The heterogeneity of goat hemoglobin: Evidence for the existence of two nonallelic and one allelic α chain structural genes

528

CO?IIMUNICATIONS REFERENCES

1. SZENT-GY~RGYI, A. G., “The Structure md Function of Muscle,” Vol. II, Academic Press, New York, (1960). 2. BAILEY, K., &o&em. J. 43, 271 (1948). 3. HOLTZER, A. M., CLARK, R., AND LOWEY, S., Biochemistry 4, 2401, (1965). 4. WOODS, E. F., J. Mol. Biol. 16, 581 (1966). 5. MUELLER, H., Biochem. Zeit. 346, 300 (1966). 6. EBASHI, S., AND KURODA, A., (Tokyo) J. Biochem. 681, 107 (1965). 7. BAILEY, K., Biochem. J. 49, 23 (1951). 8. KOMINZ, D. R., SAAD, F., GLADNER, J. A., AND LAKI, K., Arch, Biochem. Biophys. 70, 16 (1957) 9. ASAI, H., (Tokyo) J. Biochem. 60, (1962). 10. 001, T., MIHASHI, K., AND KOBAYASHI, H., Arch. Biochem. Biophys. 98, 1 (1962). 11. BODWELL, C. E., KOMINZ, D. R. AND RJNTLEY, B. J., Biochem. Biophys. Res. Commun. 21, 210 (1965). 12. NOELKEN, M., AND HOLTZER, A. N., “Biochemistry of Muscular Contraction,” Little Brown and Company, Boston, (1964). SHozo IIDA TATSUO 001 Department of Physics Faculty of Science Nagoya Universi t,y Nagoya, Japan Received May 17, 1967 Accepted June 23, 1967

The Heterogeneity

of Goat

Evidence

the Existence

for

Nonallelic cy Chain

and One Structural

Hemoglobin: of Two

Allelic Genes’

That hemoglobin polymorphism occurs in the domestic goat has been well established (1). The most commonly occurring hemoglobin component (Hb-A) shows a mobility in starch-gel electrophoresis (at pH 8.1) only slightly greater than that of human Hb-A, while a second hemoglobin type (Hb-B) with a mobility closely similar to that of human Hb-S may also be present. The two hemoglobins could readily be separated by chromatography on DEAE-Sephadex (2, 3); Hb-B was eluted as a separate component in front of Hb-A. The amounts of Hb-B in five heterozygous goats 1 This paper contains material that is to he utilized in a dissertation as partial fulfillment of the Ph.D. requirement for H. 11. Adams. This work was supported in part, by USPH grant H5168.

were fomld to be 20.4, 23.6, 20.0, 24.9, and 23.8fiyo (mean: 22.5%). When the globins were studied by starch-gel electrophoresis using an urea-6 mercaptoethanol-Verona1 buffer system (4), it became apparent that the difference in electrophoretic mobility was due to t,he presence of different Q chains. Analyses of the amino acid compositions of the tryptic peptides of the p chains of the hemoglobins A and B also failed to reveal any structural variation (2). It seemed, therefore, desirable to invest’igate the structures of the OLchains. The hemoglobins A and B were isolated from red cell hemolysates of two het,erozygous animals by DEAE-Sephadex chromatography, and the globins were prepared by the 2% acid-acetone procedure. The 01 and p chains were isolated by a simplified countercurrent distribution procedure as described previously (2). From 200 to 250 mg of the aminoethylated (Y chains were digested with trypsin for 2 hours at 24°C and at pH 8.9; the protein to trypsin ratio was 1OO:l. The digests were brought to pH 2.5, concentrated in vacua at 37”C, and dissolved in 10 ml of 307, acetic acid. The soluble trypt’ic peptides were separated by Dowex 50-X2 chromatography (5), while each separate peak was rechromatographed over a column of Dowex l-X2 (6). Amino acids were quantitatively determined in each isolated peptide after hydrolysis for 24 hours at 110°C in 6 N HCl using a Spinco model 120-B amino acid analyzer, modified to include a long path photometer (7). The chromatograms of the soluble tryptic peptides of the cy chains of the hemoglobins A and B (isolated from the same animal) are shown in Fig. 1. They were found to be identical except for a greater increase in quantity of peptides in zone 3 in the ol-B chain digest, and in zone 5, which is eluted at, a higher pH in the cu-B chain chromat,ogram. Material in all zones was further purified by rechromatography on columns of Dowex l-X2. Quantitative amino acid analyses of the single, pure peptides, isolated from the zones 4 and 6 through 11 of both chromatograms, indicated identical structures in the corresponding zones. From a comparison wit)h similar peptides isolated from human and bovine (Y chains (8) these peptides were identified (Fig. I); t.heir structures will be discussed in a forthcoming paper. The peptides from zones 3 could be identified as aT-4 with a composition: Asp,, Glu?, Gly,, Alan, Vah, Leul, Tyrl, Arg, (Table 1). The recovery of this peptide from the a-A digest, (24%) was considerably lower than that from the a-B digest (38%). Chromatography of zones 2 of the a-A and a-B digests on columns of Dowex l-X2 resulted in the isolation of three major components from zone 2 (a-A) and of two from zone 2 (a-B). The first component of each chromat,ogram

329

COhIiUl:I%ICATIOiVS .rPH 5

**********d-------

4 3

PH 5 4 3

4bo

600

600

1000

ml of Effluenl

FIG. 1. Separation on Dowex 50-X2 resin of (he soluble peptides from 2 hottr tryptir digests of t,he LYchains of the goat hemoglobins A attd B. The hrokett lines represettt the pH gradients observed during the developmettt of the chromatogram. was identified as olT-9’ (positions 62-08) and t,he second as a mixture of CXT-1 and olT-11, which were present in a ratio of approximately GO:40 and could be separated by a second rechromatography on a Ijowex 50-X2 column in which the pH of the elutittg buffer was kept constant but the molarit) increased (8). Repeated analyses of the third component (a-A) revealed a composition: Aspt, Thrl, Sert, G1u2, Gly,, Alaa, Valt, Leut, Tyrt, Argl (Table I), which is similar to that of LOT-4 (positions 17-31) except, for a substitution of a glycyl and an alanyl residue for eit.her a threonyl or a seryl residue. The recovery of t,his peptide was 14%. A similar peptide was not fouttd in the ol-B digest. Analyses of the peptide isolated from zone 5 (a-A) identified this pept.ide as aT-9” (positions 69-90) with a composition Asp,, Thrt, Set-z, Pro,, Gly,, Ala?, Yalt, Leus, Hisa, Lysl. A similar peptide was isolated from zone 5X (a-B) ; its composition, however, revealed the replacement of one of the fortr aspartic acid residttes by a tyrosyl residue (Table 1). We conclude from the amino acid compositions of the various tryptic peptides of the cl-A and OL-B chains, that the LY chain of Hb-A is in fact two different polypeptide chains, one with four glycyl and fortr danyl residues in positions between 17 and 31 (oI-G~‘~, Alar), and a second in which one of these glycyl and one of these alanyl residltes are

replaced by either a threottyl or a seryl residue ((Y-Glya, .41ao, Thrt, Sert). The exact location of these substitutions have not yet been det,ermined. It) is also possible t,hat additional differences are present) between these two chaitls, since the cores (posit.ions 106139) have not, been analyzed. The w chailt of Hb-B is presumably a charged variant, of one of these two o( chains (the (Y-Gly~, Alaa chain), since only one cuT--l peptide (czT-4, G1yl, Alar) was identified in its tryptic digest. This peptide was, moreover, recovered in a considerably higher yield from the a-B digest than from the CY-Adigest. An obviotts possibi1it.y which may explain that t,he heterogeneity, as observed in the two AB goats, is the occurrence of two closely linked ttottallelic, (Y chain strrtctural genes, and at, allele of one of these t.wo genes (the a-Gly,, Alal gene) ; its mutation then resulting in the replacement of one aspartic acid residtte by a tyrosyl residue. According to this hypot,hesis, the AA goat would be homozygous for two linked nonallelic genes (the a-Glyd, Alar gene and the a-Glys, Alas, Thr, Ser gene), while the AB wo111d he homoaygous for the LYGly,, Alai, Thr, Her gene atLd heterozygous for the a-Gly,, Alan ge:elle. At,tempts are at. present being tnade by selective breeding, t,o produce an animal with a homozygosit y for the allele of the a-Gly,, Ala4 gene. Sttch att animal should sytrthesize two 01 chains (cr-Glyl, Alas, hspz, Tyrt and a-Gly,, Ala:], Thrl, Serl, Aspi) in approxi-

530

COMMUNICATIONS TABLE

THE

AMINO

AUD

1

COMPOSITIONS OF THE T4 AND T9” PEPTIDES ISOLATED FROM THE TRYPTIC A AND B” OF THE cx CHAINS FROM GOAT HEMOGLOBINS -___

Dowex SO-zone elution pH

Lysine Hi&dine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Leucine Tyrosine Phenylalanine Number of A.A. Peptide number Positions in cx chain To Yield

DIGESTS

LX-A

a-A

e-B

a-A

a-B

2 3.98

3 4.05

3 4.02

3 4.20

5’ 4.30

1.00 3.04

1.00 2.97

3.81 1.03 1.95

3.00 0.96 1.66

0.89 1.90 2.12 0.95 4.77

0.92 1.97 1.92 1.01 5.18 0.96

22 T-Sh 69-90 41

22 T-9” 69-90 26

1.00 1.04 0.95 0.97 2.03

1.00 1.02

1.09 1.06

2.02

1.85

2.76 3.06 1.01 1.16 0.85

3.60 3.72 0.97 1.00 0.97

4.04 4.18 0.93 1.14 0.77

15 T-4 17-31 14

15 T-4 17-31 24

15 T-4 17-31 38

a The data are presented as residues/peptide relative to lysine or arginine. Amino acids present to less than 0.1 residue are omitted. The percent yield was calculated on the basis of the recovery after Dowex 50 and subsequent’ Dowex 1 chromatography assuming a 100% hydrolysis of the appropriate cleavage points during tryptic digestion.

mately equal amounts resulting in a Hb-A to Hb-B ratio of about 1: 1. It is noteworthy that our results parallel to some extent the observations made by Kilmartin and Clegg (9) who offered evidence for the existence of four 01chains in the horse, and to those of Rifkin et al., (10) who observed two types of LY chains in the hemoglobin of SEC mice (one with a seryl residue and a second with a threonyl residue in position 68), and one cy chain in C57BL/6 mouse hemoglobin (with an asparaginyl residue in position 68). REFERENCES 1. BERNHARDT, D., Deut. Tieriirztk. Wochschr. 71, 461 (1964). 2. HUISMAN, T. H. J., ADAMS, H. R., DIMMOCK, M. O., EDWARDS, W. C., AND WILSON, J. B., J. Bid. Chem. 242, 2534 (1967). 3. HUISMAN, T. H. J., AND DOZY, A. M., J. Chromafog. 19, 160 (1965). 4. CHERNOFF, A. I., AND PETTIT, N. M., Blood 24, 750 (1964). 5. SCHROEDER, W. A., JONES, R. T., CORMICK,

6. 7. 8.

9. 10.

V., Anal. Chem. 34, 1570 J., AND MCCALLA, (1962). SCHROEDER, W. A., AND ROBBERSOX, B., Anal. Chem. 37, 1583 (1965). JONES, R. T., AND WEISS, G., ilnal. Biochem. 9, 377 (1964). SCHROEDER, W. A., SHELTON, J. R., SHELTON, J. B., ROBBERSON, B., AND BABIN, D., Arch. Biochem. Biophys. 120, 1 (1967). KILMARTIN, J. V., AND CLEGG, J. B., Nature 213, 269 (1967). RIFKIN, D. B., RIFKIN, M. II., AND KONINGSBERG, W., Proc. X&Z. Sci. 66, 586 (1966). T. H. J. HUISMAN J. B. WILSOX H. R. ADAMS

The Division o.f Protein Chemistry Department of Biochemistry Medicul College of Georgia, and the Veterans ministration Hospital Augusta, Georgia 30902 Received May 29, 196’Y; Accepted .June 26, 1967

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