3-DPG hemoglobin association constants were estimated from the relationship between-Ps0-values and the concentration of 2,3-DPG using the equations of Baldwin (1975) and Szabo a'hd Karplus (1976) with an iterative nonlinear least squares procedure as described by Bauer et al. (1980).
Results
Hematological data and red cell phosphates Hematological data are summarized in table 1. Noteworthy, 2,3-DPG was found in high concentrations in mole erythrocytes. In addition, nucleoside-triphosphate, most likely adenosine-triphosphate (ATP), was detected.
Oxygen affinity of whole blood The effect of pH on the Oz-affinity of mole blood at varying pCO z is shown in fig. 1. A Ps0-value of 2.85 kPa (21.4 Torr) at pH 7.4 was calculated from this curve (Pco~ 4.7 kPa -- 35 Torr). The Bohr coefficient at varying Pco.~ was -0.61 (Alog Ps0/ApH).
10 w. JELKMANN et ell.
Hct (%)
44.7 + 3.0
Mean _+ SD, n = 5.
TABLE 1 Hematological data and red cell phosphates in Talpa europaea
Hb MCHC Met Hb 2,3-DPG Nucleoside-triphosphate (g/dl) (g/dl) ('!;;) (mmol/1 R B C ) (mmol/1 RBC)
16.8 _+ 0.7 37.7 _+ 1.4 < I 5.3 _+ 1.2 1.3 _+ 0.7
log P50 (k Pa)
0.7
0.6
0.5
0.4
69 71 7[3 7.5 p H
Fig. 1. CO~-Bohr effect ill mole blood: Dependency of oxygen half saturation pressure (Ps0) on pH at varying pCO 2 (37°C: kPa corresponds to 7.5 Torr).
Oxygen a.ff~nity o/" hemoglobin in solution Ps0 of "stripped" hemoglobin, as a measure o f the intrinsic O2-affinity of mole
hemoglobin, was 1.89 kPa (14.2 Torr) in the absence o f organic phosphates and
of CO_,. Here, the fixed acid Bohr coefficient was - 0 . 5 2 (fig. 2).
The effect o f 2 ,3 -DPG on the O2-affinity of hemoglobin is shown in fig. 3.
F r o m these data, association constants o f 2 ,3-DPG to mole hemoglobin (Hb4) were calculated, which were 5.8 ( + 1.4) x 103 l/tool for deoxygenated and 3.7
( + 1.6) x 102 l/mol for oxygenated hemoglobin (mean _+ SD).
Calculation of whole blood Pso j ?om 2,3-DPG hemoglobin association constants To prove whether the low interaction between 2 ,3-DPG and hemoglobin can explain the high O2-affinity o f whole blood, we have computed Ps0 of mole blood, using foregoing 2 ,3 -DPG hemoglobin association constants (Koxy, Kdc,xy) for approxi- mated intraerythrocyt ic condit ions (pH 7.2, 5.9 mmol Hb4/l, 5.3 mmol 2,3-DPG/I). Ps,, was calculated according to Bauer et al. (1980) f rom the equation proposed by
H E M O G L O B I N O X Y G E N AF F INITY 1N MOLE BLOOD 1 1
log P50
(kPa)
0.8
0.6
0.4
0.2
0
- 0 . 2
60 6.5 70 7.5 8.0
pH
Fig. 2. Fixed-acid Bohr effect in mole hemoglobin: Dependency of Ps0 on pH in the absence of CO, and of phosphates (37°C; 1 kPa corresponds to 7.5 Torr).
P50 k Pa }
4O
Q /- 3.1
24
19 q
0 2 4 6 8 7/
lO ;o [2.3-DPG 1 'mmol I
Fig. 3. Effect of 2,3-DPG on the O:-affinity of mole hemoglobin (37~C, pH 7.2; 100 mmol CI /1: hemoglobin concentration 0.3 mmol Hb4/l; in the absence of CO2; I kPa corresponds to 7.5 Torr).
where Ps0 I+DpCh gives the Ps0 in blood, Ps0 ( Dp(~ represents the intrinsic O2-affinity of hemoglobin and v is the mean concentration of free 2 ,3-DPG (0.91 mmol/ l RBC in our data).
We calculated a Ps0 of 2.80 kPa for blood, which agreed with our measured value of 2.85 kPa. Since our association constants were obtained in the absence of CO> this finding indicates that CO,, apart from the effect o f pH, had little influence on the O~-affinity o f mole red cells.
This conclusion was affirmed in experiments in which we studied the effect
12 w. JELKMANN et al.
of CO 2 on the O2-affinity of hemoglobin in solution, to which 2,3-DPG had been added similar to the intracellular concentration of the free compound. We found that at constant pH, CO 2 had no significant effect on Ps0. Ps0 was 2.99 _+ 0.09 kPa at Pco.~ 5.3 kPa (with 15.5 mmol HCO~/1), compared to 3.13 ___ 0.17 kPa without CO~ (mean ___ SD, n = 3, pH 7.2).
Note that in the absence of 2,3-DPG, CO 2 significantly (P <0.05) lowered hemoglobin O2-affinity, as P,~, was 2.31 + 0.21 kPa compared to 1.89 _+ 0.04 kPa without CO 2 (n = 3 4, pH 7.2).
Discussion
The O2-transport system in fossorial mammals is adapted to a hypoxic-hypercapnic environment in tunnels and sealed burrows, in which the inspiratory gas may contain 21 to 6°~ 02 and 0.5 to 5,°~; CO,_ (Kennerly, 1964; McNab, 1966; Ar et al.,
1977). To cope with the low ambient pO2, adaptation of red cell function may regard increases in the O2-capacity as well as in the O2-affinity of blood. In the following, we will first consider the distinctive O2-binding properties of blood in fossorial mammals and then turn to the cause of the high blood O2-affinity of moles.
Moles, Talpa europaea, possess blood with a relatively large O2-binding capacity, which is mainly due to the dense packing of hemoglobin in red cells (Bartels et al., 1969; table 1). Similarly, a large O_,-capacity can be deduced from the high hemoglobin blood level found in several fossorial rodents, namely in mole rats (Ar et al., 1977), pocket gophers (Chapman and Bennett, 1975; Lechner, 1976) and Australian murids (Withers, 1975).
The very high O2-binding affinity of mole blood has been first described by Bartels el al. (1969). These authors have found a Ps0 of 24 Torr at standard conditions (pCO2 40 Torr, pH 7.4, 37°C). Quilliam et al. (1971) report a Psi, of 17 Torr at standard conditions and of 20 Torr at in vivo mole conditions which are pH 7.21 and 35.5°C according to the authors. Our value of 21.4 Torr (2.85 kPa) at pH 7.4 and 37°C tallies with these data and affirms the observation that P> in mole blood is by 10 to 15 Torr lower than the one found in overground mammals of similar body size ( c f Scott et al., 1977; Bunn, 1980). Studies show that the O2-affinity of blood compared at standard conditions is also relatively high in the burrower armadillo (Dhindsa et al., 1971) and in several fossorial rodents (Hall, 1965; Withers, 1975; Johansen et al., 1976; Ar et al., 1977).
Eaton (1974), in measuring cardiac reactions and survival rates in hypoxia exposed rats, has provided experimental evidence for the protective value of an increased blood O2-affinity at low ambient pO 2. This finding is consistent with the evolution of high O:-affinity red cells under quite different conditions of hypoxia, which, apart from subterranean, high altitude ( c f Bunn, 1980) or underwater life (Dhindsa et al., 1974), regards also the prenatal hypoxia in the uterus (~f. Jelkmann and Bauer, 1977).
HEMOGLOBIN OXYGEN AFFINITY IN MOLE BLOOD 13
In considering the possible causes of the high O_,-affinity of mole blood, one has to regard the intrinsic hemoglobin O2-affinity, the intraerythrocytic concentration of organic phosphates, and the interaction of hemoglobin with organic phosphate.
Our results indicate that the intrinsic O2-affinity of mole hemoglobin is not different from that in mammals with low O2-affinity blood (table 2). Apparently, fossorial mammals have evolved different ways to increase their blood O2-affinity, since the burrowing mole rat Heterocephalus has hemoglobin with a high intrinsic O,-affinity (Johansen et al., 1976).
In addition, we found significant amounts of organic phosphates in mole red cells (table 1). The concentration of 2,3-DPG averaged 5.3 mmol/1 RBC, which is well in the range of 4-10 mmol/1 that is found in most mammals (Scott et al., 1977). Similar to moles, several fossorial rodents have high levels ( >4 mmol/1 RBC) of red cell 2,3-DPG (Johansen et al., 1976; Lechner, 1976; Ar et al., 1977). In this regard, an exception among burrowers is the armadillo, whose red cells contain relatively little 2,3-DPG (Dhindsa et al., 1971).
We conclude that the high O2-affinity of mole blood can be explained by a reduced interaction of hemoglobin with 2,3-DPG. As is shown in Results, the O~- affinity of blood can be calculated from 2,3-DPG association constants to oxy- and deoxyhemoglobin (Kox s, Ka~oxy), taking into consideration the intrinsic O:- affinity of hemoglobin and the intraerythrocytic concentration of unbound 2,3-DPG. A reduced effect of 2,3-DPG on hemoglobin function is suggested by a low Kdeoxy as opposed to a high Koxy value.
Interspecies comparisons support this reasoning, if one contrasts available 2,3-DPG hemoglobin association constants with the O2-affinity of the respective blood. Table 2 depicts such data on hemoglobin function in species which live under normoxic conditions and in hypoxia adapted mammalians. In comparing data on the mole with those on species with low O2-affinity blood, it is apparent that in the mole Kdeoxy is considerably lower than in the mouse, whereas Koxy is much higher than in the rabbit, for example.
Finally, we wish to regard the molecular modifications of mole hemoglobin which could explain the relatively low interaction with 2,3-DPG. X-ray crystallo- graphic studies (Arnone, 1972) show that 2,3-DPG binds to specific amino acids in the /?-chains of human adult hemoglobin, namely to NA1 Val, NA2 His, H21 His and EF6 Lys. Mutations at these binding sites may reduce the effect of phosphate on hemoglobin function, like it is seen in human fetal hemoglobin (Bauer et al.,
1968; Frier and Perutz, 1977) or in llama hemoglobin (Braunitzer et al., 1977; Bauer et al., 1980). We have determined recently the amino acid sequence of mole hemoglobin /?-chains, which will be described in detail elsewhere (Kleinschmidt et al., 1981). Briefly, it may be noted here that mole fl-chains differ from human adult/?-chains at 30 positions. However, all of the foregoing binding residues for 2,3-DPG are conserved in mole hemoglobin. On the other hand, we found several amino acid displacements in the A helix. Of significance could be the changes in positions A1 (Thr [human] --, Ser [mole]) and A2 (Pro --, Gly), which are neigh-
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HEMOGLOBIN OXYGEN AFFINITY IN MOLE BLOOD 15
boring the NA segment. As a result of this difference, the position of the contact sites for 2,3-DPG at NA1 and NA2 could be altered in mole hemoglobin. Interestingly, Frier and Perutz (1977) have attributed, in part, the low affinity of human fetal hemoglobin for 2,3-DPG to a shift of the NA segment due to a substitution in position NA3. The authors have concluded from X-ray studies that the displacement of the NA segment lengthens the distance between the phosphate and NA2 His. A similar intramolecular movement may be responsible for the low binding of 2,3-DPG to mole hemoglobin but this assumption is speculative, as the tertiary structure of the protein is unknown.
Acknowledgements
We are indebted to Dr. Harry S. Rollema, Nijmegen, who has developed the computer program for the determination of 2,3-DPG hemoglobin association constants. We also wish to thank L. Rauch for technical assistance and G. Wischka for secretarial help.
References
Ar. A., R. Arieli and A. Shkolnik (1977). Blood-gas properties and function in the fossorial mole rat under normal and hypoxic-hypercapnic atmospheric conditions. Respir. Physiol. 30 : 201 218.
Arnone, A. (1972). X-ray diffraction study of binding of 2,3-diphosphoglycerate to human deoxyhaemo- globin. Nature 237:146 147.
Baldwin, J. (1975). Structure and function of haemoglobin. Prog. Biophys. Mol. Biol. 29:225 320. Bartels, H., P. Hilpert, K. Barbey, K. Betke, K. Riegel, E. M. Lang and J. Metcalfe (1963). Respiratory
functions of blood of the yak, llama, camel, Dybowski deer, and African elephant. Am. J. Physiol. 205: 331-336.
Bartels, H., R. Schmelzle and S. Ulrich (1969). Comparative studies of the respiratory function of mammalian blood. V. Insectivora: Shrew, mole and nonhibernating and hibernating hedgehog. Respir. Physiol. 7: 278-286.
Bauer, C., I. Ludwig and M. Ludwig (1968). Different effects of 2,3-diphosphoglycerate and adenosine triphosphate on the oxygen affinity of adult and foetal human haemoglobin. L([b Sci. 7:1339 1343.
Bauer, C., R. Tamm, D. Petschow, R. Bartels and H. Barrels (1975). Oxygen affinity and allosteric effects of embryonic mouse haemoglobins. Nature 257:333 334.
Bauer, C., H. S. Rollema, H. W. Till and G. Braunitzer (1980). Phosphate binding by llama and camel hemoglobin. J. Comp. Physiol. 136: 67-70.
Beer, R., H. Bartels and H.-A. Raczkonski (1955). Die Sauerstoffdissoziationskurve des fetalen Blutes und der Gasaustausch in der menschlichen Placenta. PJliigers Arch. 260: 306-319.
Braunitzer, G., B. Schrank, A. Stangl and C. Bauer (1977). Die Regulation der H6henatmung und ihre molekulare Deutung: Die Sequenz der/~-Ketten der H/imoglobine des Schweines und des Lamas. Hoppe-Seyler~' Z. Physiol. Chem. 358: 921-925.
Bunn, H.F. (1980). Regulation of hemoglobin function in mammals. Am. Zool. 20:199 21 I. Chapman, R.C. and A.F. Bennett (1975). Physiological correlates of burrowing in rodents. Comp.
Biochem. Physiol. 51A : 599-603. Dhindsa, D. S., A. S. Hoversland and J. Metcalfe (1971). Comparative studies of the respiratory functions
of mammalian blood. VII. Armadillo (Dao,pus novemeinctus). Respir. Physiol. 13:198 208.
16 w. J E L K M A N N et al.
Dhindsa, D. S., C. J. Sedgwick and J. Metcalfe (1972). Comparative studies of the respiratory functions of mammalian blood. VIII. Asian elephant (Elephas maximus) and African elephant (Loxodonta ql?icana africana). Respir. Physiol. 14: 332-342.
Dhindsa, D. S., J. Metcalfe, A.S. Hoversland and R.A. Hautman (1974). Comparative studies of the respiratory functions of mammalian blood. X. Killer whale (Orcinus orca) and beluga whale (Delphinapterus leucas). Respir. Physiol. 20:93 103.
Eaton, J. W. (1974). Oxygen affinity and environmental adaptation. Ann. N.Y. Aead. Sci. 241 : 491 497. Ericson, A. and C.-H. de Verdier (1972). A modified method for the determination of 2,3-diphospho-
glycerate in erythrocytes. Scand. J. Clin. Lab. Invest. 29:85 90. Frier, J .A. and M.F . Perutz (1977). Structure of human foetal deoxyhaemoglobin. J. Mol. Biol. 112:
97 112. Hall, F. G. (1965). Hemoglobin and oxygen: affinity in seven species of Seiuridae. Seienee 148:1350
1351. Jaworek, D., W. Gruber and H.U. Bergmeyer (1974). Adenosin-5"-triphosphat. In: Methoden der
enzymatischen Analyse. Vol. 11, edited by H. U. Bergmeyer. Weinheim/Bergstral3e, Verlag Chemie, pp. 21472151.
Jelkmann, W. and C. Bauer (1976). What is the best method to remove 2,3-diphosphoglycerate from hemoglobin? Anal. Biochem. 75: 382--388.
Jelkmann, W. and C. Bauer (1977). Oxygen affinity and phosphate compounds of red blood cells during intrauterine development of rabbits, p[liigers Areh. 372 : 149 156.
Jelkmann, W. and C. Bauer (1978). Embryonic hemoglobins: Dependence of functional characteristics on tetramer composition, p[liigers Arch. 377:75 80.
Johansen, K., G. Lykkeboe, R. E. Weber and G. M.O. Maloiy (1976). Blood respiratory properties in the naked mole rat Heterocephalus glaher, a mammal of low body temperature. Respir. Physiol.
28: 303-314. Kennerly, T.E. (1964). Microenvironmental conditions of the pocket gopher burrow. Texas J. Sei.
16: 395--441. Kleinschmidt, q'., W. Jelkmann and G. Braunitzer (1981). H~imoglobine, XLIlI: Die Prim~irstruktur
des H~imoglobins des Maulwurfs (Talpa europaea). Hoppe-Seyler's Z. Physiol. Chem. (In press). Lechner, A.J . (1976). Respiratory adaptations in burrowing pocket gophers from sea level and high
altitude. J. Appl. Physiol. 41 : 168- 173. McNab, B.K. (1966). The metabolism of fossorial rodents: A study of convergence. Eeology 47:
712 733. Nevo, E. (1979). Adaptive convergence and divergence of subterranean mammals. Ann. Rev. Ecol. Svst.
10: 269--308. Petschow, R., D. Petschow, R. Barrels, R. Baumann and H. Bartels (1978). Regulation of oxygen
affinity in blood of fetal, newborn and adult mouse. Respir. Physiol. 35:271 282. Quilliam, T. A., J. A. Clarke and A. J. Salsbury ( 1971). The ecological significance of certain new haema-
tological findings in the mole and hedgehog. Comp. Biochem. Physiol. 40A : 89 102. Schmidt-Nielsen, K. and J. L. Larimer (1958). Oxygen dissociation curves of mammalian blood in relation
to body size. Am. J. Physiol. 195: 424-428. Scott, A. F., H. F. Bunn and A. H. Brush (1977). The phylogenetic distribution of red cell 2,3-diphospho-
glycerate and its interaction with, mammalian hemoglobins. J. Exp. Zool. 201 : 269 288. Szabo, A. and M. Karplus (1976). Analysis of the interaction of organic phosphates with hemoglobin.
Biochemistry 15:2869 2877. Withers, P.C. (1975). A comparison of respiratory adaptations of a semi-fossorial and a surface-
dwelling Australian rodent. J. Comp. Physiol. 98:193 203.
