affinity of whole blood can play an important role in the regulation of the DIURING a study of the adaptations made by pregnant ewes and their fetuses

Transcription

1 HEMOGLOBIN CHARACTERISTICS AND THE OXYGEN AFFINITY OF THE BLOODS OF DORSET SHEEP.* By M. A. NAuGHTON,t G. MESCHIA, F. C. BATTAGLIA,t A. HELLEGERS, H. HAGOPIAN and D. H. BARRON. From The Division of Biochemistry, Department of Biology, Massachusetts Institute of Technology, Cambridge, Mass. and The Department of Physiology, Yale University School of Medicine, New Haven, Connecticut. (Received for publication 15th January 1963) Two electrophoretically distinguishable hemoglobins have been identified in the bloods of pure bred Dorset sheep. The oxygen affinity of the whole blood, as judged by the oxygen pressure required to half-saturate its hemoglobin at 380 C. and ph 7-4 has been shown to be correlated with the relative proportions of the two hemoglobins. Blood containing only the hemoglobin which moves the more rapidly in the electrophoretic field has a higher oxygen affinity than the one with the lower mobility. The two hemoglobins appear to differ in the amino acid sequence in the beta chain of the molecule. DIURING a study of the adaptations made by pregnant ewes and their fetuses to the low oxygen pressure of high altitude, oxygen dissociation curves of bloods of a number of sheep of Merino stock were prepared at Morococha, Peru in the autumn of 1958 [Meschia et al., 1961 a]. The results demonstrated a lack of uniformity in the oxygen affinities of the bloods of the individual ewes, for some were half-saturated at an oxygen tension of mm. Hg at plasma ph 7.4 and 380 C., whereas to saturate others to the same degree, in the same circumstances, required an oxygen tension of mm. A similar study on the bloods of pedigree Dorsets at sea level [Meschia et al., 1961 b] revealed a comparable variation in their oxygen affinities. Differences in the oxygen affinities of the bloods of individuals of the same species, when compared at the same plasma ph, could be the result of: (1) differences in their hemoglobins, (2) differences in the concentrations of other solutes within the red cell (intracellular ph, etc.) or (3) a combination of the two circumstances. These factors by their effects on the oxygen affinity of whole blood can play an important role in the regulation of the circulation and respiration of the individual. Yet there is at present no agreement of the relative importance of these possible sources of variation. In an effort to learn something of the relative importance of the structure * This study was aided by grants from the National Institutes of Health and the Josiah Macy Jr. Foundation. t Present address: Department of Biophysics, Johns Hopkins Medical School, Baltimore, Maryland. 4 Research Fellow of the Josiah Macy Jr. Foundation. Present address: Department of Pediatrics, Johns Hopkins Medical School, Baltimore, Maryland. Senior Research Scholar, Joseph P. Kennedy Jr. Memorial Foundation Department of Gynecology-Obstetrics, Johns Hopkins Medical School, Baltimore, Maryland. VOL. XLVIII, NO

2 314 Naughton, Meschia, Battaglia, Hellegers, Hagopian and Barron of the hemoglobin and of its environment within the red cell, in the determination of the character of the oxygen dissociation curve of the blood of Dorset sheep, we have attempted to relate properties of the hemoglobin to the oxygen affinity of the blood of the same individual. Data already in the literature pointed to the existence of such a correlation. Thus, Harris and Warren [1955] have shown that in the bloods of a number of breeds of sheep there are two electrophoretically different types of hemoglobin. Huisman et al. [1958] have presented evidence that the oxygen affinity of the blood of individuals of a Dutch breed, Texel, can be correlated with the presence of one or the other of two electrophoretically distinguishable hemoglobins and, further, that the oxygen affinities of the two hemoglobins in solution differ in the same way as the oxygen dissociation curves of the whole blood, i.e., the blood with the higher oxygen affinity contains the hemoglobin with the higher affinity. A similar correlation between the oxygen affinity of the whole blood and that of the hemoglobin it contains has not been observed in a variety of human bloods [Allen et al., 1953; Prystowsky et al., 1959; Schruefer et al., 1962]. The data presented here demonstrate the existence of two electrophoretically distinguishable hemoglobins in the bloods of pedigree Dorset sheep and a correlation between the oxygen affinity of the blood of an individual and the specific hemoglobin or mixture of hemoglobins in it. In addition, they provide evidence that the two hemoglobins differ by a minor change in the amino acid sequence of the beta chain of the molecule. MATERIALS AND METHODS The fourteen ewes from which bloods for this study were taken were purchased from the Taunton Hill Farm, Newtown, Connecticut, breeders of pedigree Dorsets. They were on pasture and all apparently in good health at the time the blood samples were drawn. Dissociation Curves.-The bloods for the preparation of the oxygen dissociation curves were drawn into syringes containing a solution of potassium oxalate and sodium fluoride (2 per cent K2C204 and 0-6 per cent NaF) in such an amount that the fluid mixture contained nine parts of blood and one of anticoagulant. Following the procedure described earlier [Hellegers et al., 1959] two dissociation curves at different plasma, hydrogen ion concentrations were prepared for each blood. Four points were established on each curve. Hemoglobin.-The bloods for the preparation of the hemoglobin solutions were drawn into heparinized syringes. The red cells, separated by centrifugation, were washed four times in cold isotonic saline. The erythrocytes were then lyzed by freezing and thawing and the ghosts removed by centrifugation at 10,000 x g. for 20 min. The supernatant hemoglobin solution was used without further purification. Starch Get Ionophoresis.-Samples of each hemoglobin solution were run in starch gel ionophoresis by the method of Poulik [1957]. Analysis of the Tryptic Digests.-Tryptic digestion of hemoglobin and of the globin fraction was carried out in a ph-stat at ph 8-0 as described by Ingram [1958]. The analysis of the peptides resulting from the digestion was made on an aliquot which corresponded to 2 mg. of the original material making use of Baglioni's modification [1961] of Ingram's method [1956] for a two-dimensional combination

3 >OrigirI F ' Electrophoretic patterns; oni -tar(h.gel of the hiemioglobins in bloodis fromn Dorset S hieep. The faster-nox-ing of the slingle bann(1s is hiem--ogloblin fromy shieep 206.5, the slowerimo-inig single hand fromi- sheep 189 aind the mnixture fromi shieep 170. Fin-. 2.-Fingerprints of the hemoglobin In the three bloodis wvhose eleet rophoret ic patternis are ilustr-ated1 In fig. I A, the fast-moving (shieep 2065); B3, tihe s1ow~-mov\ing (shiee-p 189) and (C, the mixture (sheep- 1,70). rmo face page :315]

4 Hb Characteristics and 02 Affinity 315 of paper ionophoresis and chromatography-a procedure termed by Ingram " fingerprinting ". The stains used for the specific amino acids of the fingerprint peptides were: for arginine, the Sakaguchi reaction as modified by Acher; for methionine, the platinic iodide method of Toennies; for histidine, the Pauly reaction using sulfanilic acid; for tryptophan, Erlich's stain and for tyrosine, the reaction with alpha-nitroso-beta-napthol and nitric acid. Each of these reactions is described by Block et al. [1958]. Preparation of the Globin.-The globin fraction was obtained by treatment of the hemoglobin with acid acetone. Separation of the Alpha and Beta Chains of Sheep Hemoglobin.-The carboxy methyl cellulose (CMC) method of Dintzis [1961], originally used for rabbit hemoglobin, was found to work very well for the separation of the alpha and beta chains. The particular batch of carboxy methyl cellulose used was obtained from Serva, Heidelberg, Germany, Batch No , 0-72 mg./g. Other samples were tried but they either did not work or gave poor results. Amino Acid Analysis.-The amino acid composition of some of the peptides was determined by the method described by Naughton and Hagopian [1962]. RESULTS Starch Gel Ionophoresis.-Electrophoretic analysis of the hemoglobins demonstrates that, on the basis of mobility, two varieties can be recognized. The blood of some ewes contains hemoglobin of both varieties; the bloods of others contain only one. Examples of the different patterns are shown in fig. 1. The blood of sheep 2065 contains only one hemoglobin, which we have designated A, whereas the blood of sheep 189 contains a slower moving variety which we have designated B. A mixture of the two varieties is present in the blood of sheep 170. Blood samples from these three sheep were used for all of the subsequent analyses. Fingerprints of the Hemoglobins.-Fig. 2 shows the fingerprints of the tryptic digests of the hemoglobins from the three sheep. A comparison of the hemoglobins A and B, from sheep 2065 (fig. 2a) and of sheep 189 (fig. 2b) respectively, demonstrates that they differ in detail and when they are compared with the fingerprint of the hemoglobin mixture in the blood of sheep 170, it is clear that the latter contains all of the peptides present in the other two. Tracings of the individual spots of the fingerprints of the two Dorset sheep hemoglobins are presented in fig. 3; in those cases in which it was possible to establish, by staining, the presence of one or more specific amino acids in a particular peptide group the spot has been marked with key letters of identification. The difference between the two appears to be limited to the four methionine containing peptides, indicated in the figures by the cross-hatched spots; for the sake of comparison, we have included in fig. 3 a fingerprint of human hemoglobin A as prepared by Baglioni [1961]. As can be seen, there is a marked similarity between the number, the position and the response to specific amino acid stains of the peptides of the hemoglobins of man and of the sheep. Globin Analysis.-The globins obtained from hemoglobins A and B

5 316 Naughton, Meschia, Battaglia, Hellegers, Hagopian and Barron TYR' a Q 325 OX S) 21 HUMAN Hb-A :MlGD T (10~ - 0+ SHEEP 189 SHEEPP2065 m0 FIG. 3.-Tracings of the fingerprints of (1) htumna hemoglobin A, (2) sheep hemoglobin B and (3) sheep hemoglobin A. The key letters in the outlines indicate amino acids identified in the peptide represented arg =arginine; his =histidine; meth =methionine; try =tryptophan; ty2 =tyrosine.

6 Hb Characteristics and 02 Affinity 317 separated into two fractions when run on a chromatographic column of carboxy methyl cellulose (according to the method of Dintzis [1961]. The result of one such run is illustrated in fig. 4. One hundred mg. of globin were run on a 20-cm. column. The tubes correpsonding to the regions marked "alpha" and "beta" were collected, the peptide material they contained was precipitated with 5 per cent trichloracetic acid, centrifuged and then washed with ether to remove the TCA. Forty mg. of alpha fraction and 35 mg. of beta fraction were recovered. We have called these two C.750A Milliliters of EffluentEE FIG. 4 FIG. 5 :FIG. 4.-Chromatographic separation of alpha and beta chains of sheep hemoglobin. :FIGE. 5.- Tracings of the fingerprints of the alpha chains of sheep hemoglobin B (sheep 189) and sheep hemoglobin A (sheep 2065). Key letters are as in fig. 3. fractions alpha and beta respectively as their fingerprint patterns correspond very closely to those of the alpha and beta chains of the hemoglobins A and B respectively. The prints of the alpha chains are illustrated in fig. 5 and those of the beta chains in fig. 6. The fact that the four methionine staining peptides in which the differences between hemoglobins A and B are centered appear only in the fingerprints of the beta chains, suggests that the differences are limited to that part of the molecule constituted by the beta chains. The. Oxygen Affnity of the Whole Blood.-Blood samples from each of the three sheep-2065, 189 and 170 respectively-were equilibrated in tonometers at 380 C. with selected mixtures of oxygen, carbon dioxide and nitrogen. After equilibration, the ph of the whole blood, the oxygen tension in the gas of the tonometer and the percentage saturation of the hemoglobin determined. The results are presented in Table I. In an earlier report Meschia et al. [1961 b] pointed out that the oxygen dissociation curves of the bloods of sheep can be accurately described, between 10 and 90 per cent saturation, and at ph values in the physiological range, by A. V. Hill's equation [1910] modified to take into account the effect of ph. For a comparison of the oxygen affinities of the three bloods

7 318 Naughton, Meschia, Battaglia, Hellegers, Hagopian and Barron whose electrophoretic patterns are presented above, the equation can be modified to the following form: log P02 =log (PO2) 60, K2 (ph - 7-4) + -log S n 100- s for the oxygen when P02 stands for oxygen pressure in mm. Hg (PO2)50,,o.4 TR n( 0 SHEEP SHEEP 2065/3 FIG. 6.-Tracings of the fingerprints of the beta chains of the two sheep hemoglobins B (sheep 189) and A (sheep 2065). The cross-hatched areas represent the peptides associated with the differences in the mobilities of the two hemoglobins. Key letters as in fig. 3. pressure at which the hemoglobin is half-saturated at ph 7.4; K2 and n are two coefficients and S is the saturation of the hemoglobin with oxygen. A review of the data in Table I demonstrates that all three of the dissociation curves can be described by the equation, though the values of (log P02)50,74 K2 and n needed to give the best fit for the data are different for each of the three bloods (see Table II). In fig. 7, log lo- Sis plotted against

8 Hb Characteristics and O2 Affinity 319 P02 at ph 7.4, and it is clear that with respect to the positions of the lines on the graph-defined by log (PO2) 0,,.4 -the three bloods differ significantly. The slope of the lines, which depend upon the value of n, appears, within the limits of experimental error, to be the same. These results are in TABLE I.-DATA ON ADULT SHEEP BLOOD EQUILIBRATED AT 380 C. WITH MIXTURES OF 02, CO2 AND N2 ph of O Percentage Animal whole P Hg saturation blood of hemoglobin * X5 7* * * * * * * *50 41X0 70X * * TABLE II.-A COMPARISON OF CHARACTERISTICS RELATED TO THE OXYGEN AFFINITIES OF THE BLOODS OF DORSET EWES * Animal (P 2) K2 n Hemoglobin types * A * B * A+B * The symbols used for the characteristics are explained in the text and related in the equation on p agreement with earlier observations on the oxygen affinity of the blood of sheep [Meschia et al., 1961 b]. The values for K2 given in Table II fall within the range found for the bloods of other sheep and the difference in the K2 values for the bloods of sheep 2065 and 189 respectively, is well within the limits of the error of our measurements. Accordingly, the principal difference in the oxygen affinities of the three bloods considered here is represented by the position of their oxygen dissociation curves at a given ph.

9 320 Naughton, Meschia, Battaglia, H.llegers, Hagopian and Barron DISCUSSION The data in Table II and in fig. 7 demonstrate a clear correlation between the type of hemoglobin (as judged by its electrophoretic mobility) in a blood and its oxygen affinity as indicated by the position of the dissociation curve at plasma ph 7.4. This correlation is confirmed by the fact that the curve of the blood of sheep 170 which contains a mixture of the two hemoglobins, A and B, is intermediate in position between the other two. F A A+B B g P 2 at ph 7.4 FIG. 7.-The logarithm of the ratio, saturated/unsaturated hemoglobin, is plotted against the logarithm of oxygen pressure in mm. Hg. Four samples of blood are compared at ph 7.4 and 380 C.; F, fetal blood; A, blood from sheep 2065; A and B, sheep 170 and B, sheep 189. A similar correlation between the electrophoretic mobility and the oxygen dissociation curve can be observed in the whole blood of the sheep fetus. The fetal blood contains still a different type of hemoglobin [Karvonen, 1949; Drury and Tucker, 1962] from that found in the bloods of adult Dorsets and the oxygen dissociation curve also differs from the adult curves in its position at a given ph though the value of the coefficient n is the same for both adult and fetal blood [Meschia et al., 1961 b]. Further, the bloods of neonatal lambs have oxygen dissociation curves intermediate between the fetal and adult that approach the adult with the advancing age of the lamb [Barron, 1951; Meschia et al., 1961 b] and there is evidence that in this same neonatal period [Karvonen, 1949; Drury and Tucker, 1962] the fetal hemoglobin is being gradually replaced by an adult type. In short, the results which we

10 Hb Characteristics and 02 Affinity have obtained demonstrate a correlation between the position of the oxygen dissociation curve at ph 7.4 and the electrophoretic properties of the hemoglobin or hemoglobins in the blood of individual Dorsets though they provide no evidence that there is a causal relation between them. If the characteristics of the hemoglobin which determine the oxygen affinity of the whole blood in Dorsets are-like those that determine electrophoretic mobility-under genetic control [Ingram, 1961], it should be possible to breed a strain of Dorsets especially adapted to life at high altitude and able to graze on pastures at 14,000 ft. and above. Animals with a blood having a high oxygen affinity, such as the llama [Meschia et al., 1960], are well adapted to life at high altitude. Sheep 189 is well suited to life at sea level, where nearly all of its hemoglobin is saturated with oxygen in its passage through the lung capillaries, but the low oxygen affinity of its blood would appear to represent a handicap at high altitude by imposing there a greater stress upon its circulatory and respiratory systems than would be the case if the blood had a high oxygen affinity. Thus small differences in the hemoglobin may prove to have important consequences in the selection of an animal's habitat. Taking the fingerprint of human hemoglobin A as a standard for reference, it is clear from fig. 3 that those of the two sheep hemoglobins are similar to it and to each other with respect both to the number of peptides present and their general pattern of distribution. The similarity is further strengthened by matching the reactions of those peptides having the same position in the fingerprints to the specific amino acid stains. (The numbering system of the peptides in fig. 3 is that of Baglioni [1961].) Apparently, a considerable number of the peptides in the sheep hemoglobin contain one or more of the same amino acids that are found in the corresponding peptides of human hemoglobin A. Taken together, these facts suggest that there is a close similarity between the amino acid sequence in certain regions of the hemoglobin molecules of the two species, man and sheep. According to the results of our analysis the differences between the two hemoglobins of Dorset sheep may be limited to their beta chains for the fingerprints of the tryptic digests of their alpha chains (fig. 5) appear to be identical. A comparison of the two hemoglobins, A and B, suggests that a peptide with a positive reaction for methionine presen-t in the beta chain of hemoglobin A (sheep 2065) has been replaced by three methionine-positive peptides in the fingerprints of the beta chain of hemoglobin B (sheep 189). But this finding does not exclude the possibility that the substitution of a single amino acid in the beta chain is responsible for the difference between the two hemoglobins. A clear example of the way in which the substitution of one amino acid in the chain can give rise to complex changes in the peptides of its tryptic digest is given by a comparison of the fingerprints of the alpha chains of the human and sheep hemoglobins. The fingerprint of the alpha chain of the sheep hemoglobin has no free lysine (Spot No. 22 in fig. 3) and peptide Spots Nos. 20 and 21 in fig. 3 are missing in figs. 5 a and b. In the human alpha chain the appearance of these three peptides in the 321

11 322 Naughton, Meschia, Battaglia, Hellegers, Hagopian and Barron fingerprint is due to partial and complete digestion by trypsin of the amino acid sequence; glycine, histidine, glycine, lysine, lysine. The three units that appear as a consequence of complete and incomplete tryptic digestion together with their fingerprint numbers are illustrated in the scheme below: 21 gly-his-gly-lys lys Peptides Nos. 20 and 21 are also characterized by the fact that they are stained yellow with ninhydrin, due to the glycine as the N-terminal amino acid. A peptide giving a yellow color with ninhydrin and a positive reaction for histidine is present in the fingerprint of the alpha chain of the sheep hemoglobin. This peptide, labeled X in the tracings of fig. 5a was prepared in larger amounts by the methods described for rabbit hemoglobin [Naughton and Dintzis, 1962] and found to have the following composition: glycine2, histidine, glutamic acid, lysine. Accordingly, the first lysine that occurs in the peptide from the human alpha chain has been replaced in the sheep by glutamic acid and the presumption is that the substitution of this one amino acid resulted in a single spot in the fingerprint in place of the three in the fingerprint of the human hemoglobin. REFERENCES ALLEN, D. W., WYMAN, J. Jr. and SMITH, C. A. (1953). J. Biol. Chem. 203, 81. BARRON, D. H. (1951). Yale J. Biol. Med. 24, 191. BAGLIONI, C. (1961). Biochen. et Biophys. Acta. 48, 392. BLOCK, R. J., DURRUM, E. L. and GUNTER, Z. (1958). A Manual of Paper Chromatography and Paper Electrophoresis, 2nd Ed., p New York. DINTZIS, H. M. (1961). Proc. Nat. Acad. Sci. 47, 247. DRURY, A. N. and TUCKER, E. M. (1962). J. Physiol. 162, 16P. HARRIS, H. and WARREN, F. L. (1955). Biochem. J. 60, Proc. xxix. HELLEGERS, A., MESCHIA, G., PRYSTOWSKY, H., WOLKOFF, A. S. and BARRON, D. H. (1959). Quart. J. exp. Physiol. 44, 215. HILL, A. V. (1910). J. Physiol. 40, 4P. HUISMAN, T. H. J., VLIET, G. VAN and SEBENS, T. (1958). Nature, 182, 171. INGRAM, V. M. (1956). Nature, 178, 792. INGRAM, V. M. (1958). Biochem. et Biophys. Acta. 28, 539. INGRAM, V. M. (1961). Hemoglobin and its Abnormalities. Springfield: Thomas. KARVONEN, M. J. (1949). In Haemoglobin. Ed. Roughton and Kendrew. London: Butterworth.

12 Hb Characteristics and 02 Affinity 323 MESCHIA, G., PRYSTOWSKY, H., HELLEGERS, A., HUCKABEE, W., METCALFE, J. and BARRON, D. H. (1960). Quart. J. exp. Physiol. 45, 284. MESCHIA, G., PRYSTOWSKY, H., HELLEGERS, A., HUCKABEE, W., METCALFE, A. and BARRON, D. H. (1961 a). Quart. J. exp. Physiol. 46, 156. MESCHIA, G., HELLEGERS, A., BLECHNER, J. N., WOLKOFF, A. S. and BARRON, D. H. (1961 b). Quart, J. exp. Physiol. 46, 95. NAUGHTON, M. A. and HAGOPIAN, H. (1962). Anal. Biochem. 3, 276. NAUGHTON, M. A. and DINTZIS, H. M. (1962). Proc. Nat. Acad. Sci. 48, PRYSTOWSKY, H., HELLEGERS, A. E., COTTER, J. and BRUNS, P. D. (1959). Gynec. 77, 585. POULIK, M. D. (1957). Nature, 180, Am. J. Obst. SCHRUEFER, J. J. P., HELLER, C. J., BATTAGLIA, F. C. and HELLEGERS, A. E. (1962). Nature. [In the Press.]