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Hemoglobin heterogeneity in the rabbit: β-112 isoleucine → valine
ARCHIVES
OF
BIOCHEMISTRY
Hemoglobin
AND
BIOPHYSICS
Heterogeneity
168,
922-924
(1973)
in the
Rabbit:
D-112
Rabbit hemoglobin has been the subject of extensive investigation, particularly in studies of protein and hemoglobin biosynthesis. Fetal and adult hemoglobin forms have been distinguished in the rabbit (1) and the complete amino acid sequence of the (Y and p chains of the adult form has been determined (2, 3). A number of examples have been described of structural variations in rabbit hemoglobin involving both the LY and p chains. Von Ehrenstein described multiple amino acid substitutions in the rabbit LY chain (4) and suggested that translational ambiguity was the most likely cause for this finding. Other examples of multiple amino acid variation in rabbit hemoglobin, involving both the (I (5, 6) and B (7, 8) chains, appear to represent the presence of polymorphic alleles in which the amino acid substitutions are inherited aa a closely linked set. The rabbit hemoglobin /3 chain has been shown to contain a single residue of isoleucine occupying position 112 (3). Rabinovita and coworkers (9) first demonstrated that the p chains of certain rabbits contained approximately half-integral isoleucine values and suggested that this finding represented the presence of two p-chain forms, one containing isoleucine at b-112 and the other containing no isoleucine. We have carried out a survey of rabbit hemoglobins from animals acquired from a variety of sources to obtain rabbits demonstrating the isoleutine-deficient p-chain form. In the course of this investigation we identified a male New Zealand White rabbit whose hemoglobinp chains contained no detectable isoleucine. Breeding studies were carried out in which this animal was initially mated with several does all of which had a normal p-chain amino acid composition. Analysis of p chains of the F1 progeny and offspring of backcross matings suggested that the isoleucine-deficient p-chain form was the product of a D-chain allele. The amino acid compositions of the normal p chain and p-chain globin from rabbits homozygous and heterozygous for the variant globin form are indicated in Table I. These determinations demon1 This work was supported by grant from the National Institute of Arthritis, lism, and Digestive Diseases, National of Health.
@ 1973 by Academic Prcsb, of reproduction in any form
Inc. reserved.
--)
Valine’
strated nearly integral isoleucine values in the normal b chain, approximately half-integral values in @ chains of the heterozygous rabbits, and no detectable isoleucine in the B chains of the homoeygous variant animals. The amino acid composition of the p chain from the homozygous variant animals was otherwise consistent with the reported values of Best, Flamm, and Braunitaer (3) except for the apparent presence of an additional valine residue. The position occupied by isoleucine in the normal fi chain is in tryptic peptide pT-12 (3) which precipitates during trypsin digestion as the insoluble core. This material was prepared by trypsin treatment of p-chain globin from a homozygous animal, and after washing to remove soluble products, the peptide was further digested with pepsin as described by Best and coworkers (3). The peptide fragments were fractionated by a column chromatography procedure (Fig. 1). Two major peptic peptides were obtained, designated Pe-1 and Pe-2 (3). The latter was recovered in 2 forms differing by the presence or absence of glutamic acid in the carboxyl-terminal position of the peptide. The Pe-1 peptide exhibited a normal amino acid composition in accordance with the reported values of Best et al. (3). Analysis of the Pe-2 peptides (Table II) demonst,rated an additional residue of valine and the absence of isoleucine. These findings are consistent with an amino acid sequence of the @T-12 peptide as indicated in Fig. 2, in which p-112 isoleucine is replaced by valine. The structural analysis of the rabbit @ chain by Best et al. (3) demonstrated 14 amino acid exchanges to be present in comparison to the human @ chain. All but two of these exchanges could be accounted for by a single base substitution in the the amino acid exchanges at b-66 codons; (Gly:Asn) and 0-112 (Cys: Ile) required a substitution of 2 bases. The replacement of p-112 isoleucine by valine can be accounted for by a single A-+G transition, but the valine substituent also requires a two-base change for its replacement by cyteine, as occurs in human hemoglobin at p-112. Isoleucine in position 112 of the 0 chain has been identified in several strains of mice and the grey kangaroo as well as in the rabbit (14). Valine appears to occupy this position more frequently, having been identified in the pig, horse, llama, cow, sheep, and goat (14).
AM-12895 MetaboInstitutes 922
Copyright All rights
lsoleucine
923
COMMUNICATIONS TABLE AMINO
ACID
COMPOSITION
Normal Lysine Histidine Arginine Aspartic acid Threonine ,Serine (ilutamic acid Proline (ilycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine
12.00 8.98 2.77 12.12 3.78 9.76 14.00 4.01 10.99 15.32 17.57 0.95 0.76 17.77 2.78 7.73
I
OF RABHIT
GLORIN
Heterozygous 12.00 8.88 2.75 11.89 4.15 9.18 14.00 4.01 11.23 15.70 17.79 0.80 0.45 18.00 2.73 8.30
(18.01) (0.89) (17.99)
P-CHAINSO
Homozygous 12.00 9.25 3.22 11.84 3.87 9.42 14.00 4.02 10.90 15.13 18.50 1.08 0.00 17.90 2.68 7.92
(18.70) (0.51) (18.02)
variant
Expected* 12 9 3 12 4 10 14 4 11 15 18 1 1 18 3 8
(18.68) (0.00) (18.19)
a Blood from the animals was collected in heparin. The blood was centrifuged and the plasma was removed. The red cells were washed three times in isotonic saline and lysed in 2-4 vol of wat,er. The stroma were removed by centrifugation. The hemoglobin solutions were flushed with carbon monoxide and subjected to DEAE-Sephadex chromatography (10). Globin was prepared from the eluted hemoglobin by precipitation in acetone-HCl (11). The globin chains were separated by carboxymethylcellulose column chromatography as described by Dintais (12). The amino acid composition of the B chains was determined after hydrolysis of the samples in 6 N HCl at 1lO’C for 24 and 72 h under reduced pressure. The hydrolysates were analyzed in a Beckman model 120C amino acid analyzer. The indicated values are results from the 24-hr hydrolysates. The values in parentheses are from determinations after hydrolysis for 72 hr. Values are expressed as amino acid residues/globin chain. * Amino acid composition of the rabbit fl chain as reported by Best. et nl. (3). 07
-1
85
l’)C, 95
150 160 185
155
430 EFFLUENT
460 VOLUME
640
ix
SIL
8”’
(ML!
Fro. 1. Chromatographic separation of peptic pept,ides of fiT-12 from the variant rabbit globin. Purified p chain from a homozygous variant rabbit was prepared as described in the legend to Table I. The globin was digested with trypsin in pH 8.5 buffer at room temperature for 8 hr. The insoluble core material, containing the pT-12 peptide, was washed thoroughly with water, then further digested with pepsin at pH 1.2 (enzyme/protein ratio 1:50) for 2 hr at 37°C. The peptic digest was loaded onto a PA-35 resin column (0.9 X 13 cm). Elution was performed with a linear acetic acid-pyridine gradient (13). Approximately loo/;, of the coumn effluent was directed into a ninhydrin reaction coil and det,ect,or and the remainder colected for analysis.
924
COMMUNICATIONS TABLE AMINO
ACID.
COMPOSITION
OF THF,
Ps-2
Experimental Pe-2a Lysine Histidine Serine Glycine Valine Isoleucine Leucine Phenylalanine Glutamic acid
Fig.
II Cone
-
Peptide
OF THN
Number
of residues
values Pe-2b
l.CO 1.80 0.74 1.00 2.98 0.00 0.99 0.88 0.00
PF,PTIDI~;
Peptide 1.00 1.87 0.75 1.10 2.82 o.co 1.24 1.02 1.03
acid
sequence
ACKNOWLEDGMENTS We thank Dr. Junius fractionation. Miss Loyda technical assistance.
Adams for the peptide Vida provided valuable
REFERENCES 1. TYUMA, I., ENOKI, Y., AND MORIK~W~, S. (1964) Jap. J. Physiol. 14, 573. 2. BR~UNITZ~R, G., BEST, J. S., FL,~MM, U., AND SCHRANK, B. (1966) z. Physiol. Chem. 347, 207. BEST, J.S., FL~MM, U., AND BR~UNITZ~X, G. (1969) 2. Physiol. Chem. 359,563. VON EHRENSTEIN, G. (1966) Cold Spring Harbor Symp.Quant. Biol. 31, 705. HUNTER, T., IND MUNXO, A. (1969) A-ature 223, 1270. S~HAPIRA, G., BI!:NRUBI, M., MALF:NKNIA, N., .%ND REIBEL, L. (1969) Biochim. Biophys. Acta 188, 216. A. (1971) Nature New Biol. 229, 142. 7. GALIZZI, J., MALEKNI~, N., END SCHAPIR.~, 8. DF,LAUNSY, G. (1971) Biochim. Biophys. Acta 229, 712. M., FREXDMAN, M. L., FISHER, J. 9. RABINOVITZ, M., AND MAXWELL, C. R. (1969) Cold Spring Harbor Symp. Quant. Biol. 34,567. A. M., KLEIHAUER, E. F., AND HUIS10. DOZY, MAN, T. H. J. (1968) J. Chromatogr. 32, 723.
of the pT-12
1 2 1 1 2 1 1 1 1 (only
(3)
in Pe-2b)
(3)
VI and V, respectively,
shown
in
120 > > peptide.
11. ROSSI-F.\NI~;LLI, C~PUT~,
values
Pe-2
Leu-Leu-Gly-Asn-Val-Leu-Val-Val-Val-Leu-Ser-His-His-Phe-Gly-Lys-Glu 105 110 115 VPe 1 t Pe 2a < Pe 2b 2. Amino
p CHAINS
Literature
Q The peptic peptides Pe-2a and Pe-2b were obtained from fractions 1. The peptides were hydrolyzed in 6 N HCl at 110°C for 72 hr.
FIG.
VARI.ZNT
A.
A., ANTONINI, R., (1958) Biochim. Biophys.
AND
Acta
30, 608. 12. DINTZIS, H. M. (1961) Proc. Nat. Acad. Sci. USA 47, 247. 13. JONES, It. T. (1964) Cold Spring Harbor Symp. Qua&. Biol. 29, 297. 14. DAYHOFF, M. 0. (1972) Atlas of Protein Sequence and Structure, National Biochemical Research Foundation, Silver Spring, Maryland, Vol. 5. MIR SH~MSUDDIN R. GEORGE MESON (3.11%~ COHEN ROBERT G. TISSOT G IZORGE
It.
HONIG~
Department of Pediatrics The Abraham Lincoln School of Medicine, Center for Gerletics School of Basic Medical Sciences Uwiversity of Illinois Medical Center 840 South Wood Street Chicago, Illinois 60612 Received July 13, 1975
2 Recipient of a Research Career Award (AM-41188) from the National Arthritis, Metabolism, and Digestive
Development Institute Diseases.
of
OF
BIOCHEMISTRY
Hemoglobin
AND
BIOPHYSICS
Heterogeneity
168,
922-924
(1973)
in the
Rabbit:
D-112
Rabbit hemoglobin has been the subject of extensive investigation, particularly in studies of protein and hemoglobin biosynthesis. Fetal and adult hemoglobin forms have been distinguished in the rabbit (1) and the complete amino acid sequence of the (Y and p chains of the adult form has been determined (2, 3). A number of examples have been described of structural variations in rabbit hemoglobin involving both the LY and p chains. Von Ehrenstein described multiple amino acid substitutions in the rabbit LY chain (4) and suggested that translational ambiguity was the most likely cause for this finding. Other examples of multiple amino acid variation in rabbit hemoglobin, involving both the (I (5, 6) and B (7, 8) chains, appear to represent the presence of polymorphic alleles in which the amino acid substitutions are inherited aa a closely linked set. The rabbit hemoglobin /3 chain has been shown to contain a single residue of isoleucine occupying position 112 (3). Rabinovita and coworkers (9) first demonstrated that the p chains of certain rabbits contained approximately half-integral isoleucine values and suggested that this finding represented the presence of two p-chain forms, one containing isoleucine at b-112 and the other containing no isoleucine. We have carried out a survey of rabbit hemoglobins from animals acquired from a variety of sources to obtain rabbits demonstrating the isoleutine-deficient p-chain form. In the course of this investigation we identified a male New Zealand White rabbit whose hemoglobinp chains contained no detectable isoleucine. Breeding studies were carried out in which this animal was initially mated with several does all of which had a normal p-chain amino acid composition. Analysis of p chains of the F1 progeny and offspring of backcross matings suggested that the isoleucine-deficient p-chain form was the product of a D-chain allele. The amino acid compositions of the normal p chain and p-chain globin from rabbits homozygous and heterozygous for the variant globin form are indicated in Table I. These determinations demon1 This work was supported by grant from the National Institute of Arthritis, lism, and Digestive Diseases, National of Health.
@ 1973 by Academic Prcsb, of reproduction in any form
Inc. reserved.
--)
Valine’
strated nearly integral isoleucine values in the normal b chain, approximately half-integral values in @ chains of the heterozygous rabbits, and no detectable isoleucine in the B chains of the homoeygous variant animals. The amino acid composition of the p chain from the homozygous variant animals was otherwise consistent with the reported values of Best, Flamm, and Braunitaer (3) except for the apparent presence of an additional valine residue. The position occupied by isoleucine in the normal fi chain is in tryptic peptide pT-12 (3) which precipitates during trypsin digestion as the insoluble core. This material was prepared by trypsin treatment of p-chain globin from a homozygous animal, and after washing to remove soluble products, the peptide was further digested with pepsin as described by Best and coworkers (3). The peptide fragments were fractionated by a column chromatography procedure (Fig. 1). Two major peptic peptides were obtained, designated Pe-1 and Pe-2 (3). The latter was recovered in 2 forms differing by the presence or absence of glutamic acid in the carboxyl-terminal position of the peptide. The Pe-1 peptide exhibited a normal amino acid composition in accordance with the reported values of Best et al. (3). Analysis of the Pe-2 peptides (Table II) demonst,rated an additional residue of valine and the absence of isoleucine. These findings are consistent with an amino acid sequence of the @T-12 peptide as indicated in Fig. 2, in which p-112 isoleucine is replaced by valine. The structural analysis of the rabbit @ chain by Best et al. (3) demonstrated 14 amino acid exchanges to be present in comparison to the human @ chain. All but two of these exchanges could be accounted for by a single base substitution in the the amino acid exchanges at b-66 codons; (Gly:Asn) and 0-112 (Cys: Ile) required a substitution of 2 bases. The replacement of p-112 isoleucine by valine can be accounted for by a single A-+G transition, but the valine substituent also requires a two-base change for its replacement by cyteine, as occurs in human hemoglobin at p-112. Isoleucine in position 112 of the 0 chain has been identified in several strains of mice and the grey kangaroo as well as in the rabbit (14). Valine appears to occupy this position more frequently, having been identified in the pig, horse, llama, cow, sheep, and goat (14).
AM-12895 MetaboInstitutes 922
Copyright All rights
lsoleucine
923
COMMUNICATIONS TABLE AMINO
ACID
COMPOSITION
Normal Lysine Histidine Arginine Aspartic acid Threonine ,Serine (ilutamic acid Proline (ilycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine
12.00 8.98 2.77 12.12 3.78 9.76 14.00 4.01 10.99 15.32 17.57 0.95 0.76 17.77 2.78 7.73
I
OF RABHIT
GLORIN
Heterozygous 12.00 8.88 2.75 11.89 4.15 9.18 14.00 4.01 11.23 15.70 17.79 0.80 0.45 18.00 2.73 8.30
(18.01) (0.89) (17.99)
P-CHAINSO
Homozygous 12.00 9.25 3.22 11.84 3.87 9.42 14.00 4.02 10.90 15.13 18.50 1.08 0.00 17.90 2.68 7.92
(18.70) (0.51) (18.02)
variant
Expected* 12 9 3 12 4 10 14 4 11 15 18 1 1 18 3 8
(18.68) (0.00) (18.19)
a Blood from the animals was collected in heparin. The blood was centrifuged and the plasma was removed. The red cells were washed three times in isotonic saline and lysed in 2-4 vol of wat,er. The stroma were removed by centrifugation. The hemoglobin solutions were flushed with carbon monoxide and subjected to DEAE-Sephadex chromatography (10). Globin was prepared from the eluted hemoglobin by precipitation in acetone-HCl (11). The globin chains were separated by carboxymethylcellulose column chromatography as described by Dintais (12). The amino acid composition of the B chains was determined after hydrolysis of the samples in 6 N HCl at 1lO’C for 24 and 72 h under reduced pressure. The hydrolysates were analyzed in a Beckman model 120C amino acid analyzer. The indicated values are results from the 24-hr hydrolysates. The values in parentheses are from determinations after hydrolysis for 72 hr. Values are expressed as amino acid residues/globin chain. * Amino acid composition of the rabbit fl chain as reported by Best. et nl. (3). 07
-1
85
l’)C, 95
150 160 185
155
430 EFFLUENT
460 VOLUME
640
ix
SIL
8”’
(ML!
Fro. 1. Chromatographic separation of peptic pept,ides of fiT-12 from the variant rabbit globin. Purified p chain from a homozygous variant rabbit was prepared as described in the legend to Table I. The globin was digested with trypsin in pH 8.5 buffer at room temperature for 8 hr. The insoluble core material, containing the pT-12 peptide, was washed thoroughly with water, then further digested with pepsin at pH 1.2 (enzyme/protein ratio 1:50) for 2 hr at 37°C. The peptic digest was loaded onto a PA-35 resin column (0.9 X 13 cm). Elution was performed with a linear acetic acid-pyridine gradient (13). Approximately loo/;, of the coumn effluent was directed into a ninhydrin reaction coil and det,ect,or and the remainder colected for analysis.
924
COMMUNICATIONS TABLE AMINO
ACID.
COMPOSITION
OF THF,
Ps-2
Experimental Pe-2a Lysine Histidine Serine Glycine Valine Isoleucine Leucine Phenylalanine Glutamic acid
Fig.
II Cone
-
Peptide
OF THN
Number
of residues
values Pe-2b
l.CO 1.80 0.74 1.00 2.98 0.00 0.99 0.88 0.00
PF,PTIDI~;
Peptide 1.00 1.87 0.75 1.10 2.82 o.co 1.24 1.02 1.03
acid
sequence
ACKNOWLEDGMENTS We thank Dr. Junius fractionation. Miss Loyda technical assistance.
Adams for the peptide Vida provided valuable
REFERENCES 1. TYUMA, I., ENOKI, Y., AND MORIK~W~, S. (1964) Jap. J. Physiol. 14, 573. 2. BR~UNITZ~R, G., BEST, J. S., FL,~MM, U., AND SCHRANK, B. (1966) z. Physiol. Chem. 347, 207. BEST, J.S., FL~MM, U., AND BR~UNITZ~X, G. (1969) 2. Physiol. Chem. 359,563. VON EHRENSTEIN, G. (1966) Cold Spring Harbor Symp.Quant. Biol. 31, 705. HUNTER, T., IND MUNXO, A. (1969) A-ature 223, 1270. S~HAPIRA, G., BI!:NRUBI, M., MALF:NKNIA, N., .%ND REIBEL, L. (1969) Biochim. Biophys. Acta 188, 216. A. (1971) Nature New Biol. 229, 142. 7. GALIZZI, J., MALEKNI~, N., END SCHAPIR.~, 8. DF,LAUNSY, G. (1971) Biochim. Biophys. Acta 229, 712. M., FREXDMAN, M. L., FISHER, J. 9. RABINOVITZ, M., AND MAXWELL, C. R. (1969) Cold Spring Harbor Symp. Quant. Biol. 34,567. A. M., KLEIHAUER, E. F., AND HUIS10. DOZY, MAN, T. H. J. (1968) J. Chromatogr. 32, 723.
of the pT-12
1 2 1 1 2 1 1 1 1 (only
(3)
in Pe-2b)
(3)
VI and V, respectively,
shown
in
120 > > peptide.
11. ROSSI-F.\NI~;LLI, C~PUT~,
values
Pe-2
Leu-Leu-Gly-Asn-Val-Leu-Val-Val-Val-Leu-Ser-His-His-Phe-Gly-Lys-Glu 105 110 115 VPe 1 t Pe 2a < Pe 2b 2. Amino
p CHAINS
Literature
Q The peptic peptides Pe-2a and Pe-2b were obtained from fractions 1. The peptides were hydrolyzed in 6 N HCl at 110°C for 72 hr.
FIG.
VARI.ZNT
A.
A., ANTONINI, R., (1958) Biochim. Biophys.
AND
Acta
30, 608. 12. DINTZIS, H. M. (1961) Proc. Nat. Acad. Sci. USA 47, 247. 13. JONES, It. T. (1964) Cold Spring Harbor Symp. Qua&. Biol. 29, 297. 14. DAYHOFF, M. 0. (1972) Atlas of Protein Sequence and Structure, National Biochemical Research Foundation, Silver Spring, Maryland, Vol. 5. MIR SH~MSUDDIN R. GEORGE MESON (3.11%~ COHEN ROBERT G. TISSOT G IZORGE
It.
HONIG~
Department of Pediatrics The Abraham Lincoln School of Medicine, Center for Gerletics School of Basic Medical Sciences Uwiversity of Illinois Medical Center 840 South Wood Street Chicago, Illinois 60612 Received July 13, 1975
2 Recipient of a Research Career Award (AM-41188) from the National Arthritis, Metabolism, and Digestive
Development Institute Diseases.
of