Adaptations to neoteny in the salamander, Necturus maculosus. Blood respiratory properties and interactive effects of pH, temperature and ATP on hemoglobin oxygenation

Camp.

Biochem.

Phvsiol. Vol.

SOA, No.

4, pp. 495-501,

0300-9629185

1985

S3.00f0.00

‘CI 1985 Pergamon Press Ltd

Printed in Great Britain

ADAPTATIONS

TO NEOTENY

IN THE SALAMANDER, NECTURUS MACULOSUS. BLOOD RESPIRATORY PROPERTIES AND INTERACTIVE EFFECTS OF pH, TEMPERATURE AND ATP ON HEMOGLOBIN OXYGENATION ROY E. WEBER, RUFUS M. G. WELLS* Biology

Institute,

Odense

University,

and JOHN E. ROSSETT~

DK 5230 Odense

M, Denmark

(Received 28 June 1984) Abstract-l. The permanently neotenic, predominantly water-breathing salamander, Necturus maculosus exhibited a higher blood O2 affinity than in other amphibians (half-saturation 0, tension, P,, = 10.6 mmHg at P,,, = 5 mmHg, pH = 7.5 and 20°C) and an extremely high intrinsic 0, affinity in the purified hemoglobin (Hb) (P,, = 0.8 mmHg at pH 7.5) which is strongly depressed by ATP, the major erythrocytic phosphate cofactor. 2. Unlike Hb from other premetamorphosed amphibians, the Hb displayed a normal Bohr effect (A log P,,/A pH is negative at intermediate pH and increases to zero at high and low pH). 3. Increased temperature reduced the Bohr effect and shifted the pH range where it is manifested to lower pH values, whereas ATP had the opposite effects. 4. The data was used to apportion the overall oxygenation enthalpy among the component reactions, notably the intrinsic oxygenation reaction and the contributions from oxygenation-linked proton and phosphate dissociation. 5. The major Hb components were isoelectric at pH 7.c7.1 and exhibited similar O2 affinities. 6. The findings are discussed in relation to the functional and ontogenetic differentiation of amphibian Hbs and the possible underlying molecular mechanisms.

INTRODUCTION

warm and hypoxic water (Harris, 1959a,b; Guimond and Hutchison, 1972, 1976). Lenfant and Johansen (1967) have earlier reported blood O2 affinity of N. maculosus and Hazard and Hutchison (1982) have shown that its erythrocytes contain ATP as major organic phosphate modulator of 0, affinity, only 20’;; as much DPG (2,3_diphosphoglycerate, the major mammalian cofactor) and no IPP (inositol pentaphosphate, the principal phosphate cofactor in bird erythrocytes). This paper extends the blood measurements and documents the influences, singly and in combination, of pH, temperature and ATP on the 0, equilibrium of the Hb, aiming to discover adaptations to the aquatic-neotenic mode of life and to resolve the O,-binding reactions of the blood in terms of the constituent thermodynamic processes. An additional aim was to relate Hb function in permanent neotenes to the apparent dichotomy among amphibians Hbs, where adult anurans show normal Bohr effects (as in mammals) while anuran tadpoles and premetamorphosed urodeles generally exhibit reverse Bohr effects at physiological pH (Brunori et u/., 1968; Watt and Riggs, 1975; Bonaventura et ul., 1977: Jokumsen and Weber, 1980).

The amphibia were the first tetrapods that successfully invaded terrestrial habitats. Although most species are capable of extracting O2 from air, they have retained dependence on water breathing, at least in the larval stages. In contrast to the pronounced metamorphosis from tadpole to adult in the anuran frogs and toads, some urodelan salamanders and newts exhibit neotenism and retain the larval gills at sexual maturity. These forms (e.g. the salamander Necturus) accordingly are capable of trimodal gas exchange, i.e. by gills, lungs and the integumental and glossopharyngeal surfaces. It is likely that the 0, conformity and tolerance to low O2 tensions observed in aquatic salamanders (Guimond and Hutchison, 1976; Branch and Taylor, 1977) are a result of adaptations in respiratory gas transport. In contrast to the anuran amphibians, where the gas-transporting functions of the blood and the molecular control of 0, affinity of the tadpole and adult Hb have been studied in detail, few comparable investigations have been carried out in the Urodela. We report here on the blood and hemoglobin of the permanently neotenic salamander Necturus maculosus, which possesses well-developed gills while the lungs are simple and normally account for less than 10% of 0, uptake, but become more important in

*Present address: Department Auckland, New Zealand.

MATERIALS AND METHODS Specimens of the mudpuppy Necturus marulosus mwulosus (Ralinesque), about 26cm long and weighing about 120 g, were obtained from a commercial supplier, and kept at 2&22”C in well-aerated aquaria until use. The animals were anesthetized by immersion in MS 222 (0.12 g/l). The skin was then stripped anterio-ventrally to locate the heart

of Zoology, University of

495

496

ROY E. WEBER rt al

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1

loo0

0

2

3

4

1

a

10

Fig. 1. Family of Or equilibrium curves of Necturus maculosus whole blood at 20’C and CO? tensions (left to right) of 0.7, 3.7, 7.4, 22.0 and 36.7mmHg. Inset: Variation of P,, and n,, values with pH.

and blood was collected in heparinized hypodermic syringes by cardiac puncture. Oxygen equilibria of the whole blood were determined at 20’C using a hemoscan analyzer (Aminco. USA) modified as earlier described (Wells and Weber, 1983). Gas mixtures (of air or Nz and COJ were prepared with Wiisthoff mixing pumps (Bochum, FRG). Control experiments with human blood at 37C gave the expected values of P,, and n. Blood nucleoside triphosphate (NTP) was determined using Boehringer enzymic test chemicals, and by thin layer chromatography modified after Cashel e/ ~1. ( 1969). Blood acid-base status was investigated by equilibrating oxygenated and deoxygenated samples at 20 C with varying CO2 tensions (in air or NJ for at least 10 min and measuring pH using a Radiometer BMS 2 mk II and PHM 73 apparatus. Buffering capacity is expressed as AHCO,/A pH. calculating concentration from the HCO, Henderson-Hasselbalch equation ([HCO, ] = antilog (pHpK;)_ aco2 P,,,), using Severinghaus’s (1971) values for the CO, solubility coefficient (a,,?) and pK;. To prepare cofactor-free hemoglobin solutions the red cells were washed twice in 0.9% NaCl, buffered by addition of one-quarter their volume of 1 mol/l Tris, pH 7.5, and hemolysed by addition of iced distilled water and brief treatment with an MSE ultrasonic finger. Cell ghosts were spun down, the Hb was stripped of ions using MB-3 mixed ion exchanger and exhaustively dialysed against COsaturated 0.01 moljl Tris buffer, pH 7.5 containing

I

I

5 x 10~4mol/l EDTA as earlier described (Weber CI (I/.. 1983). Hb multiplicity was investigated by electrophoresis on Millipore cellulose acetate strips (pH 8.5) and by isoelectric focusing (IEF) at 5‘C in a I IO ml LKB column, containing ampholines of pH 558 and 779 (0.4”/(; of each). Hbs isolated by IEF were dialysed against Tris-EDTA (see above) and concentrated in Amicon B15 concentrators (Oosterhout. Holland) prior to oxygenation studies. Oxygen equilibria of the stripped hemolysates and tsolated components were determined using a diffusion chamber modified (Weber, 1981) after Niesel and Thews ( 1961). Effects of organic phosphates were studied by addition of freshly assayed stock solutions of the sodium salts of ATP and GTP. RESULTS

Whole blood The whole blood of Necturus maculosus displayed a high O2 affinity and a distinct Bohr effect; at pH 7.5, Po2 I k Pal 05

10

15

-t j0 -1

16 .-

Fig. 3. Davenport diagram showing HCOj/pH relationship for oxygenated (0) and deoxygenated (A) Necturus blood (calculated from data in Fig. 2).

LO

12 BXY

Deoxy

3 08

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:\ 04 Paz (mm Hgl 70

Fig. 2. Astrup

72

74 PH

16

titrations of oxy- and deoxygenated blood at 2OC.

whole

Fig. 4. Effect of added ATP on P,, and nsOvalues of stripped Nerturus Hb, dissolved in (bis-)TrisHCI buffer, ionic strength I = 0.05, pH 7.27; Hemoglobin concentration, 0.03 mM tetramers: temperature, 20 C.

Necturus hemoglobin

8

zymatically ATP.

0 0

‘, A 2

9 I

1 08

03

06 04

I

1

55

60

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65

70

75

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85

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Fig. 5. Variation of P,, and q0 with pH in stripped Necturus Hb dissolved in (bis-) Tris-HCI buffer, I = 0.05 in the absence of ATP (open symbols) and in the presence of

added ATP at a 2.09 molar excess over Hb tetramers (solid symbols), at 5°C (circles) and 2O’C (triangles); Hemoglobin concentration,

determined

NTP

to be predominantly

Hemolysate: proton, temperature and ATP qfects

5” 20°C -ATP/Hb=O ATPIHbz209

491

0.04 mM tetramers.

and Pcoz = 5 mmHg, P,, = 10.6 and 20-c 4 (=A log P,,/A pH) = -0.4 (Fig. 1). Cooperativity in 0, binding was low but increased with pH 1.4 and 1.7 at pH 7.0 and 7.7, respectively). (n50 The Haldane effect was marked as evident from significantly higher pH values in the deoxygenated than in the oxygenated blood over a wide range of Pco2 values tested (3.622.0 mmHg, Fig. 2). That the Haldane effect is predominantly due to higher bicarbonate concentrations in the deoxygenated than in the oxygenated blood, follows from the fact that the oxy-deoxy difference was pH independent (Fig. 2), whereas carbamino formation in Hb from mammalian and ectotherm vertebrates decreases as the amino-groups of the pigments become charged at low pH (Perrella et al., 1977; Weber and Lykkeboe, 1978). At pH 7.5 bicarbonate concentrations calculated from the Henderson-Hasselbalch equation amounted to 3.8 and 7.4 for oxy and deoxy blood, respectively (Fig. 3). At high pH (> 7.5) the buffering power was 11 mmol HCO;/(pH unit) and independent of oxygenation; at the lower pH values tested, it increased markedly in deoxy- but not in oxy-blood (Fig. 3). These values of arterial plasma bicarbonate and buffering capacity are low compared to other amphibians, but accord with the comparative inactivity of Necturus, its dependence on aquatic gas exchange, and resultant low internal CO* tensions (Lenfant and Johansen, 1967; Gahlenbeck and Bartels, 1970, Gatz et al., 1974; Boutilier and Toews, 1981). In one pooled blood sample (from 3 specimens) we found a Hb concentration of 0.56 mM, predicting an 0, carrying capacity of 5.0 vol.%, and DPG and NTP (nucleoside triphosphate) levels of 0.3 and 1.0 mM, respectively, reflecting an erythrocytic NTP/Hb ratio of 1.8. Thin-layer chromatography resolved the en-

Stripping the Hb resulted in a large increase in OX affinity. At 20°C and pH 7.3 the P,, of the cofactorfree Hb was 1.2 mmHg, compared with 12 mmHg in the fresh blood. Addition of ATP decreased 0, affinity but the P,, values (- 2.0 and 3.5 mmHg at ATP/HB = 2 and 10, respectively; Fig. 4) remained much lower than in the whole blood. The Bohr factor varied inversely with temperature and directly with ATP. Under the conditions of measurement 4 values at pH 7.0-7.5 and 5°C were -0.62 and -0.66 in the absence and presence of ATP, respectively; at 20°C the corresponding values were -0.24 and -0.58 (Fig. 5). ATP also increased the amplitude of the Bohr effect (4amp,, the maximum pH-induced change in log P,,; cfi Sick and Gersonde, 1974) and the pH range where the Bohr effect is operative (A pH,) while 4, bamp, and A pH, were significantly greater at 20 than at 5°C (cf. Fig. 5). Cooperativity fell with phosphate removal, but was restored by addition of ATP (Figs 4 and 5). Cooperativity was clearly pH dependent, showing a maximum near pH 6.5 in the absence of ATP, and near pH 7.0 in the presence of ATP i.e. under the same conditions where the Bohr effect was greatest (Fig. 5). Decreased temperature (from 20 to 5°C) similarly increased the pH values where the Bohr effect and cooperativity were maximal. These observations reflect a tight linkage between the hetero- and homotropic interactions underlying the Bohr effect and cooperativity respectively in Necturus Hb. The pH variation of P,, of the stripped Hb in the presence and absence of added ATP and at two temperatures (Fig. 5) provides an opportunity to apportion the overall enthalpy among the component processes (the intrinsic heat of oxygenation, the heat of 0, solution, and the heats of proton and phosphate binding). Figure 6 shows the effects of pH on the apparent enthalpy of oxygenation, A Hdpp, calculated from the temperature induced change in log P,, (Fig. 5) using the van? Hoff isochore (CL Garby and Meldon, 1977): A Hdpp = 2.303 R (A log P,,),,

: ~,/-

---_____-II: ATP/Hb=

2 09

1

,,_,.,...,.._..,,

I11 (=1-m./"

"L.,, .'... ,,,,,,,,,,~,,,~,,,,,,...,....."' "".....i ........ I 60

I 6.5

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70

75

80

PH

Fig. 6. Oxygenation enthalpies of Necfurus Hb, calc;llated from the van7 Hoff isochore for 5%2O”C (see Fig. 5) and their pH dependence. (Further explanation in the text).

ROY E.

498

WEBER

et

al.

Hbs and the closely similar p1 values of Hbs 11, III and IV precluded isolation of the components in pure form for functional characterization. Two fractions of the hemolysate retrieved from the IEF column, which predominantly contained either Hb II or Hb III (Fig. 7), however, showed similar Oz affinity and ATP sensitivity to the unfractionated hemolysate (Fig. 8). these

DISCUSSION

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L5 r----l

r-!-l

40

50

ho

70

80

90

Froctlonnumber

Fig. 7. Isoelectric focusing profile of Necturus tal bars, fractions pooled for oxygenation Fig. 8).

Hb. Horizonstudies (see

R is the gas constant (8.314 x lO’J/‘C/mol) and T the absolute temperature. For the stripped Hb the temperature dependence of O2 affinity, AHapp(Fig. 6, curve I) is greatest at low and high pH, where the Bohr effect is small or absent, showing that the lower values at intermediate pH may be ascribed to the heat of ionization of oxygenlinked acid groups, which subtract from the exothermic oxygenation reaction proper. The AH value for the cofactor-free Hb at pH > 8 accordingly indicates an intrinsic heat of oxygenation (AH,) of about -63 kJ/mol (Fig. 6). This value includes about _ 13 kJ/mol attributable to the heat of solution of 0,. The AH values for the Hb in the presence of ATP show similar pH dependence (Fig. 6, curve III) but lower absolute values, reflecting decreases due to endothermic dissociation of ATP. This is consistent with a reduced effect of ATP on 0, affinity as temperature increases (see Fig. 5) upon oxygenation of the Hb molecules. The heat of ATP release corresponds to the difference between curves I and II (i.e. curve III in Fig. 6) which shows a broad maximum at pH 7.5 to 7.8. At higher pH values, it decreases as expected due to decreased ionization of the cationic groups at the phosphate binding site. We are unable to explain the decrease at pH below 7.5, which may, however, be related to interdependence of the heats of proton and ATP binding to the Hb.

where

The blood 0, affinity here reported for N. muculosus (P,, = 11mmHg at pH 7.5 and 2O.C) appears to be the highest recorded for urodelan amphibians, which generally have higher affinities than the air breathing anurans (cf. Gahlenbeck and Bartels, 1970: Gatz et al., 1974; Boutilier and Toews, 1981 Burggren and Wood, 1981). The P,, is even lower than in the Lake Titicaca frog where an exceptionally high affinity correlates with an ability to skin-breathe in hypoxic water (Hutchison et al., 1976). The high 0, affinity in Necturus blood correlates with its dependence on aquatic 0, uptake, and represents an adaptation to O,-poor water in which Necturus may be encountered (Harris, 1959a,b). It moreover aligns with the observation that Necturus “with little difficulty“ (Guimond and Hutchison, 1976) can reduce water PO, to 40mmHg and lower. Apart from being adaptive to low enuironmenral O2 levels, the high blood 0, affinity may be adaptive to low nrterial0, tension, which may result from mixing of oxygenated and deoxygenated blood in the undivided ventricles of amphibian hearts. In blood of the skin-breathing urodele, Cryptobranchus alleganiensis, high 0, affinity and high cooperativity appear to offset loss of OZ saturation when arterialized and venous blood mix as a result of a perforated atria1 septum and the lack of spiral values in the conus arteriosus (Boutilier and Teows. 1981).

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Hb componen try Electrophoresis on cellulose acetate at pH 8.5 indicated the presence of two major Hb components that migrate anodally and a minor component with higher anodal migration rate. Preparative IEF showed that the major components (II and III in Fig. 7) account for some 90% of the total Hb, that these components are isoelectric near pH values of 7.1 and 7.0, and that the hemolysates contain at least three other, minor Hbs (I, IV and V, see Fig. 7) with p1 values near 7.8, 6.9 and 6.2. The meager amounts of

I

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65

70

75

80

85

PH

Fig. 8. P,, and n,, values of Hb fractions II (triangles) and III (diamonds) separated by isoelectric focusing (see Fig. 7) dissolved in (bis-)Tri-HCI buffer (I = 0.05) in the absence of ATP (open symbols) and in the presence of ATP (ATP/Hb 5 9; closed symbols). Circles, unfractionated hemolysate. Hemoglobin concentration, 0.03 mM, 20 C.

Necturus hemoglobin The significance of the high affinity in N. maculosus stands revealed by its low arterial 0, tension of 35 mmHg (Lenfant and Johansen, 1967) compared to other amphibians (Gatz et al., 1974; Boutilier and Toews, 1981). Combined with our whole blood O2 equilibria (Fig. 1) this value indicates an arterial O2 saturation of about go:?/,.Our blood P,, value is lower than that (14.6 mmHg at PcoZ of 6 mmHg) reported by Lenfant and Johansen (1967). The difference presumably relates to differences in acclimation history, given that hypoxic exposure increases blood O2 affinity in fish and neotenic adults of Ambystoma tigrinum via decreased allosteric effects of NTP (Wood and Johansen, 1972; Weber et al., 1975; Wood et al., 1982) and that decreased temperature decreases erythrocyte ATP levels in N. maculosus and j4mbystoma tigrinum (Hazard and Hutchison, 1982; Wood et al., 1982). Despite the high O2 affinity in the blood. that in the stripped Hb is more than ten times greater (P,, = 0.8 mmHg at pH 7.5) revealing ample scope for ATP modulation of blood O2 affinity. The observed difference between blood and Hb affinities are, however, not solely due to phosphates as evident from the higher OZ affinities in Hb solutions containing saturating ATP levels than in the blood (compare Figs I and 4). Other factors contributing to the difference will be higher Hb concentrations in the erythrocytes, and lower erythrocytic pH values than those measured in the whole blood and plasma (Wood et al., 1982). As shown (Figs 5 and 6) ATP decreases AH,,, reflecting heat absorption as the triphosphate is displaced from the Hb upon oxygenation. Thus at pH 7.5, AH is -52 kJ/mol 0, in the absence of ATP and -32 kJ/mol O2 in the presence of ATP (under the conditions of measurement, see Fig. 6). If it can be assumed that the heats of proton movements are unaffected by the ATP reaction (cf. Benesh et ai., 1969), then it follows that the enthalpy for ATP liberation is + 20 kJ/mole O2 bound and f 80 kJ per mole ATP liberated from the tetrameric Hb. This value is greater than corresponding values of +46 to + 57 for the dissociation of DPG from human Hb at pH 7.3 at a molar DPG/Hb ratio of 4: 1 (Benesch et al., 1969; Hedlund and Lovrien, 1974). Since ATP decreases the overall temperature sensitivity of PsO, the higher ATP levels in the blood of warm-acclimated than cold-acclimated Necturus (Hazard and Hutchison, 1982) imply greater stabilization of loading and unloading O2 tensions under fluctuating temperatures in the warm-acclimated specimens. Insofar as blood ATP concentration increases with water Po, (as observed in teleosts and Amb+rstoma--cited above), this similarly implies lower temperature sensitivities in normoxia- than in hypoxia-acclimated animals, that will curb the reduction in O2 affinity at high temperature. Higher levels of erythrocytic ATP in warm- than in cold-acclimated specimens, however, also mean that the temperature-effect in temperature-acclimated animals will be greater than in those acutely exposed to temperature change. This is opposite to the mtraspecrfic responses observed m teleosts hke the brown bullhead, ~cfuf~r~ ~e~~los~s (Grigg, 1969) and in Rana esculenta (Gahlenbeck and Bartels, 1968)

499

where blood 0, affinity increases following warm acclimation. It -is moreover at variance with the evolutionary trends indicated by lower temperature effects found in species that experience large temperature fluctuations (Johansen and Weber, 1976). Warm acclimation, however, increases ATP concentration in the antarctic fish Fagothenia borchgrecinki, living in constantly well-oxygenated waters, where the resultant 0, affinity decrease enhances tissue Ok-unloading (Wells and Jokumsen, 1982; Tetens et al., 1984). In the primitive bowfin fish, Amia caba, a conspicuously large temperature effect correlates with the fact that high temperature increases air-breathing which is better served by reduced 0, affinity (Johansen and Lenfant, 1972). That the temperature effect in Necturus cannot be similarly exploited follows from the drastic reduction in arterial O2 saturation from 35 to 10 mmHg when the animal is exposed to air (Lenfant and Johansen, 1967). In showing a normal Bohr effect (negative value of Alog P,,/ApH) Necturus Hb resembles those in adult frogs (c#: Runa esculenta, R. cates~eiffna, Xenopus laecis, BE&O~ar~~nemis and Pipa ~~~ae-Brunor~ et al., 1968; Aggarwal and Riggs, 1969; Jokumsen and Weber 1980; Vieira et al., 1982) rather than the Hbs of anuran tadpoles (Watt and Riggs, 1975) and the premetamorphosed urodele, Amphiuma means (Bonaventura et a/., 1977), which exhibit reverse (positive) Bohr factors in the absence of organic phosphates. Brunori et al. (1968) have shown that in R. esculenta the pH dependence of Hb0, affinity can be fitted satisfactorily to the same model applicable to mammalian Hb, assuming that the group responsible for the alkaline Bohr effect is an imidazole and that for the acid Bohr effect is a carboxyl group. The Bohr effect of lectures Hb decreases with increasing temperature (Fig. 5) as in horse Hb, where the phenomenon can be explained by the difference between these two groups in heat of ionization, which for each group, however, remains the same in oxy- and deoxyHb (Antonini et al., 1965). The pH-induced change in AH of -31 kJ/mol (from about - 63 kJ/mol at pH 8.0 to - 32 kJ/mol at pH 6.8; see Fig. 6) can be related to the Bohr factor (-0.7 at 5°C) which indicates that 0.7 protons are released per 0, molecule bond. This indicates that the heat of reaction per proton is about -44 kJ/mol, which compares with heats of ionization of about - 29 and -46 kJ/mol for histidine imidazole groups and z amino groups, respectively (Reeves and Rahn, 1979) i.e. the groups which are responsible for most of the alkaline Bohr effect in human Hb (Kilmartin, 1977). The heat of ionization of the Bohr groups of Necturus Hb thus appears to correspond closely with that of n-terminal Z-NH: despite the observations (De Witt and Ingram, 1967. Maruyama et al., 1980; Watt et al., 1980) that these groups are acetylated in Hb of tadpole and adult frogs, whereby they cannot contribute to the Bohr effect. In anuran tadpole Hb, the reverse Bohr effect at physiological pH, correlates with the substitution and acetylation of the amino acid residues mainly responsible for the alkaline Bohr effect in man, whereas hjstidine H21 of the 8 chain, which accounts for half of the reverse Bohr effect in human Hb appears to

ROY E. WEEER rt

500

have a higher pK (Watt et al., 1980; Perutz and Brunori, 1982). The alkaline Bohr effect in stripped Necturus Hb is large (attaining -0.70 at 5°C) compared to other amphibians, where weak Bohr effects are attributabie to the presence of a glycine residue at position FGl of the fl chain; in Xenopus Hb where glutamic acid at this position contributes to a strong salt bridge in the deoxy state, the Bohr factor is pronounced (-0.56) (Perutz and Brunori, 1982). It would thus be of particular interest to determine amino acid sequences for Necturtcs Hb. The finding of a normal Bohr effect in ~ect~rl~.~ Hb, indicates that the reverse effect found in anuran tadpoles and premetamorphosed urodela cannot be related to the water breathing habit per se or to the larval state per se. Linkage with neoteny moreover appears to be ruled out by the findings that the Bohr effect in whole blood of the salamander Dicumptodon ensures is unaltered by the larva~adult transformation (Wood, 1971) and that a reverse Bohr effect is found in Hb of the adult salamander, Pieurodeles waltii (Flavin et al., 1983). Understanding of the physiological and adaptational significance of the different patterns of pH sensitivity must await measurement of in &o arterial and venous pH and PO, values in different stages. The multiplicity of the Hb in Eeccurus similarly invites study of the ontogenetic deveiopment in Hb patterns of permanent neotenes given that adult and larval hemoglobins in P. w&ii are synthesized in different red cell populations (Flavin et ul., 1982). Acknowlellkentents-Supported by Danish Natural Science Research Council. We thank Finn Jorgensen, Odense, for giving us the experimental animals, and Renee Stgvring for technical

assistance.

~1.

globin

with oxygen

and carbon

monoxide.

Camp.

Bin-

them. Physiol. 24, 5 19-526. Branch L. C. and Taylor D. H. (1977) Physiological and behavioural responses of larval spotted salamanders (Antbystoma maculatum) to various concentrations of oxygen.

Camp. ~iochetn. Physiof. %A, 269-274. Cashel M., Lazzarini R. A. and Kalbacher B. (1969) An improved method for thin layer chromatography of nucleotide mixtures containing ‘?P-labelled orthophosphatc.

J. Chromaf. 40, 103-109. Dewitt W. and Ingram V. M. (1967) Acetylated peptide chains in bullfrog hemoglobins. Biochrm. Bi0phy.s. W.Y.

Comm. 27, 23624 I. Flavin M.. Ton That H., Deparis P. and Duprat A. M. (1982) Hemoglobin switching in the salanXUIder Pieurodeles walilii. lmmunofluorescence detection of larval and adult hemoglobins in single erythrocytes. Wilhelm

Roux’s Arch. 191, 185-190. Flavin M., Thillet J. and Rosa J. (1983) Oxygen equilibrium of larval and adult hemoglobins of the salamander.

Pieurode~e~~ u,al~l~~.Camp. Biochem. Physioi.

75A, 8 l-85.

Gahlenbeck H. and Barteis H. (1968) Temperaturadap~tion der SauerstoffaffinitZt des Blutes von Rana esculenfa. L. Ztschr. rergi. Physiol. 59, 232-240. Gahlenbeck H. and Bartels H. (1970) Blood gas transport properties in gill and lung forms of the axolotl (Am-

bysroma mexicanurn). Respir. Physioi. 9, 175- 182. G&y L. and Meldon J. (1977) The Respiratory Funcrions qj”Broods pp. l-282. Plenum, New York. Gatz R., Grawford E. C. and Piiper J. (1974) Respiratory properties of the blood of a lungless and gill-less salamander, Desmognuthus fiscus. Respir. Physiol. 20,

33-41. Grigg G. C. (1969) Oxygen equilibrium curve of the blood of the brown bullhead, Icralurus nebulosus. Crimp. Binthem. Physiol. 29, 120% 1223. Guimond R. W. and Hutchison V. H. (1972) Pulmonary. branchial and cutaneous gas exchange in the mud puppy, Nocturus maculosus (Rafinesque). Comp. Biochem. Phys-

iol. 42A, 367-392. REFERENCES

Aggarwai S. J. and Rigss A. (1969) The hemoglobins

of the bull frog, RUHQ eu~~~~~j~~~. I. Purification, amino acid composition and oxygen equilibria. J. hiof. C&m. 244, 2372-2383. Antonini E., Wyman J., Brunori M., Fronticelli C., Bucci E. and Rossi-Fanelli A. (1965) Studies on the relations between molecular and functional properties of hemoglobin. V. The influence of temperature on the Bohr effect in human and horse hemoglobin. J. biot. Chem. 240, 1096-l 103. Benesh R. E., Benesch R. and Yu C. I. (1969) The oxygenation reaction of hemoglobin in the presence of 2,3 diphosphoglycerate. Effect of temperature, pH, ionic strength and hemoglobin concentration. Biochemistry 8,

2567-2571. Bonaventura C., Sullivan B., Bonventura J. and Bourne S. (1977) Anion modulation of the negative Bohr effect of hemoglobin from a primitive amphibian. NQWP 265, 474-476. Boutilier R. G. and Towes D. P. (198 1) Respiratory properties of blood in a strictly aquatic and predominantly skin-breathing urodele, Cryptobranchus alleganiensis. Re-

spir. Ph~ysiol.46, 161-176. Burggren W. W. and Wood S. C. (1981) Respiration and acid-base balance in the salamander Amb~s~oma Eigrinutn: influence of temperature acclimation and metamorphosis. J. camp. Physioi. 144, 241-246. Brunori M., Antonini E., Wyman J., Tentori L., Vivaldi G. and Carta S. (1968) The hemoglobin of amphibia. VII. Equilibrium and kinetics of the reaction of frog hemo-

Guimond R. W. and Hutchison V. H. (1976) Gas exchange of the giant salamanders of North America. In Respirurion of’ Amphibious Vertebrates (Edited by Hughes. G. M.). pp. 313-318. Academic Press, London. Harris J. P. (1959a) The natural history of Necrurus: I. Habitats and habits. Field Lab. 27, I l--20. Harris J. P. (1959b) The natural history of Nrrturus: II. Respiration. Field Lub. 27, 71-77. Hazard E. S. and Hutchison V. H. (1982) Distribution of acid-soluble phosphates in the erythrocytes of selected species of amphibians. Camp. Biochem. P&sictl, 73A, 111-124. Hedlund B. E. and Lovrien R. (1974) Thermodynamics of 2,3-diphosphoglycerate association with human oxy- and deoxyhemoglobin. Biochem. biophys. Res. Commun. 61,

859-867. Hutchison V. H., Haines H. B. and Engbretson G. (1976) Aquatic life at high altitude: Respiratory adaptations in the lake Titicaca frog. Telmuf(lb~u.~cukeus. Respir. Ph,wioi.

27, 115-129. Johansen proach

K. and Lenfant to the adaptability

C. (1972) A comparative apof 02-Hb affinity. In Os.:gen

Afinit,v of Hemoglobin and Red Cell Acid-base Starus (Edited by Rsrth M. and Astrup P.). pp. 750-780. Munkgaard, Copenhagen. _ Johansen K. and Weber R. E. (1976) On the adaotabilitv of haemoglobin function to environmental con&ions: In

perspectives in ~xper~rnen~a~Zo&gy

Vol. I, Zooiog~.

(Edited by Spencer Davis P.), pp. 219-234. Pergamon, Oxford. Jokumsen A. and Weber R. E. (I 980) Haemoglobin-oxygen binding properties in the blood of Xenopus 1aeai.v.with special reference to the influence of aestivation and of

Necfurus temperature and salinity acclimation. J. exp. Biol. 86, 19-37. Kilmartin J. V. (1977) The Bohr effect of human hemoglobin. TIBS, Notlember 1977, 247-250. Lenfant C. and Johansen K. (1967) Respiratory adaptations in selected amphibians. Respir. Physiol. 2, 247-260. Maruyama T., Watt K. W. K. and Riggs A. (1980) Hemoglobins of the tadpole of the bullfrog Ranu catesbeiunu. Amino acid sequence of the c( chain of a major component. J. biol. Chem. 255, 3285-3293. Niesel W. and Thews G. (1961) Ein neues Verfahren zur Aufnahme der schnellen und genauen Sauerstohbindungskurve des blutes und konzentrierter Hamoglobinlosungen. Pfltigers Arch. Ges. Physiol. 273, 380-395. Perrella M., Guglielmo G. and Mosca A. (1977) Determination of the equilibrium constants for oxygen-linked CO? binding to human hemoglobin. FEBS Let/. 78, 287-290. Perutz M. and Brunori M. (1982) Stereochemistry of cooperative effects in fish and amphibian hemoglobins. Nafurr 299, 421426. Reeves R. B. and Rahn H. (1979) Patterns in vertebrate acid-base regulation. In Evolution qf‘ Respiratory Processes, A Comparatioe Approach. Series Lung Biology in Health and Disease (Edited bv Wood S. C. and Lenfant C.). Vol. 13, pp. 225-252. Marcel Dekker, New York. Severinghaus J. W. (1971) Carbon dioxide solubility and first dissociation constant (pK’) of carbonic acid in plasma and cerebrospinal fluid: Man. In Handbook of Respiration and Circularion (Edited by Altman P. L. and Dittmer D. S.), pp. 218-219. Fed. Am. Sot. Exp. Biol.. Bethesda. Sick H. and Gersonde K. (1974) Ligand dependent Bohr effect of Chironomus hemoglobins. Eur. J. Biochem. 72, 2Oll206. Tetens V., Wells R. M. G. and DeVries A. L. (1984) Antarctic fish blood: Respiratory properties and the effects of thermal acclimation. J. exp. Biol. 109, 265-279. Vieira H. F.. Vieira M. L. C.. Meirelles N. C. and Focesi A., Jr (1982) Some functional and structure properties of

hemoglobin

501

Bufus poracnemis and Pipa pipae hemoglobins. Comp. Biochem. Physiol. 73A, 197-200. Watt K. W. K., Maruyama T. and Riggs A. (1980) Hemoglobins of the tadpole of the bullfrog, Rana catesbeiana. Amino acid sequence of the p chain-of the major comuonent. J. biol. Chem. 255. 32943301. Watt K. W. K. and Riggs A. (1975) Hemoglobins of the tadpole bullfrog, Rana ca~esbeiunu. Structure and function of the isolated components. J. biol. Chem. 250, 59345944. Weber R. E. (1981) Cationic control of O2 affinity in lugworm erythrocruorin. Nature 292, 386-387. Weber R. E. and Lykkeboe G. (1978) Respiratory adaptations in carp blood. Influences of hypoxia, red cell organic phosphates, divalent cations and CO? on hemoglobinoxygen affinity. J. camp. Physiol. 128, 127-137. Weber R. E., Lykkeboe G. and Johansen K. (1975) Biochemical aspects of the adaptation of hemoglobin-oxygen affinitv of eels to hveoxia. Life Sci. 17, 134551350. Weber R. E., Wells R. M. G.’ and Rossetti J. E. (1983) Allosteric interactions governing oxygen equilibria in the hemoglobin system of the spiny dogfish, Squulus wanfhias. J. exp. Biol. 103, 109-120. Wells R. M. G. and Jokumsen A. (1982) Oxygen binding properties of hemoglobins from antarctic fishes. Comp. Biochem. Physiol. 71B, 469-473. Wells R. M. G. and Weber R. E. (1983) Oxygenational properties and phosphorylated metabolic intermediates in blood and erythrocytes of the dogfish, Syuulus ucanrhias. J. exp. Biol. 103, 95-108. Wood S. C. (1971) Effects of metamorphosis on blood respiratory properties and erythrocyte adenosine triphosphate level of the salamander Dicamp/odon cnsatus (Eschscholtz). Respir. Physiol. 12, 53-65. Wood S. C. and Johansen K. (1972) Adaptation to hypoxia via increased HbOz affinity and decreased red cell ATP concentration. Nature 237, 278-279. Wood S. C., Hoyt R. W. and Burggren W. W. (1982) Control of hemoglobin function in the salamander, Ambystomu tigrinum. Molec. Biol. 2, 263-272.