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The hemoglobin of the crossopterygian fish, Latimeria chalumnae (Smith)
ARCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSICS
163, 728-734 (1974)
The Hemoglobin
of the Crossopterygian
Latimeria Subunit
&alumnae
Structure
JOSEPH BONAVENTURA,2
(Smith)
and Oxygen
Equilibria’
ROBERT G. GILLEN,
Department of Zoology, Uniuersity Received
of
February
Fish,
AND
AUSTEN RIGGS”
Texas, Austin, Texas 78712 19, 1974
The crossopterygian fish, Latimeria chalumnue, has a single tetrameric hemoglobin which is composed of Ly- and @-like chains. The cy chains but not the @chains are blocked at their NH,-termini. The sequence of the first 15 residues of the p chain indicates that it is homologous with the /3 chains of other fish and vertebrate hemoglobins. The oxygen affinity of Latimeria hemoglobin is unusually high, with a P,, of only 1.2 mm Hg at pH 7.0 in 0.05 M Bis-Tris buffer. In 0.1 M inorganic phosphate buffer at the same pH the hemoglobin shows increased cooperativity, n increases from 1.1 to 1.6, and a lower oxygen affinity, with a P,, of 4.0 mm Hg. Cooperative interactions and P,, values are substantially reduced above pH 8.
The coelacanth, Latimeria chalumnae, is the only living member of the order crossopterygii, members of which are believed to have given rise to the Amphibia (1). This proposed relationship is consistent with studies showing that nitrogen metabolism and ion regulation in Latimeria are more like those of Elasmobranchs. Dipnoi, and Amphibia than of teleost fish (2, 3). Recent studies (4, 5) have shown that Latimeria hemoglobin has a high oxygen ’ This work was supported in part by United States Public Health Service Research Grant GM-05818, NSF Grant GB-27937X, and by Robert A. Welch Foundation Grant F-213. ’ United States Public Health Service Predoctoral trainee under Training Grant GM-00836; Present address, Duke University Marine Laboratory, Beaufort, NC 28516. a United States Public Health Service Predoctoral trainee under Training Grant GM-00836; Present address, Department of Biochemistry and Biophysics, Texas A and M University, College Station, TX 77843. ‘To whom requests should be addressed; Supported by United States Public Health Service Research Career Development Award, 5 K 03.GM038086.
affinity and that it appeared to consist of two kinds of polypeptide chains. The present experiments confirm and extend these results with a further examination of the hemoglobin structure and measurement of oxygen binding both in the presence and absence of phosphate. EXPERIMENTAL
PROCEDURE
In the course of these studies, blood samples from two specimens of Lutimeria chalumnue were used. Sample I was obtained from Dr. M. G. Larbaight and Dr. Emile Zuckerkandl in March, 1967. The titrated blood (10 ml) was kept on ice continuously from the time of collection at Grand Comore to the initial processing at the Institut Pasteur de Madagascar. Centrifuged, packed cells were taken up in 3 vol of 0.9% NaCl, whereupon hemolysis occurred. The preparation was centrifuged at 4°C for 10 min at 4000 rpm. The supematant was centrifuged again at 0°C for 55 min at 8000 rpm to remove stroma. This hemolysate was kept continuously on ice throughout its shipment from Tananarive to France and thence to Texas. Upon arrival in Austin, Sample I (129 mg in 8.4 ml) was saturated with CO, centrifuged, and dialyzed overnight against CO-saturated 0.1 M phosphate of pH 7.5. Sample II was obtained as packed red cells from one of two specimens of Latimeria caught during the 1972 French-British-American expedition to the Comores Islands, and shipped on ice to Texas. The cells
LATIMERIA
HEMOGLOBIN
of sample II were washed three times with 0.9% NaCl without any hemolysis, and were lysed with 0.001 M SEPHADEX G-100 Tris, pH 7.5. After concentration by pressurefiltraCHROMATOGRAPHY tion (Diaflo apparatus, UM-10 membrane, Amicon, Inc., Lexington, MA), chromatography of each hemolysate was carried out on a 3 x 68-cm column of IO Sephadex G-100. Vertical starch gel electrophoresis 0 at2t30nm was performed on the CO-hemoglobins as described I by Smithies (6), and on the globins as described by . at 540 nm Miiller (7). Globin was prepared as described previ5 ously (8). Polyacrylamide gel electrophoresis was done ii! at pH 9.2 or 8.6 in Tris-EDTA-borate buffer (Bulletin s: b No. 134, E.C. Apparatus Co., Philadelphia). Gels z containing 8 M urea were also used. Oxygen equilibria were measured at 20°C according to the method of Riggs and Wolbach (9). Sedimentation velocity measurements were made with, a Beckman Model E Ultracentrifuge as previously described (8). Separation of unoxidized polypepride chains from 30 mg of Sample II was done essentially as described for carp hemoglobin (lo), using a linear gradient between 0.2 M formic acid and 1.5 M formic acid in TUBE NUMBER 0.02 M pyridine. Amino acid analyses were performed FIG. 1. Gel chromatography of hemolysate from with a Beckman Model 120 Analyzer (8). Tryptic Latimeria chalumnae. Conditions: 3 x 68-cm column peptide maps were made from the isolated polypepof Sephadex G-100 equilibrated and eluted with tide chains as previously described (10). The amino acid sequence of the NH,-terminal segment of the /3 CO-saturated 0.2 M NaCl at 4’C; fraction size, 2.8 ml. The bars indicate the pools taken for fractions I and chain was determined by applying unfractionated II; all the hemoglobin was in fraction II. globin to a Beckman Sequencer Model 890 for automatic Edman degradation (11). This procedure yields only the &chain sequence. We have found that the cy globin, and behaved as a single component chain is blocked. The thiazolinone derivatives of each on electrophoresis of the CO-hemoglobin at residue were converted manually to the corresponding gels (Fig. 2a). phenylthiohydantoins (11) and identified either by pH 9.2 on polyacrylamide Figure 2a shows that both the unfracgas-liquid chromatography using a Beckman GC-45 tionated hemolysate and the GlOO purified chromatograph and/or by thin layer chromatography hemoglobin appeared as single electrophoas previously described (12).
:I3
t t t
RESULTS
Chromatography of the Sample I hemolysate of Latimeria on Sephadex GlOO (Fig. 1) showed that essentially all of the hemoglobin eluted as a single zone. This major zone was preceded by a small zone of nonheme protein. The elution volume of the hemoglobin zone corresponded to that expected for a tetrameric hemoglobin molecule. On either side of the hemoglobin zone small shoulders of 540-nm absorbing material were present. If these shoulders correspond to minor hemoglobin components of slightly different molecular size, they constitute less than 1% of the total hemoglobin. Chromatography and electrophoresis of samples I and II gave essentially the same results on Sephadex GlOO: Zone II (Fig. 1) contained virtually all the hemo-
retie components at pH 9.2. Starch gel electrophoresis of the globin at pH 1.9 (Fig. 2b) showed only a single band for Latimeriu globin but some material stayed at the origin. Electrophoresis of the globin at pH 3.7 and in 8 M urea at pH 8 gave single bands. These results suggest that the electric charge on the (Y and /3 chains may be very similar. Latimeria oxyhemoglobin in 0.2 M NaCl behaved as a single component in the ultracentrifuge (Fig. 3). The s20.Wvalue was 4.4 for a 0.4% oxyhemoglobin solution in 0.02 M NaCl at 20”. These results indicate that Latimeria hemoglobin is largely tetrameric under these conditions. The constituent polypeptide chains of Latimeria globin from Sample II were readily separated by the same chromatographic procedures used for carp globin
730
BONAVENTURA,
GILLEN
a. pH 9.2, Hemoglobin
AND
RIGGS
b. pH1.9,Globin
FIG. 2. Electrophoresis of hemoglobins and globins. (a) Polyacrylamide gel, pH 9.2; from left to right, human CO-hemoglobin A, Latimeria CO-hemolysate, fraction I (Fig. l), zone between fraction I and II, fI ,act ion II. (b) IStarch gel, pH 1.9; on left, human globin A; on right, Latimeria globin (both slots).
(10). The amino acid composition of these chains are given in Table I. Summation of the amino acid compositions of the (Y and /3 chains obtained from Sample II is generally consistent with the earlier results obtained on the amino acid composition of the intact globin from Sample I. Tryptic peptide patterns obtained from the isolated a and P-chains (Fig. 4) show that they are quite dissimilar. Intact globin was sequenced on the Beckman Protein Peptide Sequencer for 15 residues. The results (Table II) show that the unblocked polypeptide chain is quite similar to the B chains of teleost hemoglobins. Of particular interest is the similarity of the first eight residues with the corresponding sequences of the carp and Rio Grande cichlid @ chains. In both Latimeria and the carp, a tryptophanyl residue is found at position 3. Additionally, position 15 is occupied by tyrosine whereas tryptophan appears to be present at this position in all other vertebrate hemoglobins and myoglobins so far examined including the hemoglobins of the lamprey, Petromyzon (12), and the annelid worm, Gl.ycera dibrunchiata (15). Results of the oxygen equilibrium mea-
surements (Fig. 5) show that in the absence of phosphate, Latimeria hemoglobin has a very high oxygen affinity, and pronounced Bohr and phosphate effects. The oxygen affinity is among the highest reported for any fish hemoglobin (16). The value of n in Hill’s equation decreases from a maximal value to 1.6 at low pH to 1.0-1.1 above pH 8 in 0.1 M phosphate buffer. In the absence of phosphate the value of n decreases to about 1 at about 1 pH unit lower than its presence. The shape of the oxygen-binding curves are pH-dependent. In the presence of phosphate this hemoglobin is much more sensitive to pH between pH 7 and 8 at low degrees of oxygenation than at high levels. At a specified degree of oxygenation, Y, the pH dependence, A log pO,/A pH is a measure of the number of protons released per oxygen bound (17). The data indicate that as oxygenation proceeds protons are preferentially released from Latimeria hemoglobin protein at low degrees of OXygenation in this pH range. DISCUSSION
The data of Wood et al. (4) confirm our earlier observations of a Bohr factor of
LATIMERIA
731
HEMOGLOBIN TABLE AMINO THE
ACID
COMPOSITION
HEM~CLOBIN
Lysine Histidine Arginine Aspartat e Threonine’ Serine’ Glutamate Proline Glycine Alanine Half-cystined Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine
I
OF THE OF
12.4 8.67 1.89 11.9 9.17 5.51 11.8 3.66 5.52 14.2 1.73 11.7 5.73 18.5 3.03 9.14
LY AND
@ CHAINS
OF
Latimeria chalumnae”
13.2 9.04 4.61 11.4 11.2 7.95 14.8 3.51 4.83 10.3 1.09 11.0 0.89 6.35 19.3 3.83 6.45
25.6 17.7 6.50 23.3 20.4 13.5 26.6 7.17 10.4 24.5 2.82 22.7 0.89 12.1 37.8 6.86 15.6
26.5 17.6 6.87 22.8 21.0 13.8 27.7 8.93 9.43 24.0 3.33 22.4 0.84 12.1 39.8 6.45 14.8
“Averages of results of analyses obtained at 24., 48., 72., and (except for the intact globin) 96.hr hydrolysis in 6 N HCI under vacuum at 110°C. Molecular weights for the cy and P chains are assumed to be identical to those in human hemoglobin, 15.126 and 15,924, respectively. Numbers are residues per mole. Tryptophan was not determined. However. one residue was found in the NH, terminal segment of the fl chain. *The (Yand B chains were obtained from Latimeria hemoglobin Sample II; the unfractionated globin from Sample I. ‘Determined by extrapolation to zero time, d Determined as cysteic acid after 24.hr hydrolysis of a performic acid-oxidized sample.
about PO.55 between pH 7.0 and 8.0 for Latimeria hemoglobin in 0.1 M inorganic phosphate (18). Their data also confirm that n drops to near unity at or above pH 8 (Ref. 18 and this paper). The results presented here (Fig. 5) clearly show that the pH dependence of the oxygen affinity is decreased in the absence of phosphate. In addition, Latimeria hemoglobin becomes noncooperative at a lower pH in the absence of phosphate than in its presence. It is remarkable that the effect of a 4-fold excess of ATP over hemoglobin on the oxygen affinity of Latimeria hemoglobin is reported by Wood et al. to be small compared to the effect of 0.1 M phosphate: At pH 6.65, the Alog P,,, = 0.24 on going from
732
BONAVENTURA.
GILLEN
AND
RIGGS
FIG. 4. Tryptic peptide patterns of the o( and @chains of Latimeria hemoglobin. chain.
COMPARISON OF THE NH,-TERMINAL
TABLE II @ CHAIN SEQUENCE OF Latimeria OTHER ANIMALS
Left: (Y chain; Right: B
HEMOGLOBIN WITH THOSE OF VARIOUS
Yield (%) 12
76 64 88 15 40 26 -
54 58 64
8
31 26 20
9
12 13 14 15
Position 12345678 Latimeria
10
11
fl
Carp P (10) Rio Grande cichlid @(13) Human 6 (14) Human a (14) Petromyzon (12) Carp o((14) Myoglobin (14) Glycera monomer (15)
Tris buffer to Tris buffer + ATP. Under the same conditions, their data show that Alog PC0 = 0.73 on going from Tris buffer to phosphate buffer (4). In contrast, ATP has been reported to shift log P,, by about 1 log unit in several other fish hemoglobins (10, 13, 19). Our observations do not indicate significant heterogeneity in Latimeria hemoglobin and are consistent neither with the early report of heterogeneity on the basis of
solubility measurements (20) nor with recent results (5) obtained with isoelectric focusing. It should be noted, however, that the resolution of our electrophoretic technique is not as great as that possible with isoelectric focusing. Weber et al. (5) found about six hemoglobin components by isoelectric focusing, all very close to one another. The possibility exists that not all of these components represent hemoglobins with different primary structures since dis-
LA7’IMERIA
HEMOGLOBIN
733
version to the amino acids in the hydrolysate to the n-butyl-N-trifluoroacetyl esters, followed by gas-liquid chromatography. Previous studies (4, 5) of the functional properties of Latimeria hemoglobin have suggested placing the coelacanth in an evolutionary position between elasmobranchs and holosteans. Specialization of functional properties to suit particular environmental conditions make such phylo2.0-genetic assignments based on functional properties difficult. Sharks and their allies diverged quite early from the ancestors of bony fishes and have undergone extensive evolutionary changes (21). Comparison of the functional properties may be of greater FIG. 5. Oxygen equilibria of Latimeria hemorelaglobin. Dependence of log P,, and n on pH. Log P,, is value in assessing the evolutionary the 0, pressure (mm Hg) required for 50% oxygenationships among the teleostomes: the Diption. Open circles: measurements in 0.1 M phosphate noi, Crossopterygii, and Actinoptergii at pH 6, 7, and 8, and 0.1 MTris-HCI at pH 9.0; closed whose relationship is still unsolved (22). circles: measurements in 0.1 M NaCl, 0.05 M Bis-Tris Among the teleostomes, the lungfishes, adjusted to pH indicated, except the data at pH 8 and crossopterygians are usually believed which were obtained in 0.05 M Tris, 0.1 M NaCl. to have a closer evolutionary relationship based on morphological and paleontological observations (reviewed in 22 and 23). sociation products similar to those noted Latimeria is unusual in that its hemogloafter freezing may have been present. Weber et al. (5) reported on the basis of bin appears to contain only one major electrophoresis on polyacrylamide gels con- component: both lungfishes (Oldham, J., and Riggs, A., unpublished) and teleosts taining sodium dodecyl sulfate that Latime(18) frequently have multiple hemoglobin ria hemoglobin contains two polypeptide chains of slightly different molecular components present in appreciable weight (15,400 and 16,300). The ratio of amount. Latimeria hemoglobin is also unusual in that one of its hemoglobin chains these two weights is 0.945 which is almost The amino acid seidentical to the ratio of 0.950 for weights of lacks methionine. quence of the first 15 residues of the the aand /3 chains of human hemoglobin. We, therefore, believe that the two chains P-chain of Latimeria hemoglobin suggests which they observed are the (Y and p chains a close relationship between higher bony of the hemoglobin. fishes and this only surviving crossopteryThe amino acid composition of the iso- gian. Remarkably, the first seven residues lated polypeptide chains indicates that the of Latimeria myoglobin (24) appear to be (Y chain is devoid of methionine and that identical with those of the ,f3 chain of the /3 chain contains only a single methiLatimeria hemoglobin and appear to be onyl residue (Table I). The agreement be- quite different from other known myoglotween the analysis of the intact globin of bins. Sample I and the sum of the (Y and /3 chain ACKNOWLEDGMENTS analyses is generally within the errors of We thank Dr. M. G. Larbaight and Dr. Emile measurement. The agreement with the Zuckerkandl for sample I, Mr. Paul K. Brown for analysis by Weber et al. (5) is less satisfacfacilitating its shipment, and the participants of the tory. The lack of agreement is perhaps due French-British-American 1972 Expedition to the to methodological differences. Their analyComores for sample II. We thank Mrs. Marie Ervin for sis was carried out on a hydrolysate of the amino acid analyses and Mrs. Florence Waddill hemoglobin, not globin, and involved con- for operating the sequencer.
734
BONAVENTURA,
GILLEN
REFERENCES 1. THOMPSON, K. S. (1969) Biol. Reo. 44, 91. 2. PICKFORD, G. E., AND GRANT, F. B. (1967) Science 155,568. 3. BROWN, G. W., AND BROWN, S. G. (1967) Science 155,570. 4. WOOD, S. C., JOHANSEN, K., AND WEBER, R. E. (1972) Nature (London) 239, 283. 5. WEBER, R. E., BOL, J. F., JOHANSEN, K., AND WOOD, S. C. (1973) Arch. Biochem. Biophys. 154, 96. 6. SMITHIES, 0. (1959) Biochem. J. 71, 585. 7. MULLER, C. J. (1960) Nature (London) 186, 643. 8. BONAVENTURA, J., AND RIGGS, A. (1968) J. Biol.
Chem.243,980. 9. RIGGS, A., AND WOLBACH, R. (1956) J. Gen. Ph,ysiol. 39,585. 10. GILLEN, R. G., AND RIGGS, A. (1972) J. Biol. Chem.
247,6039. 11. EDMAN, P., AND BEGG, G. (1967) Eur. J. Biochem. 1, 80. 12. LI, S. L., AND RIGGS, A. (1970) J. Riol. Chem. 245, 6149. 13. GILLEN, R. G., AND RIGGS, A. (1973) Arch. Biothem. Biophys. 154, 348.
AND
RIGGS
14. DAYHOFF, M. V. (1972) Atlas of Protein Sequence and Structure, pp. D56-D85, National Biomedical Research Foundation, Silver Spring, MD. 15. IMAMURA, T., BALDWIN, T. O., AND RIGGS, A. (1972) J. Biol. Chem. 247, 2785. 16. SULLIVAN, B., AND RIGGS, A. (1971) in Respiration and Circulation (Altman, P. L., Ed.), pp. 1922194, Federation of American Societies for Experimental Biology, Washington, DC. 17. WYMAN, J. (1948) Aduan. Protein Chem. 4, 407. 18. RIGGS, A. (1970) in Fish Physiology (Hoar, W. S., and Randall, D. J., eds.), pp. 209-252, Academic Press, New York. 19. GILLEN, R. G., AND RIGGS, A. (1971) Comp. Biothem. Physiol. 38B, 585. 20. DE PRAILAUNE, S. (1955) C. R. Sot. Biol. 149,655. 21. YOVNG, J. Z. (1962) The Life of Vertebrates, Oxford Univ. Press, New York. 22. MOY-THOMAS, J. A. (1971) Palaeozoic Fishes, Saunders, Philadelphia, Pa. 23. ROMER, A. S. (1968) Notes and Comments on Vertebrate Paleontology, Univ. of Chicago Press, Chicago, IL. 24. CHAUVET, J.-P., AND ACHER, R. (1972) Fed. Eur. Biochem. Sot. Letters 28, 16.
OF BIOCHEMISTRY
AND
BIOPHYSICS
163, 728-734 (1974)
The Hemoglobin
of the Crossopterygian
Latimeria Subunit
&alumnae
Structure
JOSEPH BONAVENTURA,2
(Smith)
and Oxygen
Equilibria’
ROBERT G. GILLEN,
Department of Zoology, Uniuersity Received
of
February
Fish,
AND
AUSTEN RIGGS”
Texas, Austin, Texas 78712 19, 1974
The crossopterygian fish, Latimeria chalumnue, has a single tetrameric hemoglobin which is composed of Ly- and @-like chains. The cy chains but not the @chains are blocked at their NH,-termini. The sequence of the first 15 residues of the p chain indicates that it is homologous with the /3 chains of other fish and vertebrate hemoglobins. The oxygen affinity of Latimeria hemoglobin is unusually high, with a P,, of only 1.2 mm Hg at pH 7.0 in 0.05 M Bis-Tris buffer. In 0.1 M inorganic phosphate buffer at the same pH the hemoglobin shows increased cooperativity, n increases from 1.1 to 1.6, and a lower oxygen affinity, with a P,, of 4.0 mm Hg. Cooperative interactions and P,, values are substantially reduced above pH 8.
The coelacanth, Latimeria chalumnae, is the only living member of the order crossopterygii, members of which are believed to have given rise to the Amphibia (1). This proposed relationship is consistent with studies showing that nitrogen metabolism and ion regulation in Latimeria are more like those of Elasmobranchs. Dipnoi, and Amphibia than of teleost fish (2, 3). Recent studies (4, 5) have shown that Latimeria hemoglobin has a high oxygen ’ This work was supported in part by United States Public Health Service Research Grant GM-05818, NSF Grant GB-27937X, and by Robert A. Welch Foundation Grant F-213. ’ United States Public Health Service Predoctoral trainee under Training Grant GM-00836; Present address, Duke University Marine Laboratory, Beaufort, NC 28516. a United States Public Health Service Predoctoral trainee under Training Grant GM-00836; Present address, Department of Biochemistry and Biophysics, Texas A and M University, College Station, TX 77843. ‘To whom requests should be addressed; Supported by United States Public Health Service Research Career Development Award, 5 K 03.GM038086.
affinity and that it appeared to consist of two kinds of polypeptide chains. The present experiments confirm and extend these results with a further examination of the hemoglobin structure and measurement of oxygen binding both in the presence and absence of phosphate. EXPERIMENTAL
PROCEDURE
In the course of these studies, blood samples from two specimens of Lutimeria chalumnue were used. Sample I was obtained from Dr. M. G. Larbaight and Dr. Emile Zuckerkandl in March, 1967. The titrated blood (10 ml) was kept on ice continuously from the time of collection at Grand Comore to the initial processing at the Institut Pasteur de Madagascar. Centrifuged, packed cells were taken up in 3 vol of 0.9% NaCl, whereupon hemolysis occurred. The preparation was centrifuged at 4°C for 10 min at 4000 rpm. The supematant was centrifuged again at 0°C for 55 min at 8000 rpm to remove stroma. This hemolysate was kept continuously on ice throughout its shipment from Tananarive to France and thence to Texas. Upon arrival in Austin, Sample I (129 mg in 8.4 ml) was saturated with CO, centrifuged, and dialyzed overnight against CO-saturated 0.1 M phosphate of pH 7.5. Sample II was obtained as packed red cells from one of two specimens of Latimeria caught during the 1972 French-British-American expedition to the Comores Islands, and shipped on ice to Texas. The cells
LATIMERIA
HEMOGLOBIN
of sample II were washed three times with 0.9% NaCl without any hemolysis, and were lysed with 0.001 M SEPHADEX G-100 Tris, pH 7.5. After concentration by pressurefiltraCHROMATOGRAPHY tion (Diaflo apparatus, UM-10 membrane, Amicon, Inc., Lexington, MA), chromatography of each hemolysate was carried out on a 3 x 68-cm column of IO Sephadex G-100. Vertical starch gel electrophoresis 0 at2t30nm was performed on the CO-hemoglobins as described I by Smithies (6), and on the globins as described by . at 540 nm Miiller (7). Globin was prepared as described previ5 ously (8). Polyacrylamide gel electrophoresis was done ii! at pH 9.2 or 8.6 in Tris-EDTA-borate buffer (Bulletin s: b No. 134, E.C. Apparatus Co., Philadelphia). Gels z containing 8 M urea were also used. Oxygen equilibria were measured at 20°C according to the method of Riggs and Wolbach (9). Sedimentation velocity measurements were made with, a Beckman Model E Ultracentrifuge as previously described (8). Separation of unoxidized polypepride chains from 30 mg of Sample II was done essentially as described for carp hemoglobin (lo), using a linear gradient between 0.2 M formic acid and 1.5 M formic acid in TUBE NUMBER 0.02 M pyridine. Amino acid analyses were performed FIG. 1. Gel chromatography of hemolysate from with a Beckman Model 120 Analyzer (8). Tryptic Latimeria chalumnae. Conditions: 3 x 68-cm column peptide maps were made from the isolated polypepof Sephadex G-100 equilibrated and eluted with tide chains as previously described (10). The amino acid sequence of the NH,-terminal segment of the /3 CO-saturated 0.2 M NaCl at 4’C; fraction size, 2.8 ml. The bars indicate the pools taken for fractions I and chain was determined by applying unfractionated II; all the hemoglobin was in fraction II. globin to a Beckman Sequencer Model 890 for automatic Edman degradation (11). This procedure yields only the &chain sequence. We have found that the cy globin, and behaved as a single component chain is blocked. The thiazolinone derivatives of each on electrophoresis of the CO-hemoglobin at residue were converted manually to the corresponding gels (Fig. 2a). phenylthiohydantoins (11) and identified either by pH 9.2 on polyacrylamide Figure 2a shows that both the unfracgas-liquid chromatography using a Beckman GC-45 tionated hemolysate and the GlOO purified chromatograph and/or by thin layer chromatography hemoglobin appeared as single electrophoas previously described (12).
:I3
t t t
RESULTS
Chromatography of the Sample I hemolysate of Latimeria on Sephadex GlOO (Fig. 1) showed that essentially all of the hemoglobin eluted as a single zone. This major zone was preceded by a small zone of nonheme protein. The elution volume of the hemoglobin zone corresponded to that expected for a tetrameric hemoglobin molecule. On either side of the hemoglobin zone small shoulders of 540-nm absorbing material were present. If these shoulders correspond to minor hemoglobin components of slightly different molecular size, they constitute less than 1% of the total hemoglobin. Chromatography and electrophoresis of samples I and II gave essentially the same results on Sephadex GlOO: Zone II (Fig. 1) contained virtually all the hemo-
retie components at pH 9.2. Starch gel electrophoresis of the globin at pH 1.9 (Fig. 2b) showed only a single band for Latimeriu globin but some material stayed at the origin. Electrophoresis of the globin at pH 3.7 and in 8 M urea at pH 8 gave single bands. These results suggest that the electric charge on the (Y and /3 chains may be very similar. Latimeria oxyhemoglobin in 0.2 M NaCl behaved as a single component in the ultracentrifuge (Fig. 3). The s20.Wvalue was 4.4 for a 0.4% oxyhemoglobin solution in 0.02 M NaCl at 20”. These results indicate that Latimeria hemoglobin is largely tetrameric under these conditions. The constituent polypeptide chains of Latimeria globin from Sample II were readily separated by the same chromatographic procedures used for carp globin
730
BONAVENTURA,
GILLEN
a. pH 9.2, Hemoglobin
AND
RIGGS
b. pH1.9,Globin
FIG. 2. Electrophoresis of hemoglobins and globins. (a) Polyacrylamide gel, pH 9.2; from left to right, human CO-hemoglobin A, Latimeria CO-hemolysate, fraction I (Fig. l), zone between fraction I and II, fI ,act ion II. (b) IStarch gel, pH 1.9; on left, human globin A; on right, Latimeria globin (both slots).
(10). The amino acid composition of these chains are given in Table I. Summation of the amino acid compositions of the (Y and /3 chains obtained from Sample II is generally consistent with the earlier results obtained on the amino acid composition of the intact globin from Sample I. Tryptic peptide patterns obtained from the isolated a and P-chains (Fig. 4) show that they are quite dissimilar. Intact globin was sequenced on the Beckman Protein Peptide Sequencer for 15 residues. The results (Table II) show that the unblocked polypeptide chain is quite similar to the B chains of teleost hemoglobins. Of particular interest is the similarity of the first eight residues with the corresponding sequences of the carp and Rio Grande cichlid @ chains. In both Latimeria and the carp, a tryptophanyl residue is found at position 3. Additionally, position 15 is occupied by tyrosine whereas tryptophan appears to be present at this position in all other vertebrate hemoglobins and myoglobins so far examined including the hemoglobins of the lamprey, Petromyzon (12), and the annelid worm, Gl.ycera dibrunchiata (15). Results of the oxygen equilibrium mea-
surements (Fig. 5) show that in the absence of phosphate, Latimeria hemoglobin has a very high oxygen affinity, and pronounced Bohr and phosphate effects. The oxygen affinity is among the highest reported for any fish hemoglobin (16). The value of n in Hill’s equation decreases from a maximal value to 1.6 at low pH to 1.0-1.1 above pH 8 in 0.1 M phosphate buffer. In the absence of phosphate the value of n decreases to about 1 at about 1 pH unit lower than its presence. The shape of the oxygen-binding curves are pH-dependent. In the presence of phosphate this hemoglobin is much more sensitive to pH between pH 7 and 8 at low degrees of oxygenation than at high levels. At a specified degree of oxygenation, Y, the pH dependence, A log pO,/A pH is a measure of the number of protons released per oxygen bound (17). The data indicate that as oxygenation proceeds protons are preferentially released from Latimeria hemoglobin protein at low degrees of OXygenation in this pH range. DISCUSSION
The data of Wood et al. (4) confirm our earlier observations of a Bohr factor of
LATIMERIA
731
HEMOGLOBIN TABLE AMINO THE
ACID
COMPOSITION
HEM~CLOBIN
Lysine Histidine Arginine Aspartat e Threonine’ Serine’ Glutamate Proline Glycine Alanine Half-cystined Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine
I
OF THE OF
12.4 8.67 1.89 11.9 9.17 5.51 11.8 3.66 5.52 14.2 1.73 11.7 5.73 18.5 3.03 9.14
LY AND
@ CHAINS
OF
Latimeria chalumnae”
13.2 9.04 4.61 11.4 11.2 7.95 14.8 3.51 4.83 10.3 1.09 11.0 0.89 6.35 19.3 3.83 6.45
25.6 17.7 6.50 23.3 20.4 13.5 26.6 7.17 10.4 24.5 2.82 22.7 0.89 12.1 37.8 6.86 15.6
26.5 17.6 6.87 22.8 21.0 13.8 27.7 8.93 9.43 24.0 3.33 22.4 0.84 12.1 39.8 6.45 14.8
“Averages of results of analyses obtained at 24., 48., 72., and (except for the intact globin) 96.hr hydrolysis in 6 N HCI under vacuum at 110°C. Molecular weights for the cy and P chains are assumed to be identical to those in human hemoglobin, 15.126 and 15,924, respectively. Numbers are residues per mole. Tryptophan was not determined. However. one residue was found in the NH, terminal segment of the fl chain. *The (Yand B chains were obtained from Latimeria hemoglobin Sample II; the unfractionated globin from Sample I. ‘Determined by extrapolation to zero time, d Determined as cysteic acid after 24.hr hydrolysis of a performic acid-oxidized sample.
about PO.55 between pH 7.0 and 8.0 for Latimeria hemoglobin in 0.1 M inorganic phosphate (18). Their data also confirm that n drops to near unity at or above pH 8 (Ref. 18 and this paper). The results presented here (Fig. 5) clearly show that the pH dependence of the oxygen affinity is decreased in the absence of phosphate. In addition, Latimeria hemoglobin becomes noncooperative at a lower pH in the absence of phosphate than in its presence. It is remarkable that the effect of a 4-fold excess of ATP over hemoglobin on the oxygen affinity of Latimeria hemoglobin is reported by Wood et al. to be small compared to the effect of 0.1 M phosphate: At pH 6.65, the Alog P,,, = 0.24 on going from
732
BONAVENTURA.
GILLEN
AND
RIGGS
FIG. 4. Tryptic peptide patterns of the o( and @chains of Latimeria hemoglobin. chain.
COMPARISON OF THE NH,-TERMINAL
TABLE II @ CHAIN SEQUENCE OF Latimeria OTHER ANIMALS
Left: (Y chain; Right: B
HEMOGLOBIN WITH THOSE OF VARIOUS
Yield (%) 12
76 64 88 15 40 26 -
54 58 64
8
31 26 20
9
12 13 14 15
Position 12345678 Latimeria
10
11
fl
Carp P (10) Rio Grande cichlid @(13) Human 6 (14) Human a (14) Petromyzon (12) Carp o((14) Myoglobin (14) Glycera monomer (15)
Tris buffer to Tris buffer + ATP. Under the same conditions, their data show that Alog PC0 = 0.73 on going from Tris buffer to phosphate buffer (4). In contrast, ATP has been reported to shift log P,, by about 1 log unit in several other fish hemoglobins (10, 13, 19). Our observations do not indicate significant heterogeneity in Latimeria hemoglobin and are consistent neither with the early report of heterogeneity on the basis of
solubility measurements (20) nor with recent results (5) obtained with isoelectric focusing. It should be noted, however, that the resolution of our electrophoretic technique is not as great as that possible with isoelectric focusing. Weber et al. (5) found about six hemoglobin components by isoelectric focusing, all very close to one another. The possibility exists that not all of these components represent hemoglobins with different primary structures since dis-
LA7’IMERIA
HEMOGLOBIN
733
version to the amino acids in the hydrolysate to the n-butyl-N-trifluoroacetyl esters, followed by gas-liquid chromatography. Previous studies (4, 5) of the functional properties of Latimeria hemoglobin have suggested placing the coelacanth in an evolutionary position between elasmobranchs and holosteans. Specialization of functional properties to suit particular environmental conditions make such phylo2.0-genetic assignments based on functional properties difficult. Sharks and their allies diverged quite early from the ancestors of bony fishes and have undergone extensive evolutionary changes (21). Comparison of the functional properties may be of greater FIG. 5. Oxygen equilibria of Latimeria hemorelaglobin. Dependence of log P,, and n on pH. Log P,, is value in assessing the evolutionary the 0, pressure (mm Hg) required for 50% oxygenationships among the teleostomes: the Diption. Open circles: measurements in 0.1 M phosphate noi, Crossopterygii, and Actinoptergii at pH 6, 7, and 8, and 0.1 MTris-HCI at pH 9.0; closed whose relationship is still unsolved (22). circles: measurements in 0.1 M NaCl, 0.05 M Bis-Tris Among the teleostomes, the lungfishes, adjusted to pH indicated, except the data at pH 8 and crossopterygians are usually believed which were obtained in 0.05 M Tris, 0.1 M NaCl. to have a closer evolutionary relationship based on morphological and paleontological observations (reviewed in 22 and 23). sociation products similar to those noted Latimeria is unusual in that its hemogloafter freezing may have been present. Weber et al. (5) reported on the basis of bin appears to contain only one major electrophoresis on polyacrylamide gels con- component: both lungfishes (Oldham, J., and Riggs, A., unpublished) and teleosts taining sodium dodecyl sulfate that Latime(18) frequently have multiple hemoglobin ria hemoglobin contains two polypeptide chains of slightly different molecular components present in appreciable weight (15,400 and 16,300). The ratio of amount. Latimeria hemoglobin is also unusual in that one of its hemoglobin chains these two weights is 0.945 which is almost The amino acid seidentical to the ratio of 0.950 for weights of lacks methionine. quence of the first 15 residues of the the aand /3 chains of human hemoglobin. We, therefore, believe that the two chains P-chain of Latimeria hemoglobin suggests which they observed are the (Y and p chains a close relationship between higher bony of the hemoglobin. fishes and this only surviving crossopteryThe amino acid composition of the iso- gian. Remarkably, the first seven residues lated polypeptide chains indicates that the of Latimeria myoglobin (24) appear to be (Y chain is devoid of methionine and that identical with those of the ,f3 chain of the /3 chain contains only a single methiLatimeria hemoglobin and appear to be onyl residue (Table I). The agreement be- quite different from other known myoglotween the analysis of the intact globin of bins. Sample I and the sum of the (Y and /3 chain ACKNOWLEDGMENTS analyses is generally within the errors of We thank Dr. M. G. Larbaight and Dr. Emile measurement. The agreement with the Zuckerkandl for sample I, Mr. Paul K. Brown for analysis by Weber et al. (5) is less satisfacfacilitating its shipment, and the participants of the tory. The lack of agreement is perhaps due French-British-American 1972 Expedition to the to methodological differences. Their analyComores for sample II. We thank Mrs. Marie Ervin for sis was carried out on a hydrolysate of the amino acid analyses and Mrs. Florence Waddill hemoglobin, not globin, and involved con- for operating the sequencer.
734
BONAVENTURA,
GILLEN
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247,6039. 11. EDMAN, P., AND BEGG, G. (1967) Eur. J. Biochem. 1, 80. 12. LI, S. L., AND RIGGS, A. (1970) J. Riol. Chem. 245, 6149. 13. GILLEN, R. G., AND RIGGS, A. (1973) Arch. Biothem. Biophys. 154, 348.
AND
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14. DAYHOFF, M. V. (1972) Atlas of Protein Sequence and Structure, pp. D56-D85, National Biomedical Research Foundation, Silver Spring, MD. 15. IMAMURA, T., BALDWIN, T. O., AND RIGGS, A. (1972) J. Biol. Chem. 247, 2785. 16. SULLIVAN, B., AND RIGGS, A. (1971) in Respiration and Circulation (Altman, P. L., Ed.), pp. 1922194, Federation of American Societies for Experimental Biology, Washington, DC. 17. WYMAN, J. (1948) Aduan. Protein Chem. 4, 407. 18. RIGGS, A. (1970) in Fish Physiology (Hoar, W. S., and Randall, D. J., eds.), pp. 209-252, Academic Press, New York. 19. GILLEN, R. G., AND RIGGS, A. (1971) Comp. Biothem. Physiol. 38B, 585. 20. DE PRAILAUNE, S. (1955) C. R. Sot. Biol. 149,655. 21. YOVNG, J. Z. (1962) The Life of Vertebrates, Oxford Univ. Press, New York. 22. MOY-THOMAS, J. A. (1971) Palaeozoic Fishes, Saunders, Philadelphia, Pa. 23. ROMER, A. S. (1968) Notes and Comments on Vertebrate Paleontology, Univ. of Chicago Press, Chicago, IL. 24. CHAUVET, J.-P., AND ACHER, R. (1972) Fed. Eur. Biochem. Sot. Letters 28, 16.