Studies of the functional properties of the hemoglobins ofOsteoglossum bicirrhosum andArapaima gigas

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S T U D I E S O F THE F U N C T I O N A L P R O P E R T I E S O F THE H E M O G L O B I N S O F OSTEOGLOSSUM B I C I R R H O S U M A N D A R A P A I M A GIGAS* MARIA ISABEL GALDAMES-PORTUS, ~ ROBERT W. NOBLE, 2

MARTHA FARMER,3 DENNIS A. POWERS,4 AUSTEN RIGGS, s MAURIZIO BRUNORI,~' HANS J. FYHN7 and UNNt E. H. FYHN7 Jlnstituto Nacional de Pesquisas da Amazonia, Ca~xa Postal 478 69000 Manaus, Amazonas, Brazil -'Departments of Medicine and Biochemistry, SUNY at Buffalo, Veterans Administration Hospital. Buffalo, NY 14215, U.SA.: 3Duke Umversity Marine Laboratory, Beaufort, NC 28516, U.S.A.: '~Department of Biology, Johns Hopkins Umverslty, Baltimore, MD 21218, U.S.A., ~Department of Zoology, University of Texas, Austm, TX 78712, U.S.A.: t'CNR Centre for Molecular Biology, Institutes of Chemistry and Biochemistry, Faculty of Medicine, UmversJty of Rome, Rome, Italy 71nstitute of Zoophysiology, Umversity of Oslo, P.O. Box 1051, Bhndern, Oslo 3, Norway Abstract--I. The effects of pH and organic phosphate on the equilibrmm and kinetic properties of the binding of hgands to the hemoglobins of two related Amazonian fish, Osteoglossum hwirrhosum, an obligate water breather, and .4rapamuz ,qigas, an obligate air breather, have been studied. 2. The hemoglobms of both fish exhibit a Root effect, and the minimum oxygen affinity is assocmted with the complete loss of cooperative ligand binding 3. In this low affimty, T state, there Js great subun~t heterogeneity in both hemoglobins as evidenced by Hill coefficients well below unity and biphasic carbon monoxide combination kinetics. 4. The greatest difference m the propertics of the hemoglobins of these two fish is found at high pH (above pH 8) where both hcmoglobins exhibit their highest ligand affimt~es. Here the hemoglobins differ both in the affinity and cooperatively with which they bind hgands. Arupaima hemoglobin having the lowest affimty but the highest coopcratlvity.

INTRODUCTION

note is the marked subunit heterogeneity which they display, especmlly m the kinetics of their reactions with carbon monoxide.

Osteoglossum hicirrhosum and Arapaima gigas are members of the Osteoglossidae which arc found in large numbers in the waters of the Amazon River system. The hemoglobins of these fish are of considerable interest. First, none of the hemoglobins from this primitive family of fish have been subjected to detailed functional studies. Second, these fish, though closely related, obtain oxygen from different sources. Osteoylossum is an obligate water breather with welldeveloped gills. Arapaima, on the other hand, is an obligate air breather. Thus a comparison of the properties of the hemoglobins from these two species might reveal some molecular adaptations to their different respiratory mechanisms. Finally, the hemoglobins of these fish are particularly convenient to study because they are few in number. O.steo,qlos.sum possesses a single hemoglobin while the prevalent phenotype of Arapaima has two which appear to laave~ very similar functional properties. The equilibrium and kinetics of the reactions of Osteoylossum and Arapaima hemoglobins with various ligands have been examined. The hemoglobins of both species display Root effects, i.e. their oxygen affinities are so low at pH wdues below 6.5 that they fail to be saturated with oxygen even when equilibrated with air. In this pH range the hemoglobins have very similar properties Of particular * A Portuguesc translation of this work wdl appear m ,-I c l o

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MATERIALS

Speomens of Osteoglos.~um htcirrhosum were collected m November and December 1976 during an expedition with R V Alpha Hehv m an area about 30 miles upstream on the Amazon River (Rio Sohm6es) from its .lunction with Rio Negro. The animals (body wetght 147-2500g) were procured by beach seme and gill nets at the outlet of Lago JanauacS, and m an isolated pond which was located 1.5 redes up the crcek between Rio Solim6es and Lago Janauacfi and about 500m to the west of the creek. The collccl~on pcnod was at the end of the dry season when the water level was at its lowest m lakes and rivers. The outlet of Lago Janauacti was a slowly flowing stream of murky water with sloping, muddy rtver banks. The water Icmpcraturc was 28 30 C and the pH varied from 6.5 to 7.0 (Fisher, 1978). The pond, which was crowded with fish, was about 40 × 60m m size with a depth of 20-40cm. It was partly covered by water plants and the bottom was extremely soft and muddy. The stagnant, murky water had a tempcraturc of about 30"C. and was almost anoxic The ammals wcrc usually kept in running water for only a few hours bcforc bcmg bled. Spcomens of Arapaima gtqas were obtained from a local lisherman during the same expedition. These animals, whose body weight varied from 1.5 to 3 kg, were kept in running water for varying periods of time (a few hours to scveral days) bcfore being bled.

145

MARIA ISABELGALDAMES-PORTLISeta[.

146 METHODS

Blood was obtamed from these fish by percutaneous cardiac puncture or from the caudal veto and drawn mto a cold, heparlnized glass syringe 1-100ttl of sodium heparme (500i,u./ml) in 1.7°o NaCl/5ml of blood]. Hemolysate preparation, regular disc gel electrophoresis (pH --- 8.9), and sodium dodecyl sulphate (SDS) gel electrophoresis were carried out as described by Fyhn et al. (1978). Hematocnt was measured on freshly drawn, carefully mixed blood using micro hematocrit tubes. From regular disc gels the ratio between the migration distance of a hemoglobin component and the migration distance of bovine serum albumin on the same gel was calculated and used to compare the mobility of hemoglobin components in gels of different runs. These ratios are referred to as relative mobilities. Oxygen affinities of whole blood were determmed w~th an Aminco Hem-O-Scan Apparatus. The CO., content of the gas phase was varied in order to vary the pH of the sample. These measurements were carried out immediately after obtaining a blood sample in order to minimize the metabolic alteration of the levels of organic phosphates in the cells. The pHs of blood samples equdlbrated at different CO2 tensions were determined as described by Powers et al. (1978). Prior to hemolysis the red cells were washed 3 times in 10 volumes of isotonic saline. They were then lysed by addition of approx 4 volumes of I mM Trls, pH 8. Lysis was permitted to proceed for 30 min at 4~C. The hemolysate was then made 0.1 M m NaCI and centrifuged at 28,000g for 15 min at 4°C. The hemoglobin was stripped of organic phosphate by passage first through a G-25 Sephadex column equdibrated with 1 mM TrJs, pH 8 and then through a Dintzls type delomzing column as described by Garlick et al. (1978). For all experiments on hemoglobin solutions bis-Tns and Tns buffers of tonic strengths 0.05 were used. The effect of ATP was assessed by makmg solutions 1 mM m this reagent. Oxygen equilibria were measured by the method of Allen et al. (1950) and Riggs& Wolbach (1956). Deoxygenation was accomphshed by equilibrating the hemoglobin solution with oxygen-free argon in a tonometer with an attached I cm pathlength cuvette. When affimtics were so low that complete oxygenation could not be achieved with atmospheric oxygen, the spectrum of the flllly oxygenated hemoglobin was obtamed by addmg sohd Tris to raise the pH of the solution. In all these experiments I mM EDTA was added in order to control the formation of methemoglobin in the course of the measurements. All kinetic measurements were performed with a stopped-flow apparatus of the type origmally described by Gibson & Milnes (1964). In all cases, the ionic strength of the final solution after mixing was 0.05 and when ATP was used its concentration after mixmg was I mM. The kinetic constants presented are the least squares fits to the first 65"/~ of the observed reactions. A number of the kmetic processes examined wcrc btphasic. When the rates of the two phases differed by a factor of 10 or more, the two processes could be separated graphically with no difficulty and the ratc constants computed separately. As the magnitude of the rate difference decreases this procedure becomes more difficult and uncertam, and computer techniques involving curve-fitting routines were used. In addition, when the results of the graphed procedures were spot checked by computer curve fitting, no significant inconsistencies were found. The kinetics of oxygen dissociation were measured by the pH jump procedure as described by Noble et al. (1970} Oxygenated hemoglobin in l mM Tris, pH 8, was mixed with an equal volume of a solution of duhiomte in a 0.1 ionic strength buffer of the desired pH. The final hemoglobin concentration was approx 30 llM in hemc cquival-

ents and the reaction was followed at 560 and 540nm. The kinetics of carbon monoxide, CO, combination to deoxygenated hemoglobin were measured by mixing solutions of deoxygenated hemoglobin in 0.1 ionic strength buffers of the desired pH with a solution containing a known concentration, approx 85tiM, of CO dissolved in water. After mixing, the hemoglobin concentration was approx 3/IM in heme equivalents. The reaction was followed at 420 and 435 nm. Flash photolysis of the CO derivatives of these hemoglobins was accomplished with an Ascorlight 444 flash unit capable of dissipating 200 joules in approximately 0.5 msec. The anaerobic hemoglobin solution, approx 20/~M in heme equivalents and contamlng a known concentration of CO, was placed m a water jacketed 1 cm path cylindrical cell Water at 20cC was constantly circulated through the jacket. The monitoring light was collimated and passed through the sample before entering the monochromator. The flash tube was immersed in a concentrated solution of sodium nitrite. By then monitoring the reaction at 365 nm, a wavelength which is strongly absorbed by nitrite, the photocell was effectively shielded from the output of the flash tube. The 200 joule flash was capable of fully dissociating the CO from a hemoglobm sample m this system. Partml photolysls was achieved by lowering the flash energy and partially shielding the flash tube with aluminum foil.

RESULTS Regular disc gel electrophoresis showed the hemolysates from Osteoylossum to contain one hemoglobin component (Fig. 1). No difference was found between animals from the two localities. The relative mobility of the component was 0.28 compared to bowne serum albumin. Human HbA had a relative mobdity of 0.66. After 6 weeks of storage at 5~C the electrophoretic pattern of Osteoglossum hemolysates was unchanged. A single component was also observed by isoelectric focusing (see Bunn & R i g g s , 1979). By SDS electrophoresis the molecular weight of Osteog/ossum denatured hemoglobin chains was found to be similar to the molecular weight of human denatured hemoglobin chains (14,900 vs 14,600). The hematocrit of Osteoglossum was 27 ___ 6°o (Mean + S.D., N = 4). Regular disc gel electrophoresis showed the hemolysates Irom Arapaima to contain one major component with a relative mobility of 0.41 and a minor, more anodal component (relative mobility 0.59} (Fig. 1). The major and the minor components represented 65 and 35"o, respectively, of the total hemoglobin. One of the hemolysates showed only the major component when tested by gel electrophoresis immediately after hemolysate preparation, but when tested 24 hr later, the m m o r component was present and comprised 37°,, of the hemoglobin. The minor component had not increased further in percentage when the hemolysate was rechecked 3 weeks later. Two other hemolysates were first tested about 24hr after preparation and both hemolysates then contained the minor component. A second phenotype was found in one individual by Fyhn et al. (1978) but was not used in the present study. By SDS gel electrophoresis the molecular weight of Arapamla denatured hemoglobin chains was found to be 14,500. Hematocrit values of three specimens of .4rapaima were 25, 34 and 45°,,.

Functional properties of hemoglobins

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Fig. I. Polyacrylamlde disc gel electrophoretic patterns for the hemoglobins of Osteoglos.sum and Arapamu~. The leading band m each gel is bowne serum albumin which was added as a standard. The samples in the separate gels were as follows: (a) Osteo.qh~.ssumred cell hemolysate, (b) human hemolysate, (c) the pattern obtained from one hemolysate of Arapaima blood that was electrophoresed immediately after hemolysis (see text), (d) the usual pattern observed wtth Arapaima hemolysates, (e) human hemolysate.

Oxygen binding to whole blood The binding of oxygen to the whole blood of Osteoglossum and Arapaima was measured at 30°C as a function of pH as controlled by the CO2 partial pressure (see Fig. 2). The results appear in Fig. 3 where log P5o is plotted as a function of pH. The affinity for both bloods is strongly pH dependent:

Alog Pso/ApH = - 0 . 6 between pH 6.9 and 7.8. Measurements below pH 6.9 for Osteoglossum and pH 7.3 for Arapaima are not reported since it was unlikely that oxygenation was complete at the highest oxygen tension available.

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Fig. 3. The effect of pH on the oxygen affinities of the bloods of Osteoglossum, O, and Arapaima, A, at 30°C. The logarithm of the oxygen pressure required for half saturation, log Pso (02) is plotted vs pH. The pH was controlled by the partial pressure of COz.

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The apparent cooperatmvity of this bmding reaction, as indicated by the Hill cocfficient n, is also pH dependent. At low pH, where the affinity is at a minimum, the value of Jmis approx 0.5, indicating a markedly heterogeneous set of binding sites. As the pH is increased this coefficient increases and reaches a 0 L "l--iCt J maximum value of 2 when the log Pso (02) has decreased to 0.60. The coefficient then decreases as the pH is ramsed further and levels off at a value of 1.0 and is more or less pH independent above pH 8. 20 Addition of 1 m M A T P again shifts the curve to the J right increasing the pH at whmch n ms maximal from 151 6.9 to 7.4. oJ Similar measurements were earned out on the un\ fractionated hemoglobin of Arapaima and the rcsults t) • I0 ,\ \ \ are shown in Fig. 5. Here again we find that the Hill CL~ L! • coefficient, n, and the Pso are pH dependent. At low \ pH this hemoglobin behaves much the same as that of Osteoglossum. The value of log P.~o is about 2 and ', mk the Hill coefficient is below unity, again indicating X heterogeneity in the oxygen binding sites. As the pH is raised there is a sharp increase m oxygen affinity and in the Hill coefficient until log Pso reaches a 6 m i n i m u m of 0.75 and Jmequals approx 2. The reaction pH is relatively insensitive to pH between pH 7.2 and Fig 4 The elTcct of pH on the oxygen allimty of Osteoglo~- 8.8. In the higher pH range the properties of Ara.sum hemoglobin m the presence, m. and absence. [~. of paima hemoglobin are quite unlike those of OsteoglosATP at 20 C In the lowel part of the figure, tile logarithm sum hemoglobin. The oxygen affinities differ by about of the oxygen prc.,,~urc rcqtured for half-saturation is plot10-fold and the lower affimty Arapatma hemoglobin ted as a funct2on of pH. In the upper part of the figure. has a much more cooperative oxygen binding reacthe value of J~ m the Hill equation is plotted as a function tion. Addition of 1 m M A T P has no significant effect of pH on the oxygen affinity of Arapatma hemoglobin under the conditions of these experiments. As will be seen, it does affect reaction kinetics and it ms likely that O\vgen hmdiml eqmhhria o/ hemo~llohin .solutions the insensiuvity of the equilibrium results to this The oxygeq affiqity of Osteo.qlo.s.sum hemoglobin " allosteric effector is related to the presence of 1 m M was measured as a funct)on of pH m the presence EDTA in the buffers (see Methods). and absence of I mM ATP. The results appear m Fig. 4. The abscmssa of this graph ms pH, the ordinate of the lower portion of the figure ms log Pso (Oz) whmle that of the upper portion ms the Hml[ coefficient. G n. The affinity is strongly pH dependent. In the absence of A T P the log Ps0 (0_4 varies from a low of 0.5 mm at pH 7.6 to a h~gh of about 90 mm below pH 6. To a first approximation the addttmon of 1 mM ATP shifts tfils curve to the right increasing the pH at ti~e naidpomt of the affimty transition from 6.8 to 7 3. Tiffs shift to the rmght of 0.5 pH units which I mM ATP produces is exactly the same as that found by .r Gdlen & R~ggs (1977) m their studies of five other m--~m water-breathing teleosts. It ms smaller than the similar shift observed earlier by Gdlcn & Riggs (1972) and Tan et aL (19731 flu carp hemoglobin. ATP has little or no cfl'ect bclow pH 6 or above pH 8. In the mmddle pH range ~ts cffect decreases with increasing pH with the result that mt makes the pH transmhon steeper. Above pH 7 6 stripped Osteo.qh).s.smn hemoglobin dinsplays a small, but clearly defined reverse Bohr effect. 6 -"? 8 9 Scveral hemoglobins have no~ bccn observcd to have pH similar reverse Bohr effects at alkahnc pH. and it Fig 5 The effect of pH on the oxygen atthllty of Arupaima ramses the queshon of whether this mlgfit invoh'e the hemoglobin in the presence, m, and absence, ZI, of ATP same mechanism as the m'eversc Bohr effects exhibited at 20 C 113 tile Io~er part "bt" the figure, tile Iogartthm by eel or tadpole hemoglobins (Gtllen & Riggs. 1973: of the oxygen pressure rcqturcd for half-saturatson ts Watt & Riggs, 1975). It is, of course, more evident plotted asa functlonofpH Inthc upper part of the figure. m the latter hemoglobins bccause of the lack of a the value oft1 m tile Hill equation L,. plotted as a function normal Bohr effect of pH

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Fng. 6. The effect of pH on the rate of oxygen dissociation, k, from O.steo.qlosstml hemoglobin at 20 C in the presence. B, and absence, E], of ATP. The first order rate constant for this reaction is plotted on a logaruthmic scale as a function of pH.

The kinetics o.f oxygen dissociation The rate of oxygen dissociation from Osteoglossum hemoglobin was measured as a function of pH and the results appear ,n Fig. 6. This rate constant increases approx 10-fold as the pH is lowered from 8 to about 6. The rate constant is independent of pH below pH 6 and has a value of 130/see. Addition of 1 m M A T P has no effect on the rate in the plateau region although it extends this region by increasing the rate at pH 6.1. Some effect is seen at pH 7.1 but A T P is again without apparent effect above pH 8. The pH dependence of the rate of oxygen dissociation from Arapaima hemoglobin was measured at both 20 and 30~'C, as shown in Fig. 7. At 20°C, the rate increases almost 20-fold from pH 8 to 5.5. Two data points suggest a real effect of addition of I m M A T P and this is confirmed by the 30"C data. When we compare these properties to those of Osteoylossum hemoglobin we find a rather similar pH dependence

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although the total effect is slightly less for Arapaima hemoglobin. However, the absolute rate constants for the two hemoglobins differ substantially, the minimum rate constant being 14 sec for Osteoylossum and about 25 sec for Arapaima while the maximum rates are 130 and 500/sec, respectively. Increasing the temperature from 20 to 30°C increases the rate of this reaction but has little or no effect on the pH dependence of this rate constant for Arapaima hemoglobin. The magnitude of the per cent increase in rate seems somewhat greater at higher pH than at low. However, we must view rate constants greater than 5 0 0 s e c - I with some circumspection since the 1-1.5 msec dead time of the stopped flow apparatus permits one to observe only a fraction of a kinetic process with a rate constant this high.

The kinetics oJ carbon monoxide combination The kinetics of their reactions with C O are probably the most interesting and informative functional properties of these hemoglobins. The reactions are heterogeneous over much of the pH range examined and are composed of two distinct phases each of which contributes 50 °g of the total absorbance change associated with the reaction. The pH dependences of these reaction rate constants for the hemoglobin of Osteoylossum are shown in Fig. 8. Below pH 6.3 in the absence of ATP, the rates of the two phases differ by about 30-fold and show relatively little pH dependence. As the pH is increased the rate of the slow phase increases steeply until at pH 7.2 the reaction appears to be homogeneous and can be described by a single rate constant. The rate of the fast phase varies rather little with pH, even when it becomes the only reaction rate, being 2 x 10S/M.scc at pH 5.4 and decreasing to 1.2 x 10 s at pH 7.2. Add,Iron of I m M A T P increased the pK of the transition from biphasic to monophasic kinetics, but had no significant effect on the extremes of the two rate constants. The pH dependencies of the rates of reaction of carbon monoxide with Arapaima hemoglobin are

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Fig. 9. Tile effect of pH on the rate of carbon monoxide combination, I'. at 20 C to Arapamla hemoglobin in tile presence, II, and absence. ~, of 1 mM ATE The second order rate constant for the reaction is plotted on a logarithmic scale as a function of pH. Where two values arc given at a single pH, these represent the constants associated with the separate phases of a blphasic reaction shown in Fig. 9. The pattern is remarkably similar to that found for Osteoqlossum hemoglobin. Below pH 6.5, in the absence of A T E the reaction is composed of two phases whose rate constants differ by some 10-fold, while above pH 7 there is a single phase with a rate that appears to be an extrapolation of the pH dependence of the rate of tile fast phase that occurs below pH 6.5. Again, the addition of I m M A T P increases the pK of the transition from biphasic to monophasic kinetics, but has little or no effect on the values of the maximum and minimum rates.

Flash photolysis studies on the C O combination reaction It has been reported for a n u m b e r of fish hcmo-

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globins that at low pH, where the oxygen affinity is at a minimunl, liganded binding is no longer cooperative (Tan et al., 1973; Lau et aL, 1975). This has been interpreted as resulting from a shift in the conformational equilibrium such that the molecule remains predominantly in the low affinity, T conformation even when saturated with ligand. Equilibrium data cannot establish the absence of cooperativity in ligand binding, since a reduction in the Hill coefficient can result from either a heterogeneity in the properties of the binding sites or reduction in cooperativity. This is particularly evident in the case of Osteoglossum and Arapaima hemoglobins for which the value of n is well below unity at low pH, and there is evidence for subunit heterogeneity in the C O combination kinetics. C a r b o n monoxide combination kinetics offers an excellent assay for cooperativity in ligand binding to hemoglobins. The high and low affinity states of a hemoglobin react with C O at different rates, the high affinity structure reacting faster than the low. If ligand binding induced a significant alteration in the conformational equilibrium, then a partially liganded molecule should combine more rapidly with ligand than a fully deoxygenated one. This can be tested with flash photolysis techniques by taking advantage of the photosensitivity of the C O derivative of hemoglobin. By varying the flash intensity various degrees of photolysis can be produced, yielding anything from 0 to 100",, deoxygenated hemoglobin. In our experiments the rates of recombination of C O after full and approx 20°0 photolysis were compared at several pH values. The results obtained for Osteoqlossum hemoglobin are shown in Fig. 10. At pH 6.65 in the presence of 1 m M A T P the kinetics of recombination after full and after partial flash photolysis are identical. The data are plotted on two time scales in order to

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Functional properties of hemoglobins I00

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demonstrate that this is truc for both phases of the reaction. At pH 7.06 in the absence of ATP the situation is quite different. Following full photolysis, CO recombines not in a biphasic reaction but autocatalytically. This alone is kinetic evidence of cooperativity. Furthermore, the rate of recombination of CO after partial photolysis is much faster. As the pH is increased to 8.2 and then to 9.2 the magnitude of the rate difference decreases, but recombination after partlal photolysis is always the more rapid process. Similar results were obtained for Arapaima hemoglobin. The data obtained at pH 6.6 in the presence .of 1 m M ATP and at pH 8.9 are presented in Fig. 11. .At the latter pH the cooperativity, as indicated by the rate difference, is strong: but at pH 6.6 in the presence of ATP it is absent.

Fractionation of the two components of Arapaima hemoglohin The two components of Arapaima hemoglobin were separated by isoelectric focusing using essentially the same procedure described by Weber et al. (1976) for the separation of the hemoglobins of Amia calva. An LKB II0 ml preparative isoelectric focusing column was used at 4"C with an ampholine mixture which produced a pH gradient from pH 5 to 8. After elution of the components from the column, they were dialyzed vs I mM Tris, 0.5 mM EDTA, pH 8. in order to establish whether or not the kinetic heterogeneity of the reaction of Arapaima hemoglobin with carbon monoxide at low pH resulted, even in part, from a functional difference between these two components, the kinetics of CO combination to the purified components following rapid mixmg in the stopped-flow apparatus was examined at pH 6.4. Precisely identical btphasic kinetncs were observed which were also the same as those found for the unfrachonated hemoglobin.

EfJk'cts on CO binding kinetics of cross-linking Arapaima hemoglohin w~th suherimidate Cooperativity in ligand binding to hemoglobin results from a hgand hnked transition m the quaternary structure of the molecule. Cross-linking the molecule with a bifunctional reagent can be expected to perturb such conformational transitions and thus to alter the functional properties of the molecule. Furthermore, since the reactivity of various groups on the hemoglobin molecule, as well as intergroup distances, vary with conformattonal state, the nature and therefore the effects of the reaction with such a reagent may be different when it reacts with oxygenated and with deoxygenated hemoglobin under conditions where ligand bindmg is cooperative. In order to explore this, Arapam~a hemoglobin was reacted with dimethyl subenmMate. For reaction wnth the oxygenated derivatwe 1 ml of denonized Arapaima hemoglobin (60 mg) was added to 3 ml of 0.125 M triethanolamine-HCl, pH 8.1. Dimethyl suberimidate (40 mg: Pierce Chemical Co.) was then added and the reaction allowed to proceed at 0 ' C for 3½hr. Unreacted dimethyl subenm~date was then quenched by the addition of 0.5 ml of 2 M NH,,CI. The reaction mixture was then dialysed vs 0.5 mM Tris, pH 8.5. Reaction of the dimethyl suberimtdate with deoxygenated Arapaima hemoglobin was achieved with an ndentical procedure except that the hemoglobintriethanolamine solution was deoxygenated by bubbling with argon and by adding a small amount of dithionite prior to the addition of the dimethyl suberimidate and again immediately following the addition. The kinetics of the reaction of carbon monoxide with the two chemically modified hemoglobin preparations as well as the unmodified hemoglobin were examined at pHs 7.8 and 5.6-5.8. The pseudo first order kinetic plots appear in Fig. 12. The chemical modification does not produce a large effect on the kinetics of CO combination at pH 7.8. The material modified in the oxygenated state reacts somewhat

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Fig. 12. Psuedo first order kinetic plots for the reaction at pH 5.6-5.8, open st.mbols, and pH 7.8, closed symbols, of carbon monoxide, 215 itM, with unmodified Arapamla hemoglobin, O, and 0, and Arapamul hemoglobin modified with suberimldate in the oxygenated state, [] and I , and m the deoxygenated state, © and O.

faster than the unmodified material while the material modified as the deoxygenated derivative reacts heterogeneously. At pH 5.8 the effects of these modifications are somewhat more pronounced. The unmodified hemoglobin reacts in two equal kinetic phases. The material modified as the oxygenated derivative also reacts in two roughly equal kinetic phases but the reaction rate constants, particularly that of the slower phase, are more rapid than for the unmodified hemoglobin. The slow phase of the reaction of the hemoglobin modified as the deoxygenated derivative represents not 50°. but only 30", of the total reaction. As a result the overall reaction with ligand is much more rapid and the pH dependence of the reaction of CO with this derivative appears significantly reduced. The differences in the functional properties of the two chemically modified forms of Arapaima hemoglobin support the hypothesis that at pH 8.1 the oxygenated and deoxygenated derivatives of this hemoglobin are not conformationally equivalent. The functional differences could result from differences in the nature of the chemical modifications or from differences in the extent of the modification. The exact groups modified and the nature of the cross-links produced are unknown. It is curious that reaction of the deoxygenated hemoglobin with the cross-linking reagent yields the material whose properties are least like those of the deoxy or low affinity structure of this hemoglobin. This suggests that rather than crosslinking and thus stabilizing the low affinity structure of this protein, this procedure alters groups needed for its stability and shifts the conformational equilibrium toward the higher affinity state or states. DISCUSSION

The hemoglobins of the Osteoglosslml and Arapaima exhibit Root effects and have many functional properties in common with other Root effect hemoglobins. The states of minimum oxygen affinity of these hemoglobins at low pH are associated with a loss of cooperativity in ligand binding just as has been

reported for a number of other fish hemoglobins (Tan

et al., 1973; Lau et al., 1975; Noble et al., 1975). This cannot be concluded from equlhbrium measurements because of the intrinsic heterogeneity of the ligand binding sites of these hemoglobins. However, the flash photolysis studies establish this quite conclusively. Thus the very low oxygen affinity of these hemoglobins is achieved by the elimination of cooperativity in ligand binding, presumably as a result of eliminating the ligand linked alteration in quaternary structure normally associated with cooperativity in ligand binding to hemoglobin. The properties of these molecules at low pH can therefore be taken to be those of their low affinity, T conformational states. The properties of the low affinity states of these hemoglobins are very similar. Their oxygen affinities are nearly identical, log Pso = 2 and the binding curves are heterogeneous with Hill coefficients of about 0.5 for Osteoglossmn hemoglobin and 0.6-0.7 for Arapainla hemoglobin. This binding site heterogeneity is clearly seen in the kinetics of carbon monoxide combination to these two hemoglobins. This kinetic heterogeneity is greater for Osteoglossum hemoglobin than for Arapaima and is consistent with the lower Hill coefficient for the reaction of oxygen with the former. This heterogeneity is almost certainly the result of a functional inequivalence of the ~ and /3 subunits in the T states of these hemoglobins. Such subunit heterogeneity has also been found by Pennelly et aL (1977) in the T state of the hemoglobin of the squirrel fish, Myripristis herndi. The absolute rate constants for the reactions of Arapaimu hemoglobin with ligands are consistently higher than for Osteofflossum hemoglobin. Thus Arapaima hemoglobin in the T state dissociates oxygen 4-fold faster than does Osteoglossum hemoglobin. Likewise the rate of combination of carbon monoxide with Arapaima hemoglobin is about 4-fold faster in the slow phase and 50",, faster in the rapid phase than with O.~teo.qlossum hemoglobin. Increasing the pH destabilizes the T state of both molecules, increasing ligand affinity and reestablishing cooperativity in ligand binding. This reappearance

Functional properties of hemoglobins of cooperatwe ligand binding is associated with a conversion from biphasic to monophasic carbon monoxide combination kinetics. The rate of the single phase reaction is very similar to that of the rapid phase observed at low pH. This suggests that when hgand binding is cooperative, the carbon monoxide reacts first with the rapidly reacting subunit of the T state. However, following this reaction the hemoglobin is converted to the R conformation in which the previously slowly reacting subunits can now combine rapidly with the carbon monoxide. The result is an apparently monophasic reaction which is rate limited by the combination of CO to the reactive sites of the T state. Such a reaction sequence has been postulated by Pennelly et al. (1977) to explain similar properties of squirrel fish hemoglobin. For Arapaima hemoglobin the maximum oxygen affinity which is achieved at high pH is a log Pso of about 0.8. In this pH range, where the Bohr effect is zero and organic phosphates have no effect, oxygen binding is cooperative with a Hill coefficient of about 2. Osteoglossum hemoglobin behaves quite differently. As the pH is increased, the oxygen affinity and cooperativity increase, but as the oxygen affinity increases to its maximum value, cooperativity decreases once again, as evidenced by a Hill coefficient of unity and rates of recombination of carbon monoxide after full and partial flash photolysis which, though not equal, are far more similar than those obtained under conditions in which cooperativity is maximal. Exactly the same behavior has been reported for carp hemoglobin, and has been attributed to a nearly complete stabilization of the high affinity, R state of the hemoglobin molecule, even when deoxygenated (Tan et al., 1973: Pennelly et al., 1975). This stabilization is not complete since some cooperativity remains. If one accepts this explanation for these results, then the conclusion that follows is that the major difference between these two hemoglobins is in the equilibria which exist between their conformational states in the absence of any interaction with Bohr protons or organic phosphates. This would explain not only the differences in cooperativity but also the differences m oxygen affinity observed at high pH. There is no evidence to suggest that the subunit heterogeneity observed in the T-states of these molecules also occurs in their R-states. The relevant data is sparse, but recombination of carbon monoxide after partial flash photolysis at high pH should reflect the behavior of this strt, ctural state. This reaction is not biphasic for either of these hemoglobins. The effect of ATP on the properties of these hemoglobins is consistent with its preferential binding to their T states. Isaacks et al. (1977) has recently reported that the major organic phosphate found in Arapaima erythrocytes is inositol pentaphosphate rather than ATP. Unfortunately, this information was not available when our experiments were carried out. However, we expect that the effects of these two organic phosphates will be qualitatively the same though they may differ in magnitude. This comparison of the hemoglobins of Arapaima and Osteoglossum was undertaken in part to learn what molecular adaptations in these hemoglobins might be associated with air vs water breathing. The most significant functional difference which we have

153

found in these hemoglobins is in their maximum oxygen affinities. For Osteoglossum hemoglobin this is considerably greater than for Arapaima hemoglobin. This may well be the origin of the greater oxygen affinity of Osteoglossum blood when compared to that of Arapaima blood between pH 7.5 and 7.8. However, differences in organic phosphates and organic phosphate levels could also contribute to this. There is little doubt that in t'ivo the oxygen affinity. of the blood of Arapaima is lower than that of Osteo-. glossum. Not only is this true at any particular pH value, but the pH of Arapaima blood will almost certainly be lower than that of Osteoglossum by virtue of the higher CO2 tension in the former. This results from the far less efficient exchange of CO-, in the Arapaima pseudo-hmg than ovcurs with gills. It could be suggested that Osteoglossum requires blood of a higher oxygen affinity than Arapaima since it is often forced to obtain oxygen from rather hypoxic waters. However, this would presume a great deal of physiological information which is simply not available, The mouth of the Osteoylossum is beautifully adapted to skim along the surface of the water and apparently draw the top few millimeters into the mouth and past the gills. Thus, while the oxygen tension of the water passing over the gills is unknown, it is probably significantly higher than the average 02 tension found in the lake or stream. On the other hand, the efficiency of gas exchange in the modified, lung-like swim bladder of the Arapaima is unknown. Although the adaptation in this organ is impressive when it is compared to the swim bladder of the Osteoglossum, it is clearly no rival for a mammalian lung. In addition, the average O-, tension in the Arapaima pseudo-lung and the frequency of gas phase replacement is unknown. Without knowledge of the relative arterial oxygen tensions in these two fish under various environmental conditions, speculation about the significance of differences in blood oxygen affinity seems premature. Furthermore, the oxygen affinity of the blood of Arapaima seems almost unreasonably low. If the CO_, tension in the blood of the animal is moderately high, then it seems very unlikely that O2 saturation of the hemoglobin can be achieved on passage through the lung. The animal would then be forced to operate well down on the saturation curve of its blood, but to what purpose is unclear. Therefore, although the functional differences between the hemoglobins of these two species can be described and partially explained at the molecular level, the physiological rationale for these functional differences must await further study.

Acknowledgement.s--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 Brazilians for their help and for making it possible for the R.V..41pha Hob\" to enter the upper Amazon. We wish to thank Captain Clarke and the crew for their cooperation, Additional support was provided by the Norwegmn Research Council for Science and tile Humanities (H.J. and U.E.H.F). NSF Grant DEB-76-19877 (D.A.P.), NIH Grant HL-12524 to R,W.N. NIH Grant

HL-15460 (Io J. Bonavcntura, for support of M.F.), NSF Grant PCM-76-06719 (A.R), NIH Grant BM-21314 (A.R.). and the Univcrsit.~ of Texas Research ln,;tflute (A.R.) and

154

MARIA ISABEL GALDAMES-PORTUS et ul

the National Geographic Society (D.A.P.). M. Farmer gratefully acknowlcdgcs a Duke University Rcsearch Award, no 303-3765 REFERENCES ALLEN D. W , GUTHI: K. F. & WYMAN J., JR (1950) Further studies on the oxygen equlhbr~um of hemoglobin. J biol. Chem. 187, 393-410 BUNN H F. & RIGGS A. F. (1979) The measurement of the Bohr effect of fish hemoglobins by gel electrofocusing. Comp. Biochem. Physiol. (this issue). FYHN U. E. H , FYHN H. J., DAVIS B J., POWF,RS D. A., FINK W. L. & GARUCK R. L. (1979) Hemoglobm heterogeneity of Amazoman fishcs. Comp. Biochem. Physiol. 62, 39 66. GARLICK R. L, DAVIS J., FARMER M., FYHN H. J , FYHN U. E. H., NOBLF R W , POWERS D. A., RIGGS A. & WEBI:R R E (1978) A fetal maternal shift in the oxygen equihbrmm of hemoglobin from the vwiparous caecdian,

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LAL~ H. K F., WALLACH D. E. & NOBLI R W (1975) Ligand binding properties of hemoglobin 3 of the trout, Salmo galrdnert: the occurrence of an acid Bohr effect in the absence of h e m e - h e m e interaction. J. biol. Chenl 250, 140(~ 1404. NOBEL R. W., PARKHURST L. J. & GIBSON Q H (1970) The effect of pH on the reaction of oxygen and carbon monoxide with the hemoglobin of the carp, Cyprinus carpio J. biol. Chem 245, 6628-6633. NOBLI~ R. W., PI:.NNkLLY R. R. • RIGGS A. (1975) Studies of the functional properties of the hemoglobin from the benthic fish, Antimora rostrata. Comp. Biochem. Physiol 52B, 75-81. PENNI-LLY R. R., RIGGS A. & NOI~LI- R W The kinetics and equilibria of squirrel fish hemoglobin: a Root effect hemoglobin comphcated by large s v b u m t heterogeneity. Biochim. hioph)'s. Acta 533, 120 129. PENNELLY R R., TAN-WILSON A. L. & NOBLE R. W (1975) Structural states and t r a n s m o n s of carp hemoglobin J. biol. Chem. 250, 7239-7244. POWERS D. A., FYHN H. J., FYHN U. E. H., MARTIN J. P., GARLICK R L. & WOOD S. C. (1979) A comparatwe study of the oxygen equilibria of bloods from several species of A m a z o m a n fishes. Comp. Biochem Phv.su~l. 62, 67 86. RIGGS A. & WOLBACH'R. A. (1956) Sulfhydryl groups and the structure of hemoglobin. J. gen. Physiol. 39, 585-605. TAN A. L., NOBLE R. W. & GIBSON Q. H. (1973) Conditions restricting allosterlc t r a n s m o n s in carp hemoglobin. J. biol. Chem. 248, 2880-2888. WATT K. W. K. & RiGGS A. (1975) Hemoglobins of the tadpole of the bullfrog. Rana catesheiana, structure and function of isolated components. J. biol. Chem. 250, 5934-5944. WF.BI-R R., SULLIVAN B, BONAVENTURAJ. & BONAVI-.'NTURA C. (1976) The hemoglobin system of the primitive fish. Amta cah, a: isolation and functional characterization of the indiwdual hemoglobin components. Bioctmn. blophys. Acta 434, 18 31.