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• W1993782777 abstract "The evolutionary convergence of endothermic tunas and lamnid sharks is unique. Their heat exchanger-mediated endothermy represents an interesting example of the evolutionary pressure associated with this specific characteristic. To assess the implications of endothermy for gas transport and the possible contribution of hemoglobin (Hb), we investigated the effect of temperature on the oxygen equilibria of purified isohemoglobin components V and III from the porbeagle shark (Lamna nasus). In the absence of ATP the effect of temperature on oxygen affinity is normal in both Hb III (P 50 = 0.9 and 2.2 torr at 10 and 26 °C, respectively) and Hb V (P 50 = 1.5 and 2.5 torr at 10 and 26 °C, respectively). In the presence of this effector P 50 decreases with increasing temperature in both components (P 50 at 10 and 26 °C = 9.9 and 8.4 torr (Hb III), respectively, and 9.6 and 7.4 torr (Hb V), respectively. The reverse temperature effect in the presence of ATP will reduce the risk of oxygen loss from the arterial to the venous blood by lowering the oxygen tension gradient between the blood vessels. The mechanism behind the reverse temperature effect resembles that found in the bluefin tuna (Thunnus thynnus), an endothermic teleost, thus evidencing further convergent evolution. The evolutionary convergence of endothermic tunas and lamnid sharks is unique. Their heat exchanger-mediated endothermy represents an interesting example of the evolutionary pressure associated with this specific characteristic. To assess the implications of endothermy for gas transport and the possible contribution of hemoglobin (Hb), we investigated the effect of temperature on the oxygen equilibria of purified isohemoglobin components V and III from the porbeagle shark (Lamna nasus). In the absence of ATP the effect of temperature on oxygen affinity is normal in both Hb III (P 50 = 0.9 and 2.2 torr at 10 and 26 °C, respectively) and Hb V (P 50 = 1.5 and 2.5 torr at 10 and 26 °C, respectively). In the presence of this effector P 50 decreases with increasing temperature in both components (P 50 at 10 and 26 °C = 9.9 and 8.4 torr (Hb III), respectively, and 9.6 and 7.4 torr (Hb V), respectively. The reverse temperature effect in the presence of ATP will reduce the risk of oxygen loss from the arterial to the venous blood by lowering the oxygen tension gradient between the blood vessels. The mechanism behind the reverse temperature effect resembles that found in the bluefin tuna (Thunnus thynnus), an endothermic teleost, thus evidencing further convergent evolution. True tunas and lamnid sharks are the only fish that have developed endothermy. The fact that this characteristic has evolved independently in two such different families (Scombridae and Lamnidae, respectively) makes these species choice subjects for comparison of the traits associated with endothermy (1Block B.A. Finnerty J.R. Environ. Biol. Fishes. 1994; 40: 283-302Crossref Scopus (110) Google Scholar, 2Block B.A. Finnerty J.R. Stewart A.F.R. Kidd J. Science. 1993; 260: 210-213Crossref PubMed Scopus (213) Google Scholar, 3Graham J.B. Dickson K.A. Zool. J. Linn. Soc. 2000; 129: 419-466Crossref Scopus (48) Google Scholar, 4Bernal D. Dickson K.A. Shadwick R.E. Graham J.B. Comp. Biochem. Physiol. Part A Mol. Integr. Physiol. 2001; 129: 695-726Crossref PubMed Scopus (155) Google Scholar). Endothermic fishes maintain body temperatures of up to 20 °C above ambient water temperatures, depending on species, water temperature, and activity level (5Carey F.G. Teal J.M. Comp. Biochem. Physiol. 1969; 28: 205-213Crossref PubMed Scopus (116) Google Scholar, 6Carey F.G. Teal J.M. Kanwisher J.W. Lawson K.D. Am. Zool. 1971; 11: 137-145Crossref Scopus (146) Google Scholar, 7Dewar H. Graham J.B. Brill R.W. J. Exp. Biol. 1994; 192: 33-44PubMed Google Scholar). The consequences of high core body temperatures for gas transport, ion balance, oxygen consumption, aerobic capacity, and muscle performance have been the subject of extensive studies in endothermic tuna species (for review, see Refs. 8Jones D.R. Brill R.W. Mense D.C. J. Exp. Biol. 1986; 120: 201-213Google Scholar, 9Dewar H. Graham J. J. Exp. Biol. 1994; 192: 13-31PubMed Google Scholar, 10Dickson K.A. Environ. Biol. Fishes. 1995; 42: 65-97Crossref Scopus (85) Google Scholar, 11Altringham J.D. Block B.A. J. Exp. Biol. 1997; 200: 2617-2627PubMed Google Scholar); however, little is known about these relationships in elasmobranchs. The porbeagle shark (Lamna nasus) has a core body temperature of 8–10 °C above ambient water temperature (12Carey F.G. Teal J.M. Comp. Biochem. Physiol. 1969; 28: 199-204Crossref PubMed Scopus (134) Google Scholar) and has one of the highest body temperatures among the Lamnidae (13Goldman K.J. J. Comp. Physiol. Biochem. Syst. Environ. Physiol. 1997; 167: 423-429Crossref Scopus (83) Google Scholar, 14Carey F.G. Teal J.M. Kanwisher J.W. Physiol. Zool. 1981; 54: 334-344Crossref Google Scholar, 15Anderson S.D. Goldman K.J. Copeia. 2001; 3: 794-796Crossref Scopus (25) Google Scholar). As in tunas its endothermy is brought about by a number of countercurrent heat exchangers, rete mirabilia, placed in series with the heat-producing organ (16Carey F.G. Sci. Am. 1973; 228: 36-44Crossref PubMed Scopus (65) Google Scholar, 17Graham J.B. Fish. Bull. (Wash. D. C.). 1975; 73: 219-229Google Scholar, 18Burne R.H. Philos. Trans. R. Soc. Lond.-Biol. Sci. 1923; 212B: 209-257Google Scholar). The lateral heat exchangers are placed between the surroundings and the internally located red muscle, and the major blood supply to the red muscle is directed through these via large cutaneous arteries and veins. By minimizing the heat loss via the blood, metabolically produced heat is retained within the active organ (5Carey F.G. Teal J.M. Comp. Biochem. Physiol. 1969; 28: 205-213Crossref PubMed Scopus (116) Google Scholar, 6Carey F.G. Teal J.M. Kanwisher J.W. Lawson K.D. Am. Zool. 1971; 11: 137-145Crossref Scopus (146) Google Scholar). One consequence of heat exchanger-mediated endothermy is that factors other than heat may be exchanged. The smaller the arteries and veins constituting the heat exchanger are, the more efficient the heat retention and the warmer the fish can be relative to the surrounding water. However, decreased dimensions of the heat exchanger increase the risk of oxygen diffusing from the cold arterial blood to the warm venous blood. Already in 1960 Rossi-Fanelli and Antonini (19Rossi-Fanelli A. Antonini E. Nature. 1960; 186: 895-896Crossref PubMed Scopus (69) Google Scholar) showed that temperature has virtually no effect on oxygen binding of R-state crystalline Hb 1The abbreviations used are: Hb, hemoglobin; MWC, Monod-Wyman-Changeux.1The abbreviations used are: Hb, hemoglobin; MWC, Monod-Wyman-Changeux. from the endothermic bluefin tuna (Thunnus thynnus) at pH values between 6.45 and 8.7. Further studies revealed that although temperature does not influence P 50 in the 10–30 °C temperature range, it does alter the shape of the oxygen equilibrium curve (20Carey G.F. Gibson Q.H. Biochem. Biophys. Res. Commun. 1977; 78: 1376-1382Crossref PubMed Scopus (26) Google Scholar, 21Ikeda-Saito M. Yonetani T. Gibson Q.H. J. Mol. Biol. 1983; 168: 673-686Crossref PubMed Scopus (37) Google Scholar), resulting from a normal temperature effect at low oxygen saturation and a reversed one at higher saturations. The heat exchanger-mediated endothermy represents a curious incidence of convergent evolution. In this context it is interesting to investigate whether endothermy in the porbeagle shark is associated with adaptations in the temperature sensitivities of Hb, as documented in Hb I of bluefin tuna (19Rossi-Fanelli A. Antonini E. Nature. 1960; 186: 895-896Crossref PubMed Scopus (69) Google Scholar, 20Carey G.F. Gibson Q.H. Biochem. Biophys. Res. Commun. 1977; 78: 1376-1382Crossref PubMed Scopus (26) Google Scholar, 21Ikeda-Saito M. Yonetani T. Gibson Q.H. J. Mol. Biol. 1983; 168: 673-686Crossref PubMed Scopus (37) Google Scholar). This paper reports the effects of temperature, ATP, and pH on isolated Hb components III and V of the porbeagle shark. Previous studies on hemolysate indicate that the oxygen affinity of porbeagle shark blood is temperature-insensitive (22Hazard E.S. Gibson Q.H. Am. Soc. Zool. 1984; 24 (abstr.): A120Google Scholar, 23Andersen M.E. Olson J.S. Gibson Q.H. Carey F.G. J. Biol. Chem. 1973; 248: 331-341Abstract Full Text PDF PubMed Google Scholar); however, the allosteric mechanism behind the temperature effect remains to be investigated in purified isohemoglobins. Moreover the contribution of organic phosphates needs to be investigated in light of the observation that ATP induces a reverse temperature effect in striped marlin (Tetrapturus audax) Hb (24Weber R.E. Jensen F.B. Annu. Rev. Physiol. 1988; 50: 161-179Crossref PubMed Scopus (130) Google Scholar). Heparinized blood from a porbeagle shark (L. nasus) was supplied from North Sea fishermen. Red blood cells were lysed by addition of three volumes of 0.1 m Tris buffer, pH 7.00 (25 °C). Following centrifugation for 15 min at 14,000 rpm to remove cell debris, the Hb stock solution was stored at –80 °C. Preparation of Isohemoglobins and Oxygen Binding Studies—Isolation of the individual Hbs was performed using preparative isoelectric focusing (column, type 8102, Amersham Biosciences) at 3.9–4.5 °C (Fig. 1) after CO equilibration of the hemolysate. The ampholytes (Amersham Biosciences AB) used were pH 6–8 (40%) and pH 6.7–7.7 (60%) in a sucrose gradient. The seven separated components were dialyzed against three changes of CO-equilibrated, 10 mm Hepes buffer, pH 7.7, containing 0.5 mm EDTA. Three Hb components (Hb V, IV, and III) accounted by far for most of the Hb (Fig. 1). Their isoelectric points at 16 °C were 7.58, 7.62, and 7.68, respectively. Given the small differences in isoelectric points and the fact that fish Hbs with similar electrophoretic mobility exhibit basically similar functional properties (25Weber R.E. di Prisco G. Giardina B. Weber R.E. Hemoglobin Functions in Vertebrates: Molecular Adaptations to Extreme and Temperate Environments. Springer-Verlag, Milan, Italy2000: 23-27Google Scholar), further investigations were focused on Hb V and III. Hb concentrations were measured spectrophotometrically using millimolar extinction coefficients of oxyhemoglobin: 14.37 (β) and 15.37 (α) at 542 and 577 nm, respectively. Hb solutions were concentrated where necessary by ultrafiltration using Centriprep YM-10 tubes (Amicon Bioseparations, Millipore). Oxygen binding was measured at heme concentrations of 0.29–0.30 mm in 0.1 m Hepes buffer, pH 7.304 ± 0.008. Values of pH were measured using a thermostatted BMS Mk2 capillary electrode (Radiometer, Copenhagen, Denmark) calibrated with S1500 and S1510 precision buffers (Radiometer). Oxygen binding equilibria were generated at 10, 16, 21, and 26 °C in the absence or presence of ATP (ATP/Hb4 ratio, >30) using a modified gas-diffusion chamber allowing stepwise increments of oxygen partial pressure (26Fago A. Bendixen E. Malte H. Weber R.E. J. Biol. Chem. 1997; 272: 15628-15635Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). The chamber was coupled to two cascaded Wösthoff pumps (type M201a-f and M301) for admixture of atmospheric air, O2, and pure N2 (99.998%). Based on close similarity of the temperature sensitivities of Hbs III and V, analysis of the interactive effects of pH in the absence and presence of ATP was limited to Hb III. Data Analysis—Extended Hill plots were analyzed using the MWC (two-state) model (27Monod J. Wyman J. Changeaux J.-P. J. Mol. Biol. 1965; 12: 88-118Crossref PubMed Scopus (6108) Google Scholar) S=LKTP(1+KTP)(q-1)+KRP(1+KRP)(q-1)L(1+KTP)q+(1+KRP)q(Eq. 1) where S denotes saturation, P the partial pressure of oxygen, L the allosteric constant, K T and K R the association constants for the low affinity (T, tense) and the high affinity (R, relaxed) forms, respectively, and q the number of interacting binding sites. Based on the results of Andersen et al. (23Andersen M.E. Olson J.S. Gibson Q.H. Carey F.G. J. Biol. Chem. 1973; 248: 331-341Abstract Full Text PDF PubMed Google Scholar), who performed ultracentrifugation on hemolysate of porbeagle shark blood and found the slope of the absorbance versus radius squared to be consistent with a homogenous solution of tetrameric hemoglobins, the Hb was assumed tetrameric, and a fixed value of q = 4 was imposed at all times. Non-linear least squares curve fitting was performed using the software package Mathematica® (Wolfram Research Inc., Cambridge, MA) employing the Levenberg-Marquardt method. Estimates of the standard errors of the fitted parameters were obtained from the diagonal elements of the curvature matrix associated with the fit (26Fago A. Bendixen E. Malte H. Weber R.E. J. Biol. Chem. 1997; 272: 15628-15635Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). To minimize the errors introduced by incomplete saturation or desaturation, when equilibrating with pure oxygen or nitrogen, respectively, the absorbance values at zero and full saturation were extrapolated from the data in the fitting procedure as described by Fago et al. (26Fago A. Bendixen E. Malte H. Weber R.E. J. Biol. Chem. 1997; 272: 15628-15635Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). P 50, the oxygen tension at half-saturation of the Hb, was calculated as the PO2 value at log(S/(1–S)) = 0, and n 50 was calculated as ∂log(S/(1–S))/∂logPO2 at that PO2. The median oxygen tension P m was calculated from Pm=(1/KR)[(L+1)/(Lcq+1)](1/q)(Eq. 2) where c = K T/K R (28Imai K. Allosteric Effects in Haemoglobin. Cambridge University Press, Cambridge, UK1982Google Scholar, 29Weber R.E. Malte H. Braswell E.H. Oliver R.W.A. Green B.N. Sharma P.K. Kuchumo A. Vinogradov S.N. J. Mol. Biol. 1995; 251: 703-720Crossref PubMed Scopus (46) Google Scholar). The free energy of heme-heme interaction ΔG was calculated according to Wyman et al. (30Wyman J. Gill S.J. Noll L. Giardina B. Colosimo A. Brunori M. J. Mol. Biol. 1977; 109: 195-205Crossref PubMed Scopus (42) Google Scholar). ΔG=RTln(L+1)(Lcq+1)(Lc+1)(Lcq-1+1)(Eq. 3) The difference in bond energies in the R and T states between the absence and presence of ATP was calculated as ΔG = RTln(K x,ATP/K x), where x denotes the T or R state. The heat of oxygenation was calculated according to the following relationship ΔH=2.303×R×ΔlogX/1T1-1T2(Eq. 4) where R is the gas constant, X is P 50, 1/L, 1/K T, or 1/K R, and T is the absolute temperature. ΔH app is ΔH at P 50 and includes the heat of release of effectors and solution of ligands and the heat of oxygen binding. Effects of Temperature and ATP—Extended Hill plots of the oxygen equilibria of Hbs III and V at pH 7.3 and at four temperatures (10, 16, 21, and 26 °C) each in the presence or absence of ATP are shown (Fig. 2). The MWC model was successfully fitted to the data. In all instances P 50 of the fitted curves was similar to P m, and the fitted curves were symmetrical around P 50. Both Hbs exhibit high intrinsic oxygen affinities (low P 50 values) (Table I) and small, normal temperature effects at half-saturation (ΔH = –40.1 and –20.7 kJ·mol–1 in Hbs III and V, respectively) at pH 7.3 (Table II). Increasing temperature from 10 to 26 °C increases P 50 from 0.9 to 2.2 torr (Hb III) and from 1.5 to 2.5 torr (Hb V) (Table I). The addition of ATP reduces the oxygen affinity in Hbs III and V, but less so at higher temperatures, resulting in a small reverse temperature effect in both Hbs (ΔH = 12.2 and 11.6 kJ·mol–1 in Hbs III and V, respectively) (Table II).Table IMWC and derived parameters for Hb V and IIIHb sampleTemperatureP50n50nmaxPmLog LS.E.aStandard error of the fitted parameters.Log KTS.E.aStandard error of the fitted parameters.Log KRS.E.aStandard error of the fitted parameters.ΔG°Ctorrtorrtorr-1torr-1kJ·(mol of heme)-1V101.501.931.941.442.660.219-0.6220.04170.5050.05611.42161.391.631.661.471.250.271-0.8140.3000.1500.06991.10211.461.281.281.500.7250.362-0.5700.1650.02110.07450.562262.541.221.262.680.3300.0994-1.0750.183-0.3040.02150.513V + ATP109.652.342.389.043.950.219-1.5140.02720.03130.07901.98168.522.522.538.393.330.271-1.650.0426-0.09070.06362.12217.812.302.307.663.000.362-1.520.0319-0.1340.05101.89267.442.142.157.702.180.0994-1.620.0710-0.3400.04481.76III100.8831.821.840.9231.650.148-0.6140.09390.4460.03761.30161.451.531.531.441.690.128-0.5160.02850.2630.03210.920211.941.481.492.021.100.0462-0.8090.0283-0.02250.01260.884262.161.161.192.270.0930.0791-1.050.188-0.2680.01350.384III + ATP109.892.362.399.333.840.299-1.540.0292-0.01060.07571.981610.92.332.3610.343.700.334-1.590.0266-0.09100.08681.98217.062.132.137.072.470.150-1.480.0327-0.2320.03951.69268.382.082.088.352.440.161-1.510.0249-0.3110.04341.65a Standard error of the fitted parameters. Open table in a new tab Table IIThe apparent heat of reaction, ΔHapp, for L, the T and R states, and at half-saturation oxygen tension (P50) in Hb III and V in the absence or presence of ATPHb sampleΔHLTRP50kJ·mol-1V-222-27.1-81.5-20.7V + ATP-182-4.97-38.011.6III-184-66.6-77.8-40.1III + ATP-1525.51-31.512.2 Open table in a new tab Fig. 3 shows the temperature dependence of P 50 (the van't Hoff plot) and n 50 with and without ATP present. A reverse temperature effect in the presence of ATP is seen. ATP additionally increases cooperativity expressed as n 50 (Fig. 3, lower panel), particularly at high temperature. As evident, Hb III and V show very similar oxygenation characteristics. Mechanisms of the Temperature Effect—The MWC parameters L, K R, and K T provide a mechanistic basis for the effects of temperature on the Hbs (Fig. 4 and Table I). At increasing temperature L decreases, indicating a destabilization of the T (tense) state relative to the R (relaxed) state (Fig. 4a). ATP stabilizes the T state (increases L) but only slightly affects the temperature sensitivity. Increasing temperature decreases oxygen affinity of the R and T states in Hb III and V, but more so in the R state (Fig. 4, b and c). ATP reduces the oxygen affinities of both the T and R states and their temperature sensitivities, making K T of both Hbs insensitive to temperature change, whereas K R is still somewhat reduced by increasing temperature. The effect of ATP on K R decreases with increasing temperatures, indicating that ATP also binds to the R state, particularly at low temperatures. The T state becomes less stable at high temperatures (L decreases), and the overall oxygen binding to the T state accordingly decreases. The reverse temperature effect in the presence of ATP thus stems from the combined effects of the reduction of L with increasing temperature and reduced temperature sensitivity of the T state and especially of the R state. These effects are reflected by the temperature invariance of the lower asymptotes of the extended Hill plots in Fig. 2 (constant K T values) and a change in the slope of the curve around log[S/(1–S)] = 0as L falls at higher temperatures. The change in K R is evident from the greater stability of the upper asymptotes of the curves in the presence of ATP than in its absence. Free Energies and Enthalpies of Reaction—In the absence of ATP the free energy of heme-heme interaction, ΔG, falls with increasing temperature (Table I). By stabilizing the T state, as evidenced by the temperature invariance of K T and the increase in L (Fig. 4), ATP increases the free energy of cooperativity but does so more at high than at low temperature, thus resulting in a low ATP sensitivity at high temperature. The change in ΔG mirrors the effect of temperature on L and n 50 and reflects the ATP-induced increased cooperativity. In both Hb III and V, ATP increases the apparent (overall) heat of oxygenation, ΔH app (ΔH at P 50), which becomes positive (Table II). This effect is consistent with the elevation of ΔH L, ΔH T, and ΔH R by ATP (Table II). The displacement of the lower asymptote of the Hill plots in the presence of ATP reflects the additional bond energies that stabilize the T state in the presence of the polyanionic phosphate anion (Fig. 2). The ATP-induced bond energy differences are greater in the T state than in the R state (4.8 –5.0 kJ·(mol of heme)–1 compared with 2.5–2.6 kJ·(mol of heme)–1 for both Hbs at 10 °C and pH 7.3; Table III). Additionally, the bond energy difference decreases with increasing temperature in both the T and R states. The difference in bond energy, ΔG T and ΔG R, with and without ATP is presented in Table III.Table IIIThe differences in bond energies of Hb III and V in the T or R state in the presence compared to the absence of ATPHbTemperatureΔGTΔGR°CkJ·(mol of heme)-1kJ·(mol of heme)-1V104.832.57164.631.33215.340.875263.150.210III105.042.48165.921.96213.761.18262.640.245 Open table in a new tab Effect of Changes in pH—Hill plots of oxygen equilibria of Hb III at 10 and 26 °C with and without ATP and at different pH values were fitted using the MWC model (Figs. 5 and 6). The pH sensitivities of P 50 and n 50 are shown in Fig. 7. At 10 °C Hb III shows a reverse Bohr effect between pH 8.3 and 7.5 (φ ∼ 0.5) that becomes normal at pH <7.5 (φ ∼ –0.6). At 26 °C the phosphate-free Hb show no Bohr effect in the experimental range of pH values. In the presence of ATP the Bohr effect becomes normal at both temperatures. Noticeably, the reverse temperature dependence with ATP present is marked at low pH and phases out at high pH (Fig. 7). The Bohr effect at pH ∼7.0 –7.3 in the presence of ATP is larger at 10 °C (φ ∼ –0.76) than at 26 °C (φ ∼ –0.3).Fig. 6Hill plots for oxygen equilibrium of Hb III with ATP (ATP/Hb >30) at different pH values. a, at 10 °C. b, at 26 °C. Lines represent the fitting of the MWC model to the data.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 7The effect of pH on P 50 and n 50 of Hb III at 10 (circles, ——) and 26 °C (triangles, ·······) in the absence (open symbols) or presence (filled symbols) of ATP.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The cooperativity coefficient n 50 is negatively affected by lowering pH at low temperature, whereas it is positively affected at high temperature (Fig. 7) and invariably higher in the presence of ATP than in phosphate-free solution. The n 50 values at 10 °C exceed those at 26 °C at all pH values when ATP is absent and above pH 7.3 with ATP. Mechanisms of the Bohr Effect—The allosteric control mechanisms of the Bohr effect are illustrated in Fig. 8. At 10 °C without ATP L decreases from >104 at pH 8.0 to ∼102 at a pH of 7.6, indicating a destabilization of the T state at low pH. The reverse Bohr effect at 10 °C in the absence of ATP described previously concurs with the large fall in L as pH is lowered, whereas K T and K R remain unchanged (Fig. 8). With ATP present this effect is shifted to a lower pH (6.7); the pH effect becomes normal, K T decreases, and K R drops drastically as pH falls below 7.5. At 26 °C the effect of pH on L, K T, and K R is negligible in the absence of ATP. At 26 °C ATP elevates L by stabilizing the T state, in part because of a stabilizing effect of additional proton binding to the T state, its effect being larger at low pH. Concurrently, K T becomes lower and slightly decreases at lower pH values, and K R remains low and stable irrespective of the presence ATP (Fig. 8). Free Energies of Reaction—The free energy of heme-heme interaction (ΔG) is slightly higher at 10 °C than at 26 °C over the entire pH range and in the presence of ATP than in stripped Hb (Table IV). In the absence of ATP, ΔG varies only slightly with pH. With ATP ΔG decreases at low pH (<7.5 at 10 °C) and increases at high pH (>7.0 at 26 °C) (Table IV).Table IVThe free energy of heme-heme interaction for Hb III and V at different pH values, at 10 and 26 °C, and in the presence and absence of ATPTemperatureNo ATPATPpHΔGpHΔG°CkJ·(mol of heme)-1kJ·(mol of heme)-1106.7981.496.6991.497.0460.707.0191.247.3091.307.3131.987.6451.307.6562.377.9101.388.1412.58.3021.858.2822.68.6401.938.4702.05266.5630.776.4522.196.7170.476.6892.087.0550.376.9841.817.3050.387.2911.657.6100.487.5931.537.9190.697.9861.608.2080.848.2341.558.3520.79 Open table in a new tab The MWC model, which assumes functional homogeneity between the subunits comprising the tetrameric Hb, successfully describes the oxygen binding characteristics of porbeagle shark Hb. This indicates either that the chain heterogeneity found in porbeagle shark hemolysate (23Andersen M.E. Olson J.S. Gibson Q.H. Carey F.G. J. Biol. Chem. 1973; 248: 331-341Abstract Full Text PDF PubMed Google Scholar) is not found in the purified Hb components investigated in this study or that it does not influence oxygen binding. The reverse temperature effect on isolated Hb III and V from porbeagle shark resembles that in bluefin tuna Hb I in phosphate buffer (21Ikeda-Saito M. Yonetani T. Gibson Q.H. J. Mol. Biol. 1983; 168: 673-686Crossref PubMed Scopus (37) Google Scholar). In Hbs from both species low temperature stabilizes the T state, and a reverse temperature effect on K R is seen at low pH. The difference between the two species is qualitative. In the bluefin tuna the stabilization of the T state at low temperature (high L) abolishes the T → R transition. Therefore, oxygen binds in the T state even at high oxygen saturation levels, revealing the presence of two functionally distinct subunits in the T state (21Ikeda-Saito M. Yonetani T. Gibson Q.H. J. Mol. Biol. 1983; 168: 673-686Crossref PubMed Scopus (37) Google Scholar). In porbeagle shark the T → R transition does occur, and we find no evidence for differences in the two subunits comprising the Hb. Our results thus agree with those of Andersen and co-workers (23Andersen M.E. Olson J.S. Gibson Q.H. Carey F.G. J. Biol. Chem. 1973; 248: 331-341Abstract Full Text PDF PubMed Google Scholar), who found a reverse temperature effect in hemolysate in the 5–15 °C range and a decrease in L with rising temperature. Thus it appears that the evolutionary convergence of endothermic tunas and lamnid sharks includes changes in the functional mechanisms of their main Hb components. In porbeagle shark Hb the reverse temperature effect is only seen at pH values below 7.5 and in the presence of ATP (Fig. 7). We find that the reverse temperature effect is caused largely by an increase in the allosteric constant, L, with decreasing temperature (Fig. 4). Although acting in the opposite direction, the concurrent increase in K R at pH 7.3 in the presence of ATP is insufficient to counteract the effect of L. At lower pH values the temperature effect on K R becomes reversed, and this magnifies the overall reverse temperature effect. The effect of ATP on K R is most prominent at low pH, where it increases the magnitude of the reverse temperature effect, and more or less vanishes at pH values ≥7.7 (Fig. 8). The lack of an effect of ATP at 26 °C indicates that no ATP is allosterically bound to the oxyhemoglobin at this temperature. The bond energy per salt bridge is 4–8 kJ·mol–1 (31Perutz M.F. Nature. 1970; 228: 726-739Crossref PubMed Scopus (2201) Google Scholar), which suggests that an additional salt bridge per heme group constrains the T state when ATP is present (Table III). The small changes in the differential bond energy in the R state further indicate the preferential binding of ATP to the T state relative to the R state, although additional weaker bonds may be implicated, particularly at high temperature, where the difference bond energy is below 4 kJ·mol–1. At pH 7.3 (which likely falls in the physiological intraerythrocytic range) the overall ΔH app is negative (oxygenation is exothermic) in the absence of ATP but becomes positive (oxygenation is endothermic) with ATP (Table II). The increase is due to the positive heat of ATP and proton release from the Hb during the T → R transition. The fact that ΔH T is much less negative (or positive) than ΔH R is due to a greater ATP and proton release in the T state. Thus the addition of ATP obliterates the temperature effect in the T state, whereas it persists in the R state at high pH and is reversed at pH below 7.3 (Figs. 2, 4, and 7 and Table I). In bluefin tuna the temperature effect is normal in the T state (ΔH 1 is –8.0 kJ·mol–1, and ΔH 2 is –11.0 kJ·mol–1) and reversed in the R state (ΔH 3 and ΔH 4 are 10.4 and 21.3 kJ·mol–1, respectively) at pH 7.0 (21Ikeda-Saito M. Yonetani T. Gibson Q.H. J. Mol. Biol. 1983; 168: 673-686Crossref PubMed Scopus (37) Google Scholar). In contrast, in trout Hb I, the effect of temperature on CO binding shows opposite dependence on ligand saturation; ΔH T is 6 kJ·mol–1, and ΔH R is –11.3 kJ·mol–1 (21Ikeda-Saito M. Yonetani T. Gibson Q.H. J. Mol. Biol. 1983; 168: 673-686Crossref PubMed Scopus (37) Google Scholar, 30Wyman J. Gill S.J. Noll L. Giardina B. Colosimo A. Brunori M. J. Mol. Biol. 1977; 109: 195-205Crossref PubMed Scopus (42) Google Scholar). In tench (Tinca tinca) the addition of ATP increases ΔH T (–55.8 to –30.9 kJ·mol–1) and slightly decreases ΔH R (–60.5 to –68.0 kJ·mol–1) at pH 7.35, indicating that the proton release occurs gradually and that ATP does not bind to the R state (32Jensen F.B. Weber R.E. J. Comp. Physiol. Biochem. Syst. Environ. Physiol. 1987; 157: 137-143Crossref PubMed Scopus (12) Google Scholar). Our results illustrate the complex relationship between pH and ΔH app (Fig. 7). In the presence of ATP at pH values below 7.7, ΔH app becomes increasingly positive with falling pH because of an increasing ΔH L that eventually becomes positive at pH <6.7 and an increasing ΔH R that becomes positive at pH <7.1 (Fig. 8, a and c). In spiny dogfish (Squalus acanthias), the ΔH app of stripped Hb also is pH-dependent with a value of –35 kJ·mol–1 at pH 7.0 and –44 kJ·mol–1at pH 7.9 (33Weber R.E. Wells R.M.G. Rossetti J.E. J. Exp. Biol. 1983; 103: 109-120PubMed Google Scholar). The free energies of cooperativity, ΔG, at 10 and 26 °C with ATP (Hb III) are comparable to those found in tench (6.1 kJ·(mol of Hb)–1) (34Weber R.E. Jensen F.B. Cox R.P. J. Comp. Physiol. Biochem. Syst. Environ. Physiol. 1987; 157: 145-152Crossref PubMed Scopus (61) Google Scholar). Human Hb has a higher ΔG at 25 °C (8.8 kJ·(mol of Hb)–1 at pH 7.4), close to values found at 10 °C with ATP at high pH (Hb III, Table IV) and at 16 °C with ATP (Hb V, Table I) (35Imai K. Biochemistry. 1973; 12: 798-808Crossref PubMed Scopus (130) Google Scholar). The Bohr effect in porbeagle shark Hb at 10 °C with ATP (φ ∼ –0.76) is large and similar to values for other active fishes. Albacore tuna (Thunnus alalunga) and striped marlin have Bohr factors of –1.2 and –1.0, respectively (36Cech J.J. Laurs R.M. Graham J.B. J. Exp. Biol. 1984; 109: 21-34Google Scholar, 37Dobson G.P. Wood S.C. Daxboeck C. Perry S.F. Physiol. Zool. 1986; 59: 150-156Crossref Google Scholar), whereas those obtained from the moderately active blue shark (Prionace glauca) stripped hemoglobin with and without added inositol hexaphosphate are –1 and –0.4, respectively (38Pennelly R.P. Noble R.W. Riggs A. Comp. Biochem. Physiol. 1975; 52B: 83-87Google Scholar). It is notable that the reverse Bohr effect at low temperature and in the absence of ATP extends to high pH values (7.5–8.3). The reverse Bohr effect is seen in other fish Hbs but is usually restricted to cathodic Hbs (for review, see Ref. 39Fago A. Carratore V. di Prisco G. Feuerlein R.J. Sottrup-Jensen L. Weber R.E. J. Biol. Chem. 1995; 270: 18897-18902Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), where it also disappears when ATP is present. This suggests that upon oxygenation alkaline Bohr groups change pK value in the reverse direction from normal. Possible Implications for Oxygen Transport—Allosteric cofactors such as organic phosphates and protons lower the intrinsically high oxygen affinity of Hb to values that ensure oxygen unloading at oxygen tensions that are sufficiently high for physiological needs. Compared with small Bohr effects reported in sluggish elasmobranch species (40Wells R.M.G. Weber R.E. J. Exp. Biol. 1983; 103: 95-108PubMed Google Scholar), active ones, like the porbeagle and mako sharks, have substantial Bohr effects at low temperature and in the presence of ATP (23Andersen M.E. Olson J.S. Gibson Q.H. Carey F.G. J. Biol. Chem. 1973; 248: 331-341Abstract Full Text PDF PubMed Google Scholar). The pH insensitivity on K R has been interpreted as advantageous under changing environmental oxygen tensions (34Weber R.E. Jensen F.B. Cox R.P. J. Comp. Physiol. Biochem. Syst. Environ. Physiol. 1987; 157: 145-152Crossref PubMed Scopus (61) Google Scholar). By maintaining a constant high affinity for oxygen in the R state, loading is protected during hypoxia and during exercise when the blood may become acidotic. In porbeagle shark K R falls at low temperature when pH is low, an effect not present at high temperature when environmental, dissolved oxygen concentration can be assumed to be lower and loading may become compromised. The concentration of ATP and other organic phosphates in nucleated fish red cells decreases under conditions of decreased oxygen availability, forming a regulatory mechanism that favors oxygen loading through the resulting increase in oxygen affinity. During exercise the affinity gain may, however, be offset by the decreased blood pH (Bohr effect). In elasmobranchs the presence of high urea levels may hinder oxygen affinity modulation by dampening the ATP effect as seen in stripped hemolysate of spiny dogfish (33Weber R.E. Wells R.M.G. Rossetti J.E. J. Exp. Biol. 1983; 103: 109-120PubMed Google Scholar). The presence of countercurrent heat exchangers in the porbeagle shark imposes a risk of oxygen loss from the arterial to the venous blood. However, as in the endothermic bluefin tuna, the presence of a reverse temperature effect in isolated Hb indicates that this risk is reduced. The increased oxygen affinity with rising temperature reduces the oxygen tension gradient between the arterial and venous blood. This adaptation to an efficient heat conservation mediated by heat exchangers may only be necessary in cases in which the heat exchangers are very efficient. As such the bluefin tuna and the porbeagle shark are the among the warmest species in their distantly related families, respectively, each representing very efficient countercurrent heat exchangers (6Carey F.G. Teal J.M. Kanwisher J.W. Lawson K.D. Am. Zool. 1971; 11: 137-145Crossref Scopus (146) Google Scholar, 13Goldman K.J. J. Comp. Physiol. Biochem. Syst. Environ. Physiol. 1997; 167: 423-429Crossref Scopus (83) Google Scholar, 14Carey F.G. Teal J.M. Kanwisher J.W. Physiol. Zool. 1981; 54: 334-344Crossref Google Scholar, 15Anderson S.D. Goldman K.J. Copeia. 2001; 3: 794-796Crossref Scopus (25) Google Scholar, 16Carey F.G. Sci. Am. 1973; 228: 36-44Crossref PubMed Scopus (65) Google Scholar, 41McCosker J.E. Copeia. 1987; 1: 195-197Crossref Google Scholar, 42Holland K.N. Sibert J.R. Environ. Biol. Fishes. 1994; 40: 319-327Crossref Scopus (54) Google Scholar, 43Carey F.G. Kanwisher J.W. Brazier O. Gabrielson G. Casey J.G. Pratt H.L. Copeia. 1982; 2: 254-260Crossref Google Scholar). We extend thanks to an anonymous reviewer for helpful comments on the manuscript and to A. Bang for technical assistance." @default.
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