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Equilibria and kinetic of oxygen and carbon monoxide binding to the haemoglobin of the South American lungfish,Lepidosiren paradoxa
( omp Btmhcm Pht~¢.l I ol 62A. pp 139to 143 PeltlOtllotl press Lid 1979 Pllltlcd Ill GII'III Bl'llltln
0300-96297911101-0139$0200/0
EQUILIBRIA AND KINETICS OF OXYGEN AND CARBON MONOXIDE BINDING TO THE HAEMOGLOBIN OF THE SOUTH AMERICAN LUNGFISH, LEPIDOSIREN PARADOXA* CHARLES PHELPS, J MARTHA FARMER,2 HANS J. FYHN,3 UNNI E. H. FYHN, 3 ROBERT L. GARLICK,4 ROBERT W. NOBLEs and DENNIS A. POWERS6 :Department of Biological Sciences, University of Lancaster, Lancaster LAI 4YQ, England; -'Duke University, Marine Laboratories, Beaufort, NC 28516, U.S.A.; 3Zoological Institute, University of Oslo, Blindern, Oslo 3, Norway; '*Department of Zoology, University of Texas at Austin, Austin, TX 78712, U.S.A.; 5Department of Medicine and Biochemistry, Veterans Administration Hospital, S.U.N.Y., Buffalo, NY 14215, U.S.A.; 6Department of Biology, Johns Hopkins University, Baltimore, MD 21218, U.S.A.
(Received 5 April 1978) Abstract--I. The haemoglobin of the South American lungfish Leptdosiren paradoxa has a single component. 2. The equilibria of this respiratory protein with oxygen have been investigated both in the blood and with the purified haemoglobin. There is a substantial, normal, alkahne Bohr effect and marked sensitivity to organic phosphates in the haemoglobin solutions. 3. Studies on the pH dependence of the kinetics of oxygen dissociation can be interpreted in terms of a normal Bohr effect. 4. The kinetics of combination of carbon monoxide have an unusual pH dependence. 5. These findings are discussed in terms of the two-state model of Monod et al. (1965)
INTRODUCTION The transition from water breathing to air breathing in fishes is a significant evolutionary step. Fish which show both marked structural adaptations, and sophisticated physiological modifications to this end are found mainly in tropical waters or estuaries. The South American lungfish, Lepidosiren paradoxa, is structurally the most advanced of airbreathing fishes. In this dipnoan, the air bladder has evolved into a paired lung, and internal septa subdivide the air space into successively smaller compartments ending in alveoli-like lobules, richly suffused with blood vessels. Whereas the structural and physiological adaptations are well documented, little information is available on the molecular characteristics of its haemoglobin. This paper sets out to document the oxygen equilibrium properties of the blood, and of the haemoglobin in both organic phosphatestripped and unstripped states and further, to describe some aspects of the kinetics of reaction of the haemoglobin with the ligands oxygen and carbon monoxide. METHODS AND MATERIALS Specimens of the South American lungfish (Lepidosiren paradoxa) were obtained from a local fisherman in November 1976 during an expedition with the R.V. Alpha * A Portuguese translation of this work will appear in ,'tCI~I A m ~ l Z O l l l c ( i .
139
Helix in an area about 30 miles upstream the Amazon River (Solim6es) from its junction with the Rio Negro. The collection period was at the end of the dry season when the water level was at its lowest in lakes and rivers. The animals (body weight 370-2000 g) were not aestivating when collected and were kept in aquaria for 2-6 days before being bled. Blood was obtained by cardiac puncture of ice-chilled animals and drawn into a cold heparinized glass syringe (100/d of sodium heparln (5000i.u./mll in 1.7",. NaCI/5 ml of blood) The cells were spun at 1000g in a refrigerated centrifuge (Sorvall Model RC2-B, I. Sorvail, Norwalk, CT) for 5min. The supernatant was removed and the cells washed 3 times using the same protocol by suspension in 1.7°o (w/v) NaCI containing 5 mM Tris buffer pH 8.0. The cells were then lysed by exposure to l mM Tris buffer pH8.3 at 4'C for 20min. Sodium chloride was added to a final concentration of 100mM, and the cell debris removed by centrifugation at 15,000g. Desalting was conducted on a column (110 x 1.5cm) of G-25 medium (Pharmacia Fine Chemicals, Uppsala) equilibrated in 1 mM Tris pH 8.3. "'Stripping" of haemoglobin solutions was performed on a column (15 × 2.5 cm) of mixed bed resin AG 501- × 8 (DI from Bio Rad Laboratories. Richmond, CA, on top of which were layered two separate 3 cm pads of ion exchangers viz Dowex-50W NH,~ and Dowex I-Acetate (Dow Chemical Co., MI). Flow rates of 1 fnl/min were maintained. Disc gel clectrophoresis at pH 8.9 were performed as described by Fyhn et al. (1979). From the same stained gels the ratio between the migration distance of the haemoglobin component and that of bovine serum albumin was calculated and used to effect comparisons of haemoglobin components in gels of different runs. These ratios are referred to as relative mobilities.
140
CHARLES PHELPS et al.
Oxygen equilibrium measurements were performed using glass tonometers as described by Allen et al. (1950) and Riggs & Wolbach (1956). The results were calculated as described by Nagel et al, (1965). All experiments were carried out at 20°C with haemoglobin concentrations approx 50-100/~M in haem equivalents. Oxygenation of the priorly deoxygenated solution was followed spectrophotometrically at two wavelengths, 560 and 576 nm. Gas additions were either air or pure oxygen. Equilibration times after each addition were 10-20min. Buffers used were all of ! = 0.05, with Bis-Tris in the range pH 5.25-7.3, and Tris for pH values above 7.3. Methaemoglobin formation was measured as described in Benesch et al. (1965) and was at all times less than 3~o. Measurements of oxygen equilibrium in the whole blood was performed using the Hem-O-Scan apparatus (American Instrument Co. Silver Spring, MD) described by Powers et al. (1979). Kinetic measurements were performed at 20~C with a stopped-flow rapid mixing apparatus similar to that originally described by Gibson & Milnes (1969). Solutions of haemoglobin were identical in composition to those used for equilibrium measurements. The kinetics of oxygen dissociation were measured by the pH-jump procedure as described by Noble et al. (1970). Oxygenated haemoglobin in 1 mM Tris pH 8.0 was mixed with a solution of dithionite in a buffer of desired pH, ! = 0.05. The final haemoglobin concentration was approx 30/~M in haem equivalents and the reaction was followed at both 560 and 540 nm independently. The kinetics of carbon monoxide combination to deoxygenated haemoglobins were measured by mixing solutions of deoxygenated haemoglobins in buffers of the desired pH of ionic strength I = 0.1, containing a known concentration, approx 85~uM, of carbon monoxide dissolved m water. After mixing, the haemoglobin concentration was ca. 3/aM in haem equivalents. The reaction was followed at 420 and 435 nm independently. RESULTS
AND
.~..
)
DISCUSSION
Studies on the whole blood of the animal reveal that the haemoglobin is half saturated at 8.3 mm Hg pO2 at 30°C and pH 8.06. O n re-equilibrating the blood sample in the presence of 5.6°'0 v/v CO2, the i
i
i
1.0
Fig. 2. Electrophoresis in polyacrylamide gels of the haemoglobin from Lepidosu'en paradoxa (left) and from man (right). Bovine serum albumin is the fast running marker. *g
,,~
pH of the sample fell to 7.33 and the Pso increased to 1 4 m m Hg pO2 (Fig. 1). Thus at 30°C, there is evidence of a significant Bohr effect which may be c o m p o u n d e d of both H + and C O , effects, the apparent magnitude of which for the pH range 7.33-8.06 is:
°07 o°y
0.5
A log Pso A pH
0.31.
A thorough investigation of the oxygen equilibrium on the isolated haemoglobin was undertaken. Poly0 1 2 acrylamide gel electrophoresis (Fyhn et al. 1979) log pO2 Fig. 1. Oxygen equdibrium curves for whole blood from revealed a single component. The haemoglobin has Leptdosu'en paradoxa under the conditions specified. T h e - - a- mobil,ity of 0.66 + 0.01 relative to bovine serum ordinate relates to fractional saturauon of the haemoglobin albumin, and Fig. 2 compares the electrophoretic patby oxygen and the abscissa is scaled to log pO2 values terns obtained for haemolysates from Lepidosiren parexpressed in mm Hg. a d o x a with that of man. |
!
!
!
Oxygen and carbon monoxide binding to the haemoglobm of the South American lungfish
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Fig. 3. The pH dependence of the oxygen equilibria with haemoglobin from Lepidosiren paradoxa showing the effect of "stripped" and "unstripped'" preparations. The ATP concentration was I mM. The pH dependence of the oxygen dissociation curve was measured for material that had been stripped of organic phosphates and for identical solutions to which had been added ATP to a final concentration of I mM (Fig. 3). The two curves are broadly similar except that the presence of organic phosphate has greatly decreased the affinity of the haemoglobin, as would be expected if it stabilized the deoxy conformer. The magnitude of the Bohr effect is much greater in the presence of l mM ATP and its response to pH across the pH zone from 7.00 to 8.00 is 5 times more acute in the presence of organic phosphate (Fig. 3). The equilibrium data reflect the behaviour of a haemoglobin not greatly different in response from that of human haemoglobin A. The magnitude of the Bohr effect in haemoglobin A at 25r'c in similar buffer systems is - 0 . 3 0 between pH 6.8 and 8.8, and the effect of excess organic phosphate decreases the oxygen affinity by a factor of 10 (lmai, 1974). It seems clear that whatever molecular adaptations in haemoglobins are required in the course of the water to air transition, they are complete at the level of evolution of the lungfish. The pH dependence of the rate of oxygen dissociation from the haemoglobin of Lepidosiren is shown, both in the presence and absence of 1 mM ATP. The pH dependence observed is indicative of a normal Bohr effect which is augmented appreciably by ATP. In the absence of ATP, k increases some 3-fold as the pH is lowered from between 8 and 9 6. In the presence of ATP this change is nearer to 10-fold. The absolute reaction rates are unremarkable, being more or less in the same range as those reported for mammalian haemoglobins (Fig. 4). The pH dependence of the reaction of carbon monoxide with deoxygenated Lepidosiren haemoglobin is also shown, again in the presence and absence of 1 mM ATP. This pH dependence is very
F~g. 4. The pH dependence of tile dissociation velocity constant of oxygen from oxy-haemoglobm from Lepido,stren puradoxa, showing the effect of stripped and unstripped preparations. The ATP concentration was I raM. Temperature, 20 C. unusual. In tile absence of ATP this dependence has two distract phases. Initially the rate of the reaction increases with increasing pH but at about pH 7.5 this dependence is reversed and the rate decreases sharply with further increases in pH. ATP modulates this dependence and has a significant effect on this reaction at unusually high pH values. Only at p H 9 is the effect insignificant. Thus we can conclude that there is preferential binding of ATP to the deoxystructural state of this haemoglobin molecule even at pH values between 8 and 9 (Fig. 5). It is at first surprising to observe such as ordinary pH and ATP dependcnce for the rate of oxygen dissociation while finding so extraordinary a dependence for the rate of carbon monoxide dissociation. However, it should be recalled that these two kinetic processes are controlled by very different factors in a cooperative haemoglobin molecule. This can be interpreted in terms of the two-state allosteric model (Monod et al., 1965). This proposes that subunits can assemble with two different, but equilibrated quaternary strt, ctures. The different bondings between subunits in these two structures alters the individual conformation of the subunit so that in one structure, designated R, the affinity of the subunits for ligands is higher than in the other, T, state. The binding of ligand inevitably shifts the equilibrium towards the R state, resulting in successive increases in affinity as ligation proceeds. One further postulate in this scheme is that within each quaternary structure the affinity of one st.bunit is not influenced by the ligand state of its neighbours. Thus with the present experiments the kinetic profile of oxygen dissociation while being influenced by the step in the deoxygenation process at which the protein converts from the R to the T state, is rate-limited primarily by the dissociation of oxygen from the high affinity, R state of the haemoglobin molecule. In contrast, the rate of CO combination reflects primarily the functional behaviour of thc deoxy or T state of the protein. The pH dependence of the rate constants suggest
CHARLFS PHI'LPS et al.
142
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I
•
"o.
( M . 1 sec_l) 4
I
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6
7 pH
8
9
Fig. 5. The pH dependence of the combination velocity constant between carbon monoxide and Lepulo.siren parado~a deoxyhaemoglobin The two traces show the parameter for stripped and for unstrlpped haemoglobins. The ATP concentration was 1 raM. Temperature, 20"C. that the functional properties of the deoxygenated derivative of Lepidosiren haemoglobin are sensitive to the state of ionization of a number of acid groups on the haemoglobin. GENERAL
DISCUSSION
The dipnoan hmg developed as an emergency organ in the African and South American lungfish (Protopterus, l_x,pidosiren, respectively) but pulmonary ventilation is now obligatory, as the gdls have only a vestigial function, surviving as osmoregulators. The Australian lungfish Neoceratodus is not an obligate air breather and dies out of water. One curious feature of the life style of Lepidosiren which contradicts its obligate aerobiosis, is indulged in by the male. During egg-guarding the males do not surface and it develops considerable respiratory filaments to its pelvic fins. These are first seen after mating and atrophy soon after egg hatching, when the larval gills degenerate and the yotmg begin to air breathe (Krogh, 1941). Johansen & Lenfant (1968) and Lenfant & Johansen (1968)studied the physiological adaptations involved in the transition to air breathing in bragfishes. Both the properties of the haemoglobin and the respiratory behavior of the animal suggest that Neoceratodus is a gill-breather and that the hmgs serve only an auxiliary function when the external pO2 is low. However, Lepidosiren and Protopterus use their lungs functionally, though they use their vestigial gills for much of the carbon dioxide elimination. It would be pleasing to generalize on the factors observed in transition from aquatic to air breathing, but such does not seem possible. From studies on the carbon dioxide sensitivity among teleost fish a suggestion has been made that sluggishly moving fish inhabiting stagnant waters tended to show small Bohr effects whilst active species in fast moving aerated water show large responses in oxygen affinity to small
changes in pC02. An instance of this latter is seen in the mackerel where the Bohr effect AIogP~o/ ApH = - 1 . 2 (Prosser, 1961). The values of this term for the lungfish reported by Lenfant & Johansen (1968) were: Neoceratodus - 0 . 6 2 ; Protopterus - 0 . 4 7 and Lepidosiren -0.24. Our value obtained for Lepidosiren is clearly dependent on the amount of organic phosphate bound. Thus while whole blood gave a Bohr effect of -0.31, reference to Fig. 3 reveals that for stripped haemoglobin in the pH range 7.0-8,0, the figure may be as low as -0.18, whilst in the presence of 1 mM ATP the value rises steeply to - 1.05. Oldham & Riggs (1969) report that the oxygen equilibrium for the two species of African lungfish is different, both in affinity and in Bohr response. Thus for Protopterus aethiopicus, caught in deep water lakes, the haemoglobin had a higher affinity for oxygen than Protopterus annectans, obtained from marshlands. Both fish have multicomponent haemoglobins, sigmoid oxygen equilibria, and cooperativity, expressed as n values, which are pH dependent. Haemoglobins from Protopterus aethiopicus had a Bohr effect of - 0 . 8 whilst that from Protopterus amwctans gave values of -0.54. It is not easy to support the suggestion of reduced Bohr response to stagnant water environments, and this is further revealed in the other group of animals, the Amphibia, subjected to study by Lenfant & Johansen (1967). Here the trend is reversed. Nectm'us has a Bohr effect of -0.13, Amphiuma - 0,21, and Rana catesheiana - 0.29.
Acknowledgements--This work was supported by Grant PCM-06451 from the National Science Foundation for studies aboard the R.V. Alpha Helix. We are grateful to the Brazdlans for their help and for making it possible for the R.V Alpha Helix to enter the upper Amazon We wish to thank Captain Clarke and the crew for their cooperation. Additional support was provided by the Royal Sooety and the Natural Environment Research Counol (C.F.P.). thc Norwegmn Research Councd for Socncc and
Oxygen and carbon monoxide binding to the haemoglobin of the South American lungfish the Humanities (H.J. and U.E.H.F.k NSF Grant DEB-7619877(D.A.P.), NIH Grant HL-15460 (to J Bonaventura for support of M.F.), NSF Grant PCM-76-06719 (to A. Riggs), thc National Geographic Society (D.A.P.) and NIH grant GM 21314 (to A. Riggs). M. Farmcr is a rcctpicnt of Duke University Research Award, no 303-3765.
REFERENCES
ALLEN D. W., GUTHE K. F. & WYMAN J. (1950) Further studies on the oxygen equilibrium of hemoglobin. J biol. Chem. 187, 393-410. BENESCH R., MACDUFF G. & BI'NESCH R. E. (1965) Determination of oxygen equilibria with a versatile new tonometer. Analyt. Biochem. I I, 81-87. FYHN U. E. H., FYHN H. J., DAVIS B. J., POWERS D. A., FINK W. L. & GARLICK R. L. (1979) Hemoglobin heterogeneity in Amazonian fishes. Comp. Biochem. Physiol. 62, 39-66. GIBSON Q. H. & M]LNES L. (1964) Apparatus for rapid and sensitive spectrophotometry. Biochem. J. 91, 161-171. 1MAJ K. (1974) Hemoglobin Chesapeake (92~t, arganine---, leucine). Precise measurements and analyses of oxygen equilibrium. J. biol. Chem. 249, 7607-7612.
143
JOHANSEN K. & LENFANT C. (1968) Respiration in the African lungfish If. Control of breathing. J. exp. Biol. 49, 453~.68. KROGH A. (1941) The Coolparative Physiology of Respiratory Mechanisms. University of Pennsylvania, Philadelphia. LENFANT C. & JOHANSEN K. (1967) Respiratory adaptations in selected amphibians. Resp. Physiol. 2, 247-260. LENFANT C. t~ JOHANSEN K. (1968) Respiration in the African lungfish Protopterus aethiopicus I. Respiratory properties of blood and normal patterns of breathing and gas exchange. J. exp. Biol. 49, 437-452. MONOl) J., WYMAN J. & CHANGEUX J.-P. (1965) On the nature of allosteric transitions: a plausible model. J. Molec. Biol. 12, 88 118. NAGEL R. L.. WITTENBERG J. B. & RANNEY H. M. (1965) Oxygen equilibria of the hemoglobin-haptoglobin complex. Biochem. biophys. Acta 100, 286-289. NOBEl- R. W., PARKHURST L. J. & GIBSON Q. H. (1970) The effect of pH on the reactions of oxygen and carbon monoxide with the hemoglobin of the carp, Cyprinus carpto. J. biol Chem. 245, 6628-6633. OLDHAM J. & RIGGS A. (1969) cited in A. Riggs. Properties of fish hemoglobins. In Fish Physiology, Vol. 4 (Edited by HOAR W. S. & RANDALL D. J.), pp. 245-246. Academic Press, New York (1970). RIGGS A. & WOLBACH R. A. (1956) Sulfhydryl groups and the structure of hemoglobin. J. yen. Physiol. 39, 585-605.
0300-96297911101-0139$0200/0
EQUILIBRIA AND KINETICS OF OXYGEN AND CARBON MONOXIDE BINDING TO THE HAEMOGLOBIN OF THE SOUTH AMERICAN LUNGFISH, LEPIDOSIREN PARADOXA* CHARLES PHELPS, J MARTHA FARMER,2 HANS J. FYHN,3 UNNI E. H. FYHN, 3 ROBERT L. GARLICK,4 ROBERT W. NOBLEs and DENNIS A. POWERS6 :Department of Biological Sciences, University of Lancaster, Lancaster LAI 4YQ, England; -'Duke University, Marine Laboratories, Beaufort, NC 28516, U.S.A.; 3Zoological Institute, University of Oslo, Blindern, Oslo 3, Norway; '*Department of Zoology, University of Texas at Austin, Austin, TX 78712, U.S.A.; 5Department of Medicine and Biochemistry, Veterans Administration Hospital, S.U.N.Y., Buffalo, NY 14215, U.S.A.; 6Department of Biology, Johns Hopkins University, Baltimore, MD 21218, U.S.A.
(Received 5 April 1978) Abstract--I. The haemoglobin of the South American lungfish Leptdosiren paradoxa has a single component. 2. The equilibria of this respiratory protein with oxygen have been investigated both in the blood and with the purified haemoglobin. There is a substantial, normal, alkahne Bohr effect and marked sensitivity to organic phosphates in the haemoglobin solutions. 3. Studies on the pH dependence of the kinetics of oxygen dissociation can be interpreted in terms of a normal Bohr effect. 4. The kinetics of combination of carbon monoxide have an unusual pH dependence. 5. These findings are discussed in terms of the two-state model of Monod et al. (1965)
INTRODUCTION The transition from water breathing to air breathing in fishes is a significant evolutionary step. Fish which show both marked structural adaptations, and sophisticated physiological modifications to this end are found mainly in tropical waters or estuaries. The South American lungfish, Lepidosiren paradoxa, is structurally the most advanced of airbreathing fishes. In this dipnoan, the air bladder has evolved into a paired lung, and internal septa subdivide the air space into successively smaller compartments ending in alveoli-like lobules, richly suffused with blood vessels. Whereas the structural and physiological adaptations are well documented, little information is available on the molecular characteristics of its haemoglobin. This paper sets out to document the oxygen equilibrium properties of the blood, and of the haemoglobin in both organic phosphatestripped and unstripped states and further, to describe some aspects of the kinetics of reaction of the haemoglobin with the ligands oxygen and carbon monoxide. METHODS AND MATERIALS Specimens of the South American lungfish (Lepidosiren paradoxa) were obtained from a local fisherman in November 1976 during an expedition with the R.V. Alpha * A Portuguese translation of this work will appear in ,'tCI~I A m ~ l Z O l l l c ( i .
139
Helix in an area about 30 miles upstream the Amazon River (Solim6es) from its junction with the Rio Negro. The collection period was at the end of the dry season when the water level was at its lowest in lakes and rivers. The animals (body weight 370-2000 g) were not aestivating when collected and were kept in aquaria for 2-6 days before being bled. Blood was obtained by cardiac puncture of ice-chilled animals and drawn into a cold heparinized glass syringe (100/d of sodium heparln (5000i.u./mll in 1.7",. NaCI/5 ml of blood) The cells were spun at 1000g in a refrigerated centrifuge (Sorvall Model RC2-B, I. Sorvail, Norwalk, CT) for 5min. The supernatant was removed and the cells washed 3 times using the same protocol by suspension in 1.7°o (w/v) NaCI containing 5 mM Tris buffer pH 8.0. The cells were then lysed by exposure to l mM Tris buffer pH8.3 at 4'C for 20min. Sodium chloride was added to a final concentration of 100mM, and the cell debris removed by centrifugation at 15,000g. Desalting was conducted on a column (110 x 1.5cm) of G-25 medium (Pharmacia Fine Chemicals, Uppsala) equilibrated in 1 mM Tris pH 8.3. "'Stripping" of haemoglobin solutions was performed on a column (15 × 2.5 cm) of mixed bed resin AG 501- × 8 (DI from Bio Rad Laboratories. Richmond, CA, on top of which were layered two separate 3 cm pads of ion exchangers viz Dowex-50W NH,~ and Dowex I-Acetate (Dow Chemical Co., MI). Flow rates of 1 fnl/min were maintained. Disc gel clectrophoresis at pH 8.9 were performed as described by Fyhn et al. (1979). From the same stained gels the ratio between the migration distance of the haemoglobin component and that of bovine serum albumin was calculated and used to effect comparisons of haemoglobin components in gels of different runs. These ratios are referred to as relative mobilities.
140
CHARLES PHELPS et al.
Oxygen equilibrium measurements were performed using glass tonometers as described by Allen et al. (1950) and Riggs & Wolbach (1956). The results were calculated as described by Nagel et al, (1965). All experiments were carried out at 20°C with haemoglobin concentrations approx 50-100/~M in haem equivalents. Oxygenation of the priorly deoxygenated solution was followed spectrophotometrically at two wavelengths, 560 and 576 nm. Gas additions were either air or pure oxygen. Equilibration times after each addition were 10-20min. Buffers used were all of ! = 0.05, with Bis-Tris in the range pH 5.25-7.3, and Tris for pH values above 7.3. Methaemoglobin formation was measured as described in Benesch et al. (1965) and was at all times less than 3~o. Measurements of oxygen equilibrium in the whole blood was performed using the Hem-O-Scan apparatus (American Instrument Co. Silver Spring, MD) described by Powers et al. (1979). Kinetic measurements were performed at 20~C with a stopped-flow rapid mixing apparatus similar to that originally described by Gibson & Milnes (1969). Solutions of haemoglobin were identical in composition to those used for equilibrium measurements. The kinetics of oxygen dissociation were measured by the pH-jump procedure as described by Noble et al. (1970). Oxygenated haemoglobin in 1 mM Tris pH 8.0 was mixed with a solution of dithionite in a buffer of desired pH, ! = 0.05. The final haemoglobin concentration was approx 30/~M in haem equivalents and the reaction was followed at both 560 and 540 nm independently. The kinetics of carbon monoxide combination to deoxygenated haemoglobins were measured by mixing solutions of deoxygenated haemoglobins in buffers of the desired pH of ionic strength I = 0.1, containing a known concentration, approx 85~uM, of carbon monoxide dissolved m water. After mixing, the haemoglobin concentration was ca. 3/aM in haem equivalents. The reaction was followed at 420 and 435 nm independently. RESULTS
AND
.~..
)
DISCUSSION
Studies on the whole blood of the animal reveal that the haemoglobin is half saturated at 8.3 mm Hg pO2 at 30°C and pH 8.06. O n re-equilibrating the blood sample in the presence of 5.6°'0 v/v CO2, the i
i
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1.0
Fig. 2. Electrophoresis in polyacrylamide gels of the haemoglobin from Lepidosu'en paradoxa (left) and from man (right). Bovine serum albumin is the fast running marker. *g
,,~
pH of the sample fell to 7.33 and the Pso increased to 1 4 m m Hg pO2 (Fig. 1). Thus at 30°C, there is evidence of a significant Bohr effect which may be c o m p o u n d e d of both H + and C O , effects, the apparent magnitude of which for the pH range 7.33-8.06 is:
°07 o°y
0.5
A log Pso A pH
0.31.
A thorough investigation of the oxygen equilibrium on the isolated haemoglobin was undertaken. Poly0 1 2 acrylamide gel electrophoresis (Fyhn et al. 1979) log pO2 Fig. 1. Oxygen equdibrium curves for whole blood from revealed a single component. The haemoglobin has Leptdosu'en paradoxa under the conditions specified. T h e - - a- mobil,ity of 0.66 + 0.01 relative to bovine serum ordinate relates to fractional saturauon of the haemoglobin albumin, and Fig. 2 compares the electrophoretic patby oxygen and the abscissa is scaled to log pO2 values terns obtained for haemolysates from Lepidosiren parexpressed in mm Hg. a d o x a with that of man. |
!
!
!
Oxygen and carbon monoxide binding to the haemoglobm of the South American lungfish
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Fig. 3. The pH dependence of the oxygen equilibria with haemoglobin from Lepidosiren paradoxa showing the effect of "stripped" and "unstripped'" preparations. The ATP concentration was I mM. The pH dependence of the oxygen dissociation curve was measured for material that had been stripped of organic phosphates and for identical solutions to which had been added ATP to a final concentration of I mM (Fig. 3). The two curves are broadly similar except that the presence of organic phosphate has greatly decreased the affinity of the haemoglobin, as would be expected if it stabilized the deoxy conformer. The magnitude of the Bohr effect is much greater in the presence of l mM ATP and its response to pH across the pH zone from 7.00 to 8.00 is 5 times more acute in the presence of organic phosphate (Fig. 3). The equilibrium data reflect the behaviour of a haemoglobin not greatly different in response from that of human haemoglobin A. The magnitude of the Bohr effect in haemoglobin A at 25r'c in similar buffer systems is - 0 . 3 0 between pH 6.8 and 8.8, and the effect of excess organic phosphate decreases the oxygen affinity by a factor of 10 (lmai, 1974). It seems clear that whatever molecular adaptations in haemoglobins are required in the course of the water to air transition, they are complete at the level of evolution of the lungfish. The pH dependence of the rate of oxygen dissociation from the haemoglobin of Lepidosiren is shown, both in the presence and absence of 1 mM ATP. The pH dependence observed is indicative of a normal Bohr effect which is augmented appreciably by ATP. In the absence of ATP, k increases some 3-fold as the pH is lowered from between 8 and 9 6. In the presence of ATP this change is nearer to 10-fold. The absolute reaction rates are unremarkable, being more or less in the same range as those reported for mammalian haemoglobins (Fig. 4). The pH dependence of the reaction of carbon monoxide with deoxygenated Lepidosiren haemoglobin is also shown, again in the presence and absence of 1 mM ATP. This pH dependence is very
F~g. 4. The pH dependence of tile dissociation velocity constant of oxygen from oxy-haemoglobm from Lepido,stren puradoxa, showing the effect of stripped and unstripped preparations. The ATP concentration was I raM. Temperature, 20 C. unusual. In tile absence of ATP this dependence has two distract phases. Initially the rate of the reaction increases with increasing pH but at about pH 7.5 this dependence is reversed and the rate decreases sharply with further increases in pH. ATP modulates this dependence and has a significant effect on this reaction at unusually high pH values. Only at p H 9 is the effect insignificant. Thus we can conclude that there is preferential binding of ATP to the deoxystructural state of this haemoglobin molecule even at pH values between 8 and 9 (Fig. 5). It is at first surprising to observe such as ordinary pH and ATP dependcnce for the rate of oxygen dissociation while finding so extraordinary a dependence for the rate of carbon monoxide dissociation. However, it should be recalled that these two kinetic processes are controlled by very different factors in a cooperative haemoglobin molecule. This can be interpreted in terms of the two-state allosteric model (Monod et al., 1965). This proposes that subunits can assemble with two different, but equilibrated quaternary strt, ctures. The different bondings between subunits in these two structures alters the individual conformation of the subunit so that in one structure, designated R, the affinity of the subunits for ligands is higher than in the other, T, state. The binding of ligand inevitably shifts the equilibrium towards the R state, resulting in successive increases in affinity as ligation proceeds. One further postulate in this scheme is that within each quaternary structure the affinity of one st.bunit is not influenced by the ligand state of its neighbours. Thus with the present experiments the kinetic profile of oxygen dissociation while being influenced by the step in the deoxygenation process at which the protein converts from the R to the T state, is rate-limited primarily by the dissociation of oxygen from the high affinity, R state of the haemoglobin molecule. In contrast, the rate of CO combination reflects primarily the functional behaviour of thc deoxy or T state of the protein. The pH dependence of the rate constants suggest
CHARLFS PHI'LPS et al.
142
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Fig. 5. The pH dependence of the combination velocity constant between carbon monoxide and Lepulo.siren parado~a deoxyhaemoglobin The two traces show the parameter for stripped and for unstrlpped haemoglobins. The ATP concentration was 1 raM. Temperature, 20"C. that the functional properties of the deoxygenated derivative of Lepidosiren haemoglobin are sensitive to the state of ionization of a number of acid groups on the haemoglobin. GENERAL
DISCUSSION
The dipnoan hmg developed as an emergency organ in the African and South American lungfish (Protopterus, l_x,pidosiren, respectively) but pulmonary ventilation is now obligatory, as the gdls have only a vestigial function, surviving as osmoregulators. The Australian lungfish Neoceratodus is not an obligate air breather and dies out of water. One curious feature of the life style of Lepidosiren which contradicts its obligate aerobiosis, is indulged in by the male. During egg-guarding the males do not surface and it develops considerable respiratory filaments to its pelvic fins. These are first seen after mating and atrophy soon after egg hatching, when the larval gills degenerate and the yotmg begin to air breathe (Krogh, 1941). Johansen & Lenfant (1968) and Lenfant & Johansen (1968)studied the physiological adaptations involved in the transition to air breathing in bragfishes. Both the properties of the haemoglobin and the respiratory behavior of the animal suggest that Neoceratodus is a gill-breather and that the hmgs serve only an auxiliary function when the external pO2 is low. However, Lepidosiren and Protopterus use their lungs functionally, though they use their vestigial gills for much of the carbon dioxide elimination. It would be pleasing to generalize on the factors observed in transition from aquatic to air breathing, but such does not seem possible. From studies on the carbon dioxide sensitivity among teleost fish a suggestion has been made that sluggishly moving fish inhabiting stagnant waters tended to show small Bohr effects whilst active species in fast moving aerated water show large responses in oxygen affinity to small
changes in pC02. An instance of this latter is seen in the mackerel where the Bohr effect AIogP~o/ ApH = - 1 . 2 (Prosser, 1961). The values of this term for the lungfish reported by Lenfant & Johansen (1968) were: Neoceratodus - 0 . 6 2 ; Protopterus - 0 . 4 7 and Lepidosiren -0.24. Our value obtained for Lepidosiren is clearly dependent on the amount of organic phosphate bound. Thus while whole blood gave a Bohr effect of -0.31, reference to Fig. 3 reveals that for stripped haemoglobin in the pH range 7.0-8,0, the figure may be as low as -0.18, whilst in the presence of 1 mM ATP the value rises steeply to - 1.05. Oldham & Riggs (1969) report that the oxygen equilibrium for the two species of African lungfish is different, both in affinity and in Bohr response. Thus for Protopterus aethiopicus, caught in deep water lakes, the haemoglobin had a higher affinity for oxygen than Protopterus annectans, obtained from marshlands. Both fish have multicomponent haemoglobins, sigmoid oxygen equilibria, and cooperativity, expressed as n values, which are pH dependent. Haemoglobins from Protopterus aethiopicus had a Bohr effect of - 0 . 8 whilst that from Protopterus amwctans gave values of -0.54. It is not easy to support the suggestion of reduced Bohr response to stagnant water environments, and this is further revealed in the other group of animals, the Amphibia, subjected to study by Lenfant & Johansen (1967). Here the trend is reversed. Nectm'us has a Bohr effect of -0.13, Amphiuma - 0,21, and Rana catesheiana - 0.29.
Acknowledgements--This work was supported by Grant PCM-06451 from the National Science Foundation for studies aboard the R.V. Alpha Helix. We are grateful to the Brazdlans for their help and for making it possible for the R.V Alpha Helix to enter the upper Amazon We wish to thank Captain Clarke and the crew for their cooperation. Additional support was provided by the Royal Sooety and the Natural Environment Research Counol (C.F.P.). thc Norwegmn Research Councd for Socncc and
Oxygen and carbon monoxide binding to the haemoglobin of the South American lungfish the Humanities (H.J. and U.E.H.F.k NSF Grant DEB-7619877(D.A.P.), NIH Grant HL-15460 (to J Bonaventura for support of M.F.), NSF Grant PCM-76-06719 (to A. Riggs), thc National Geographic Society (D.A.P.) and NIH grant GM 21314 (to A. Riggs). M. Farmcr is a rcctpicnt of Duke University Research Award, no 303-3765.
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JOHANSEN K. & LENFANT C. (1968) Respiration in the African lungfish If. Control of breathing. J. exp. Biol. 49, 453~.68. KROGH A. (1941) The Coolparative Physiology of Respiratory Mechanisms. University of Pennsylvania, Philadelphia. LENFANT C. & JOHANSEN K. (1967) Respiratory adaptations in selected amphibians. Resp. Physiol. 2, 247-260. LENFANT C. t~ JOHANSEN K. (1968) Respiration in the African lungfish Protopterus aethiopicus I. Respiratory properties of blood and normal patterns of breathing and gas exchange. J. exp. Biol. 49, 437-452. MONOl) J., WYMAN J. & CHANGEUX J.-P. (1965) On the nature of allosteric transitions: a plausible model. J. Molec. Biol. 12, 88 118. NAGEL R. L.. WITTENBERG J. B. & RANNEY H. M. (1965) Oxygen equilibria of the hemoglobin-haptoglobin complex. Biochem. biophys. Acta 100, 286-289. NOBEl- R. W., PARKHURST L. J. & GIBSON Q. H. (1970) The effect of pH on the reactions of oxygen and carbon monoxide with the hemoglobin of the carp, Cyprinus carpto. J. biol Chem. 245, 6628-6633. OLDHAM J. & RIGGS A. (1969) cited in A. Riggs. Properties of fish hemoglobins. In Fish Physiology, Vol. 4 (Edited by HOAR W. S. & RANDALL D. J.), pp. 245-246. Academic Press, New York (1970). RIGGS A. & WOLBACH R. A. (1956) Sulfhydryl groups and the structure of hemoglobin. J. yen. Physiol. 39, 585-605.