Heterogeneity of Amino Acid Sequence in Hippopotamus ...

Vol. 253, No. 24, Issue of December Prrnted m U S A.

Heterogeneity Cytochrome

25, pp 8957-8961,1978

of Amino

Acid

Sequence

in Hippopotamus

c* (Received for publication, January 23, 1978)

Richard From

B. Thompson,*

the Department

Dennis

of Biochemistry

Borden,

George

and Molecular

E. Tarr,

Bzology,

The amino acid sequences of the cytochromes c from over 75 different eukaryotic species have been determined to date (1, 2), including 18 mammals, 15 nonmammalian chordates, 4 insects, 1 mollusc, 4 protists, 6 fungi, and 26 higher plants. This represents the most extensive compilation available for any one set of orthologous proteins. It has been used as a model for the study of evolutionary variability of proteins (3-15), while the numerous primary structure variants of the same spatial conformation are particularly convenient for structure-function studies (5, 16-20). Remarkably, in this extensive survey of amino acid sequences there have been very few reports of heterogeneity, even though as many as 79% of the residues have been observed to vary among the cytochromes c of different species (1, 2). As previously reported (21), mules and hinnies carry equal proportions of horse and donkey cytochromes c, a result of their hybrid origin and a phenomenon of no immediate evolutionary significance. Possible cytochrome c heterogeneity among mammals has also been detected in the case of the human protein (22), but this is likely to have resulted from * This work was supported by Grants GM 19121 and HL 11119 from the National Institutes of Health and Grant DEB 76-81694 from the National Science Foundation to E.M. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact. $ Present address, Department of Biochemistry, School of Chemical Sciences, University of Illinois, Urbana, Ill. 61801. 5 To whom reprint requests should be addressed.

University,

Evanston,

Illinois

60201

transpeptidation during chymotryptic digestion. Other cases of the presence of more than one cytochrome c are those of the mouse (23-25), the carp (26), the box elder (27), and baker’s yeast (28, 29) proteins. For the carp and box elder cytochromes c, one position was occupied by 2 different residues in approximately equal proportions. Since the proteins were prepared from a large number of individuals, the populations were probably either entirely heterozygous as a result of controlled breeding, or a mixture of populations had been employed. In the cases of the mouse somatic and testicular cytochromes c and the baker’s yeast iso-l and iso- cytochromes c, the proteins differ in many positions and are obviously the products of independently evolving genes that had duplicated and separated early in evolutionary history. As reported below, hippopotamus cytochrome c prepared from four hearts contained 10.6% of Residue 3 as isoleucine, the rest being valine, indicating that one of the four animals was heterozygous in the cytochrome c gene. This is the first report of heterogeneity in cytochrome c arising spontaneously in a wild population. As such, it is a condition of the normal evolutionary variation of protein structure. EXPERIMENTAL

PROCEDURES

Cytochrome c (1.6 g) was prepared from four hearts of Hippopotamus amphibius obtained from the Kruger National Park, Republic of South Africa, according to the procedure of Margoliash and Walasek (30). Trypsin (L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated) and chymotrypsin (three times crystallized) were from Worthington, Staphylococcus aureus protease (31,32) was from Miles Laboratories and the reagents used for Edman degradation were Pierce “Sequanal grade.” Pyridine was redistilled over p-toluenesulfonyl chloride. All other chemicals were reagent grade. Amino acid compositions were determined with a Durrum D-500 automatic amino acid analyzer following hydrolysis in 6 N HCl at 1lO’C in uacuo for 20 to 72 h. Preparation and Isolation of Peptides-For both tryptic and chymotryptic digestions, 16 pmol of cytochrome c in 20 ml of 0.1 M NH4HC01 were treated twice with 6 mg of enzyme, at 0 and 3 h. Digestion was stopped by immersion in boiling water for 3 min, after 6 h for trypsin and 12 h for chymotrypsin. The digests were centrifuged and the supernatants were lyophilized. The chymotryptic digest was chromatographed on a column (1 X 100 cm) of Dowex 50-X2 (Bio-Rad, equal amounts of 80 to 200 and 250 to 325 wet mesh beads), according to Margoliash and Smith (33). The column was eluted successively with 500 ml of 0.2 M pyridine acetate, pH 3.1, a linear gradient (total of 4 liters) to 2.0 M pyridine acetate, pH 5.0, and 500 ml of 8.0 M pyridine acetate, pH 5.6. Fractions (5 ml) were assayed by the fluorescamine (Roche) reaction according to Bohlen et al. (34), pooled, concentrated by rotary evaporation, and lyophilized. The pooled fractions, designated C-l to C-14, were subjected to peptide mapping on cellulose thin layer plates (20 X 20 cm) (Brinkmann) (35, 36). Peptides were detected by reaction with ninhydrin, purified by either chromatography or electrophoresis on Whatman No. 3MM paper according to their peptide map positions (33, 36), eluted with water, and lyophilized. Purity was assessed from amino acid composition. The presence of tryptophan was detected by the Ehrlich reaction on the peptide maps (37). The chymotryptic heme peptide was not eluted by the pyridine

8957

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The amino acid sequences of chymotryptic and tryptic peptides of Hippopotamus amphibius cytochrome c were determined by a recent modification of the manual Edman sequential degradation procedure. They were ordered by comparison with the structure of the hog protein. The hippopotamus protein differs in three positions: serine, alanine, and glutamine replace alanine, glutamic acid, and lysine in positions 43, 92, and 100, respectively. Since the artiodactyl suborders diverged in the mid-Eocene some 50 million years ago, the fact that representatives of some of them show no differences in their cytochromes c (cow, sheep, and hog), while another exhibits as many as three such differences, verifies that even in relatively closely related lines of descent the rate at which cytochrome c changes in the course of evolution is not constant. Furthermore, 10.6% of the hippopotamus cytochrome c preparation was shown to contain isoleucine instead of valine at position 3, indicating that one of the four animals from which the protein was obtained was heterozygous in the cytochrome c gene. Such heterogeneity is a necessary condition of evolutionary variation and has not been previously observed in the cytochrome c of a wild mammalian population.

and E. Margoliashg

Northwestern

Heterogeneity

8958

of Hippopotamus

Cytochrome

Ace+yl-Gly-Asp-~~-Glu-Ly.-Gly-Lya-Lys-Ils-~~-~al-Gln-Ly.-Cys-Ala-Gln-C

c Sequence

s-His-Thr-V&l-

HEME

1

Glu-Lys-Gly-Gly-Lyr-His-Lys-Thr-Gly-Pro-Asn-Leu-His-Gly-Leu-Phe-Gly-Arg-Lys-

30

Thr-Gly-Gln-~-Pro-Gly-Phe-Ser-Tyr-Thr-A~p-Ala-Asn-Lys-Asn-Lyr-Gly-Ile-Thr-

40

FIG. 1. Amino acid sequence of hippopotamus cytochrome c. The positions at which this protein differs from hog cytochrome c are underlined. The heterogeneity at position 3 is indicated by the isoleucine above the line.

50

Trp-Gly-Glu-Glu-Thr-Leu-Met-Glu-Tyr-Leu-Glu-~~-Pro-Lys-Ly~-Tyr-Ile-Pro-

60 Gly-Thr-Lys-Met-Ile-Phe-Ala-Gly-IIe-Lys-Ly~-Lyr-Gly-Glu-Arg-~-Asp-Leu-Ile-Ala-

90

60 Tyr-

Leu-Lys-z-

Ala-Thr-

Asn-GluCOOH

100

104

RESULTS

The amino acid compositions and positions in the final sequence of the chymotryptic and of the tryptic peptides isolated, as well as chromatogram of the chymotryptic and tryptic digests of the protein, are given in the Supplementary Material at the end of the paper,’ while Fig. 1 lists the amino acid sequence of hippopotamus cytochrome c. The amino acid sequences of all peptides studied were determined by the Edman sequential degradation procedure, and the relative ’ Portions of thii paper (including Figs. 1, 2, and 3 and additional Table I) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 RockviIle Pike. Bethesda, Md. 20014. Request Document No. 78M-114, cite autho&), and include a check or money order for $1.20 per set of photocopies.

positions of the chymotryptic peptides were decided by comparison with the known amino acid sequences of the cytochromes c of other mammalian species. The only residue positions which showed differences from the corresponding positions of hog cytochrome c (45) were Residues 43 (serine replacing alanine), 92 (alanine replacing glutamic acid), and 100 (glutamine replacing lysine). Table I lists the amino acid composition of hippopotamus cytochrome c as determined by amino acid analysis, and demonstrates that it is the same as that derived from the amino acid sequence. The second major fraction from the Dowex 50 column chromatography of the initial tryptic digest (Fraction 5) showed on peptide mapping a major acidic peptide and a faint, chromatographically more mobile spot nearby. Amino acid analysis showed that Fraction 5 consisted of the NH*terminal pentapeptide of the protein. The two spots were partially separated by paper chromatography. The slower fraction (5A) had essentially the same composition as the original, while the faster (5B) contained instead of 1 valine, approximately equal amounts of valine and isoleucine (see Table II). Together, valine and isoleucine amounted to 1 full TABLE I Amino acid composition of hippopotamus cytochrome c The values in the “From analysis” column represent average or extrapolated values from duplicate acid hydrolysates of the whole protein for 24, 48, and 72 h. Cystine was not accurately determined by this procedure, and proline was not accurately read by the amino acid analyzer employed. The values in the “From amino acid seof quence ” column are based on the results of Edman degradation the chymotryptic and tryptic peptides isolated. N.D., not determined. From amino acid Residues From analysis Wq”.SlVX res~dues/molecule 8 Aspartic acid 8.19 Threonine 7.98 8 2.43 2 Serine 12.59 12 Glutamic acid N.D. 4 Proline 14.10 14 Glycine 6.46 6 Alanine 2 N.D. l/2-Cystine 3 Valine 3.03 2 Methionine 1.67 5.91 6 Isoleucine 6.22 6 Leucine 4.06 4 Tyrosine 4.08 4 Phenylalanine 3 Histidine 2.89 16.95 17 Lysine 2.12 2 Arginine 1 Tryptophan N.D.


acetate buffers. The red-colored portion of the column was removed and eluted with 2.5 N NH40H, diluted, and dried in a rotary evaporator. Peptide maps and amino acid composition indicated no further purification was required. The heme was removed by reaction with o-nitrophenylsulfenyl chloride (38) and the released cysteinyl residues carboxymethylated with iodoacetate (39). The tryptic digest of 16pmol of cytochrome c was chromatographed on a column of Dowex 50, as described above for the chymotryptic digest. Only seven fractions were purified. One of these (Fraction 5) contained the two NH*-terminal pentapeptides (see “Results”). To confirm the heterogeneity at Residue 3 and to obtain a peptide containing Residues 34 to 36, a tryptic digest of 15 mg (1.2 pmol) of the protein was chromatographed on a column (0.4 X 15 cm) of Beckman PA-35 resin eluted with two successive linear gradients from 0.05 to 0.2 M pyridine acetate, pH 3.25 (total volume 50 ml), and from 0.2 M pyridine acetate, pH 3.25, to 2.0 M pyridine acetate, pH 5.2 (total volume 250 ml). Fractions of 1.3 ml were collected, assayed with fluorescamine, and processed as described above for the chymotryptic fragments. The chymotryptic NH*-terminal decapeptide was digested with Staphylococcus aureus protease yielding two fragments. That containing Residues 5 to 10 was subjected to Edman degradation. The tryptic NH?-terminal pentapeptide was treated with 0.1 N acetic acid at 100°C for 7 h (36) and the mixture was subjected to Edman degradation to establish the sequence of Residues 2 to 5. Edman Degradation-The procedure of Tarr (40) was used, except that 5-ml centrifuge tubes, larger reagent volumes, and Nz stream drying were employed. The heme peptide, residues 11 to 26, was degraded on a Beckman 890C Sequencer. Phenylthiohydantoin amino acids were identified by thin layer chromatography on Eastman 6060 silica gel plates (5 X 5 cm) with solvent V of Jeppson and Sjoquist (41). The plates were sprayed with 0.1% ninhydrin in ethanol and heated for 5 min at 110°C to develop the characteristic colors of the different derivatives (42, 43). The phenylthiohydantoin derivative of arginine was identified with phenanthrenequinone (44). For some peptides, after the penultimate residue was identified as the phenylthiohvdantoin derivative. the COOH-terminal residue was identified by akin0 acid analysis without acid hydrolysis.

Heterogeneity

of Hippopotamus

TABLE II Amino acid composition of NH&erminal pentapeptide hippopotamus cytochrome c Fraction” Residues

Cytochrome

c Sequence

8959

far, the camel (49) and the guanaco (50), species which are in the same family (Camelidae), are identical and differ by 2 residues from the hog protein. This remarkable variability in the number of structural differences among the cytochromes c of species whose lines of evolutionary descent are thought 5A 5B 5 to have all diverged in the mid-Eocene, some 50 million years residues/molecule ago, indicates that the rate of accumulation of evolutionary Aspartic acid 1.07 1.03 1.07 protein variations is not constant, even in fairly closely related 1.24 1.21 Glutamic acid 1.24 groups. Recent analyses of statistical phylogenetic trees deGlycine 0.89 0.91 0.94 0.91 0.95 0.45 Valine rived for several different proteins have shown that this is the Isoleucine 0.09 0.03 0.50 case in general. It was further demonstrated that the rate of 0.89 0.76 Lysine 0.89 residue change is similarly not constant for one protein in the n The peptide fractions analyzed are described in the text. same line of descent during different evolutionary intervals, or for different proteins in the same species in the same residue. The rest of the amino acid composition was unevolutionary interval (14, 51). changed. This increase in the isoleucine content, taken toOf the three differences between hog and hippopotamus gether with the corresponding decrease in the valine content, cytochromes c, the changes at Residues 100 and 92 are not demonstrates that the original column chromatographic fracremarkable, occurring in positions which have already shown tion contained a mixture of two NHz-terminal pentapeptides. nine and eight different amino acids, respectively, in the One had the valine commonly found in position 3, while the proteins of other species, of which vertebrates show 5 and 6 other carried an isoleucine in that position, causing a slight different residues. In contrast, the change at Residue 43 is so increase in chromatographic mobility on paper. Calculating far unique, the hippopotamus protein being the only one to from the relative yields and the valine and isoleucine contents carry a serine in that position. The only other residue found of the two fractions separated by paper chromatography, a at position 43 in vertebrates is alanine, while valine, threonine, value of 10.6% is obtained for the proportion of isoleucineand the glutamic acid also occur in the proteins of noncontaining peptide. This value compares well with the value vertebrate species (1, 2). of 6.7% isoleucine detected in the analysis of Fraction 5, since The most interesting aspect of this study is the discovery the latter is apt to be less accurate, being based on a smaller that one of the four animals from which the protein was analytical sample. Six other tryptic peptides were isolated prepared was probably heterozygous in the cytochrome c from the preliminary tryptic digest and their compositions are locus, since approximately 12% of the protein carried an listed in the Supplementary Material. isoleucine rather than a valine at position 3, and differential In an attempt to confii this heterogeneity, 2 pmol of the gene expression does not seem to occur in allelic cytochromes chymotryptic NH*-terminal decapeptide were digested with c. In the case of the mule and the hinny, horse and donkey trypsin and another 2 pmol were treated with S. aureus cytochromes c are present in equal amounts (21). protease. The resulting fragments were separated by paper Cytochrome c being a protein subject to slow evolutionary electrophoresis and their amino acid compositions did not change (3, 4, 6, 11, 14, and 52), the likelihood of observing indicate the presence of the expected isoleucine variant. heterozygosity is small (7)) even though such variations must Whether this resulted from the separation of the isoleucineoccur if a population is to evolve at all in this gene. Of the containing decapeptide during ion exchange chromatography other cases in which cytochromes c of different primary strucor the separation of the isoleucine-containing fragments durtures have been observed in a single species, the presence of ing paper electrophoresis is uncertain. However, the two 1.3iso-l and iso- cytochromes c in baker’s yeast (28,29) is clearly ml fractions which contained most of the tryptic NHz-terminal the product of a long process in which the two genes have pentapeptide eluted from the Beckman PA-35 resin column evolved independently to structures differing by some 21 (Fraction T-l, see Supplementary Material) showed on amino residues (1). The maintenance of two such proteins in the acid analysis an increasing ratio of isoleucine to valine from same cell must be the result of some biological requirement the front to the back of the chromatographic peak. The pooled (53-57). The recent observation (23-25) that mice carry a material yielded a peptide map identical to that of Fraction 5 cytochrome c in testicular tissue that differs by some 13 of the initial tryptic digest, showing both pentapeptides. Folresidues from the protein in the rest of the animal represents lowing treatment with 0.1 N acetic acid, Edman degradation a similar case of functional specialization, presumably of yielded the phenylthiohydantoins of aspartic acid, valine, and sperm cells. isoleucine at the fust cycle and only that of glutamic acid at The heterogeneity observed in carp (26), box elder (27), and the second. These properties are precisely those expected for mule and hinny (21) cytochromes c is of a different type. In a mixture of the two postulated pentapeptides and it is therethe case of the carp, the fish from which the protein was fore considered that the heterogeneity at Residue 3 is conextracted were either hybrids or a mixed population of two firmed despite our inability to observe it in the chymotryptic varieties, a West and an East European carp. The difference digest. occurs at Residue 4, which is either a glutamic or an aspartic acid, adjacent to the valine-isoleucine interchange found at DISCUSSION Residue 3 in the hippopotamus protein. In the case of the box The cytochromes c of the cow (46), the sheep (47), and the elder, the seeds were obtained from England and from Italy, and here again the heterogeneity observed at Residue 112 hog (45) are identical even though the cow and the sheep are (alanine or serine) may have resulted from the presence of in the same family of artiodactyls (Bovidae), while the hog is seedlings of two subspecies. A much better documented case in a different family (Suidae) classified in a different suborder. is that of the mule and hinny (21). The cytochrome c prepared It was therefore surprising to find that the cytochrome c of from both have equal proportions of threonine and serine at the hippopotamus, classified in the same suborder as the hog, to a mixture of equal amounts of but in a different family (Hippopotamidae) (48), differed in as position 47, corresponding many as three residue positions from the hog protein. The the horse and donkey proteins, respectively. In these three cytochromes c of the only other artiodactyls investigated so cases, the observed heterogeneities result either from the of


8960

Heterogeneity

of Hippopotamus

Acknowledgments-We National Park, Republic employed in this study analyses.

are grateful to Dr. A. Brynard, Kruger of South Africa, for the hippopotamus hearts and Dr. J. F. Beecher for the amino acid REFERENCES

E. (1976) in Handbook ofBiochem1. Borden, D., and Margoliash, istry and Molecular Biology (Fasman, G. D., ed) 3rd Ed, Vol. 3, pp. 268-277, Chemical Rubber Co. Press, Cleveland 2. Dayhoff, M. O., ed (1972) Atlas ofProtein Sequence and Structure, Vol. 5; (1973) Suppl. 1; (1976) Suppl. 2, National Biomedical Research Foundation, Bethesda, Md. 3. Fitch, W. M. (1976) J. Mol. Euol. 8, 13-41 4. Maraoliash. E.. and Smith. E. L. (1965) in Evoluin~ Genes and P&eins ‘(B&on, V., and Vogel, H. J., eds) pp. 221-242, Academic Press, New York 5. Margoliash, E., and Schejter, A. (1966) Adu. Protein Chem. 21, 113-286 6. Fitch, W. M., and Margoliash, E. (1967) Science X5,279-284 7. Margoliash, E., Fitch, W. M., and Dickerson, R. E. (1969) Brookhaven Symp. Biol. 21, 259-305 8. Kimura, M. (1968) Nature 217,624-626 9. King, J. L., and Jukes, T. II. (1969) Science 164, 788-798 10. Smith, E. L. (1970) in The Enzymes, (Boyer, P. D., ed) 3rd Ed, Vol. 1, pp. 267-339, Academic Press, New York 11. Margoliash, E. (1972) Harvey Lect. 66, 177-247 12. Fitch, W. M. (1973) Annu. Reu. Genet. 7, 343-380 13. Boulter, D. (1973) Syst. 2001. 22, 549-553 C. H. (1976) Fed. Proc. 35,2092-2097 14. Fitch, W. M., and Langley, 15. Wu, T. T., Fitch, W. M., and Margoliash, E. (1974) Annu. Reu. Biochem. 43,539-566 16. Margoliash, E., Ferguson-Miller, S., Brautigan, D. L., and Chaviano. A. H. (1976) in ProceedinEs of the Third John Znnes Symposium, 1976’(Markham, R.,and Horne, R. W., eds) pp. 145-165, Elsevier, New York 17. Margoliash, E., Ferguson-Miller, S., Kang, C. H., and Brautigan, D. L. (1976) Fed. Proc. 35, 10 18. Urbanski, G. J., and Margoliash, E. (1977) J. Zmmunol. 114, 1170-1180 R. E., and Timkovich, R. (1975) in The Enzymes 19. Dickerson, (Boyer, P. D., ed) 3rd Ed, Vol. 11, pp. 397-547, Academic Press,

c Sequence

New York 20. Ferguson-Miller, S., Brautigan, D. L., and Margoliash, E. (1979) in The Porphyrins (Dolphin, D., ed) in press 21. Walasek, 0. F., and Margoliash, E. (1977) J. Biol. Chem. 252, 830-834 H., and Smith, E. L. (1963) J. Biol. Chem. 238, 22. Matsubara, 2732-2753 23. Hennig, B. (1975) Eur. J. Biochem. 55, 167-183 24. Carlson, S. S., Mross, G. A., Wilson, A. C., Mead, R. T., Wolin, L. D., Bowers, S. F., Foley, N. T., Muijsers, A. O., and Margoliash, E. (1977) Biochemistry 16, 1437-1442 25. Goldberg, E., Sberna, D., Wheat, T. E., Urbanski, G. J., and Margoliash, E. (1977) Science 196, 1010-1012 26. Guertler, L., and Horstmann, H. J. (1970) Eur. J. Biochem. 12, 48-57 27. Brown, R. H., and Boulter, D. (1974) Biochem. J. 137,93-100 28. Slonimski, P. P., Acher, R., P&e, G., Sels, A., and Somlo, M. (1965) in Mbcunismes de Rtgulution des Actiuit&s Celluluires chez les Microoorgunismes, pp. 435-461, Centre National de la Recherche Scientifique, Paris 29. Sherman, F., Tabor, H., and Campbell, W. (1965) J. Mol. Biol. 13,21-39 30. Margoliash, E., and Walasek, 0. F. (1967) Methods Enzymol. 10, 339-348 31. Drapeau, G. R., Boily, Y., and Houmard, J. (1972) J. Biol. Chem. 247,6720-6726 32 Houmard, J., and Drapeau, G. R. (1972) Proc. Nutl. Acad. Sci. U. S. A. 69,3506-3509 33 Margoliash, E., and Smith, E. L. (1962) J. Biol. Chem. 237, 2151-2160 34 Bohlen, P., Stein, S., Dairman, W., and Udenfriend, S. (1973) Arch. Biochem. Biophys. 155, 213-220 35 Ingram, V. M. (1958) Biochim. Bcophys. Actu 28, 539-545 36. Nolan, C.. and Maraoliash. E. (1966) J. Biol. Chem. 241. 1049-1059 37. Easley, C. W. (1965) Biochim. Biophys. Actu 107, 386-388 38. Fontana, A., Veronese, F. M., and Bucco, E. (1973) FEBS Lett. 32, 135-138 39. Cresttield, A. M., Moore, S., and Stein, W. H. (1963) J. Biol. Chem. 238,622-627 40. Tarr, G. E. (1975) Anal. Biochem. 63,361-370 41. Jeppson, J., and Sjoquist, J. (1967) Anal. Biochem. 18, 264-269 42. Nolan, C., Fitch, W. M., Uzzell, T., Weiss, L. J., and Margoliash, E. (1973) Biochemistry 12,4052-4060 43. Roseau, G., and Pantel, P. (1969) J. Chromutogr. 44, 392-395 44. Easley, C., Zegers, B. J. M., and DeVijlder, M. (1969) Biochim. Biophys. Actu 175, 211-213 45. Stewart, J. W., and Margoliash, E. (1965) Can. J. Biochem. 43, 1187-1206 46. Nakashima, T., Higa, H., Matsubara, H., Benson, A. M., and Yasunobu, K. T. (1966) J. Biol. Chem. 241, 1166-1177 47. Smith, E. L., and Margoliash, E. (1964) Fed. Proc. 23, 1243-1247 48. Romer, A. S. (1966) Vertebrate Paleontology, 3rd Ed, University of Chicago Press, Chicago 49. Sokolovsky, M., and Moldovan, M. (1972) Biochemistry 11, 145-149 50. Niece, R. L., Margoliash, E., and Fitch, W. M. (1977) Biochemistry 16,68-72 51. Langley, C. H., and Fitch, W. M (1974) J. Mol. Evol. 3, 161-177 E., and Fitch, W. M. (1968) Ann. N. Y. Acud. Sci. 52. Margoliash, 151, 359-381 53. Sels, A. A., Fukuhara, H., P&e, G., and Slonimski, P. P. (1965) Biochim. Biophys. Actu 95,486-502 54. Fukuhara, H., and Sels, A. (1966) J. Mol. Biol. 17, 319-333 55. Fukuhara, H. (1966) J. Mol. Biol. 17, 334-342 56. Sherman, F., and Stewart, J. W. (1971) Annu. Reu. Genet. 5, 257-296 57. Dethmers, J., and Ferguson-Miller, S. (1977) Fed. Proc. 36, 727 58. Dickerson, R. E.. Takano. T.. Eisenbera. D.. Kallai. 0. B.. Samson, L., Cooper, A:, and Margoliash, E. (197i) J. Biol. &em. 246, 1511-1535 59. Swanson, R., Trus, B. L., Mandel, N., Mandel, G., Kallai, 0. B., and Dickerson, R. E. (1977) J. Biol. Chem. 252, 759-775 60. Fitch, W. M., and Markowitz, E. (1970) Biochem. Genet. 4, 579-593


breeding of hybrids or the sampling of different subspecies. In contrast, the heterogeneity observed in hippopotamus cytochrome c appears to be an authentic spontaneous evolutionary event. It is not known what proportion of the hippopotamus population sampled carries the mutant allele. The occurrence of an isoleucine at Residue 3 has not been observed in any of the 18 cytochromes c from mammals previously studied (1, 2). Among the 33 vertebrates examined, an isoleutine is found in that position only in some bird cytochromes c (1, 2). Although the exchange of an isoleucine for valine would appear to be a conservative substitution, the phylogenetic segregation of isoleucine 3 may mean that this substitution is functionally significant. Moreover, two of the other three positions at which the hippopotamus protein differs from hog cytochrome c (Residues 92 and 100) are spatially close to Residue 3, all three of them being located on the top rear surface of the molecule (58,59). This localization may be of biological importance. It could also be related to the fact that the cytochrome c gene shows only a small number of concomitantly variable codons, or covarions (60). When one of these undergoes a change, this decides which other spatially or functionally related residues are capable of undergoing evolutionary variation. This process would explain why the cytochrome c of the hippopotamus exhibits changes in some of the same surface locations as do the bird cytochromes c in comparison to the hog protein (Residues 3, 15, 33, 35, 44, 62, 89, 92, 100, 103, and 104 for the bird proteins and Residues 3, 43, 92, and 100 for the hippopotamus protein).

Cytochrome

Heterogeneity

of Hippopotamus

Cytochrome

c Sequence

8961


LEGEND PEPTlDE

TO

FIGURE

CLEAVAGE

3. POINTS.

,OENT,F,CATlON

OF

RESIDUES