- Email: [email protected]
Rabbit hemoglobin
BIOCHIMICA ET BIOPHYSICA ACTA
712 BBA
35801
RABBIT HEMOGLOBIN T H E H E T E R O G E N E I T Y OF THE/5-CHAIN
J. D E L A U N A Y , N. M A L E K N I A AND G. S C H A P I R A
Instztut de Pathologie Mol$cula,re*, 24, rue du Faubourg Saint-Jacques, Parzs I4e (France) (Received N o v e m b e r 5th, 197 o)
SUMMARY
Two new allelomorphs of rabbit hemoglobin fiT5 peptide are described. One variant contains glutamine and asparagine while the other contains serine and histidine. Both variants can be separated on ion-exchange resin with a pyridine-acetic acid as well as a pyridine-formic acid gradient.
INTRODUCTION
VON EHRENSTEIN1 has reported that in the rabbit hemoglobin a-chain, a neutral amino acid can replace another neutral amino acid in six positions. He suggested that these multiplicities were due to translational variations. We demonstrated however that the ambiguity in position 29 (valine or leucine) in the peptide aT4 originated from an allelomorphism2, 3. Our results were consistent with those obtained by HUNTER AND MUNRO4. The question arises: is such a polymorphism also present in the r-chain? Vow EHRENSTEIN has reported fingerprint differences1. There are some discrepancies between the r-chain peptide amino acid compositions given by NAUGHTONAND DINTZlS~ and by BRAUNITZER et al. ~. Isoleucine is known to be inconstant in the r-chain 7. Cysteine appears to be inconstant**. METHODS
Hemoglobin was purified by starch electrophoresis in a barbital KCN buffer. Heine and globin were separated at --20 ° by the acetone-HC1 procedure, a- and rchains were separated according to the method of DINTZlS8. r-Chain was digested * I n s t i t u t d ' U n i v e r s i t 6 , Groupe U I 5 de l ' I n s t i t u t N a t i o n a l de la Sant~ et de la R e c h e r c h e M6dicale, L a b o r a t o i r e associ6 a u Centre N a t i o n a l de la R e c h e r c h e Scientifique. ** I n s e p a r a t i o n of a- a n d r - c h a i n s according to t h e m e t h o d o f DINTZlS 8, we noticed two slightly s e p a r a t e d r - c h a i n s ; a m i n o acid a n a l y s i s of initial a n d final p a r t s of t h e p e a k s h o w e d a difference in c y s t e i n e c o n t e n t ( u n p u b l i s h e d data).
B~och,m. Bzophys. Acta, 229 (1971) 712-715
RABBIT HEMOGLOBIN
7~t3
by trypsin at 37 ° and pH 8.6. The soluble peptides were separated by chromatography on a Technicon resin P 4 with a pyridine-formic acid gradient (o.I M, pH 3.5-2 M, pH 5) which proved to be capable of separating the two variants of the aT4 peptide of rabbit hemoglobin a-chain, whereas the pyridine-acetic acid gradient did not give the same resolution. We also used a Spinco PA 35 ion-exchange resin with a pyridine-acetic acid gradient (0.2 M, pH 3.1-2 M, pH 5) according to the method of JONES9. Further purification of peptides was carried out on a Biorad AG I-X2 ionexchange resin with a gradient a-picoline-N-ethylmorpholine-pyridine-acetic acid of decreasing pH's by the method of SCHROEDER et al. 1°. In one case it was found necessary to rechromatograph one particular peak on a Biorad AG 5oW-X2 ionexchange resin with a pyridine-acetic acid gradient. Complete hydrolysis of peptides was performed with 6 M HC1 for 22, 36 and 72 h at IiO ° under vacuum. Analyses of the amino acid composition of the hydrolysate was achieved with a Beckman Spinco 12o C amino acid analyzer.
Fig. i. r-chain peptide map. Top figure, homozygous rabbits A; middle figure, homozygous rabbits B; bottom figure, heterozygous rabbits AB.
RESULTS We first studied the chromatograms of the r-chain from fifteen rabbits chosen at random. Whatever the resin or the developer, we found three types of chromatograms. Fig. I represents the three types obtained with a Beckman PA 35 resin and a pyridine-acetic acid developer. Peak A is present on chromatogram A (top figure), but not on chromatogram B (middle figure). Peak B is present on chromatogram B, but not on chromatogram A. Both peaks, although smaller, are present on chromatogram AB (bottom figure). Peaks A and B could represent two variants of the same peptide. They were purified, and the amino acid composition determined (Table I). The two corresponding peptides differ by two positions: A contains Glx and Asx, B contains His and Ser. Biochim. Biophys. Acta, 229 (197I) 7t2-715
714
j. DELAUNAY et al.
TABLE I AMINOACID COMPOSITIONOF THE VARIANTSA AND B Amino acid
A
B
Lys His NH3 Arg Asx Thr Ser Glx Pro Gly Ala Cyg Val Met Ile Leu Tyr Phe
o.93
o.93 o.96
2.94
2.13
2.7o 1.91 0.82 1.o8 2.o6
4.o8 0.97 0.93 1.o9 1.94
1.07 0.76
o 98 0.97
0.74
0.94
2.43
2.32
OF
THE PEPTIDE fiT5
I t is concluded t h a t these two peptides, fiT 5 (Asn, Gin) a n d fiT 5 (Ser, His), represent two v a r i a n t s of t h e p e p t i d e fiT5. O n l y t h e p e r m u t a t i o n between Set a n d Asn is possible on the basis of a single nucleotide change in t h e codon. T h u s His m u s t necessarily p e r m u t e with Glu or Gln. On the same basis however, only the p e r m u t a t i o n w i t h Gln is possible (Table II). The p e r m u t a t i o n s seem therefore to b e : Asn ~ Ser a n d Gln ~ His. T h e fact t h a t we never o b s e r v e d a n y different e l e c t r o p h o r e t i c m o b i l i t y of t h e whole hemoglobin on s t a r c h seems also to eliminate t h e presence of A s p a n d Glu in v a r i a n t A. H i s t i d i n e is a n e u t r a l residue at p H 9.3 a n d t h u s does n o t change t h e electrophoretic m o b i l i t y on starch. On the o t h e r hand, histidine b e h a v e s as a basic residue at low p H . This p r o b a b l y explains w h y the p e p t i d e flT5B a n d flT5A s e p a r a t e easily w i t h a p y r i d i n e - a c e t i c acid developer which is known, in m a n y cases, to give no s e p a r a t i o n of p e p t i d e s with identical charges. TABLE II P O S S I B L E P E R M U T A T I O N S B E T W E E N T H E R E S I D U E S D I F F E R I N G IN T H E VARIANTS A
PEPTmE fiT5
CAU
HiSCAC
GAU Asp(~AC
AAU ASnAAC
GAA GlUGAG
CAA GlncAG
+
+
--
+
--
+
--
UCU UCC
UCA SerucQ AGU AGC
Bzochzm. Biophys. Acta, 229 (I971) 712-715
AND B
OF
715
RABBIT HEMOGLOBIN
One of the ambiguities likely fits with the discrepancy in position 5o between the sequence given by BRAUNITZER et al.8: Phe~t Phe42 Glu4~ Ser44 Phe45 Gly46 Asp47 Leu4s Ser49 Ser~o Alast Ash52 Alas3 Va154 Met55 Asn~e Asn57 Pro~8 Lysso
and the one established according to DINTZIS8: Phe41 Phe42 Glx43 Ser~4 Phe4~ Gly4e Asx,~ Leu48 Ser49 Asxso Alas1 Asx52 Alas2 Va154 Met55 Leu58 Asx57 Pross Lyss9
We did not find the other discrepancy observed by these authors in position 56 (Asn or Leu, respectively). But neither BRAUNITZER et al. nor DINTZIS mentioned any His in the peptide fiT5. Six rabbits were found with peptide A (Asn, Gln), six with peptide B (Ser, His) and only three with both peptides. In rabbits AB, the two peptides were present in equal amounts. After purification we obtained twice as much peptide B (IiO nmoles) from rabbit B fl-chain as from rabbit AB fl-chain (50 nmoles). These values are consistent with the possibility of two allelic forms of the fl-chain structural gene. We have not done any breeding experiment; however, these results make allelomorphism quite likely. The apparent inconsistency between the equal frequency of both alleles A and B in the population and the low proportion of heterozygous rabbits AB may be due to the small number of rabbits studied. ACKNOWLEDGMENTS
We wish to thank Dr. D. Labie for helpful discussion. REFERENCES I G. VON EHRENSTEIN, Cold Spring Harbor Symp. Quant. Biol., 31 (1966) 705 . 2 G. SCHAPIRA, J. C. DREYFUS AND N. MALEKNIA, Biochem. Biophys. Res. Commun., 32 (1968) 558 • 3 G. SCHAPIRA, M. BENRUBI, N. MALEKNIAAND L. REIBEL, Biochim. B*ophys. Acta, 188 (1969) 216. 4 T. HUNTER AND A. MUNRO, Nature, 223 (197 o) 127o. 5 M. A. NAUGHTON AND H. M. DINTZlS, Proc. Natl. Acad. Sci. U.S., 48 (1962) 1822. 6 G. BRAUNITZER, S. B. BEST, F. ULRICH AND B. SCHRANK,Z. f. Physiol. Chem., 347 (1966) 207. 7 M. RAmNOVlTZ, M. L. FREEDMAN, J. M. FISHER AND C. R. MAXWELL, Cold Spring Harbor Symp. Quant. Biol., 34 (1969) 567. 8 H. M. DINTZlS, Proc. Natl. Acad. Sci. U.S., 47 (1961) 247. 9 R. T. JONES, Cold Spring Harbor Syrup. ~uant. Biol., 29 (1964) 297. IO W. A. SCHROEDER, R. T. JONES, J. CORNICK AND K. MCCALLA, Anal. Chem., 34 (I962) 1571. II R.V. ECK AND M. O. DAYHOFF,Atlas of Protein Sequence and Structure, National Biochemical Research Foundation, Silverspring, Maryland, 1966.
Biochim. Bzophys. Acta, 229 (1971) 712-715
712 BBA
35801
RABBIT HEMOGLOBIN T H E H E T E R O G E N E I T Y OF THE/5-CHAIN
J. D E L A U N A Y , N. M A L E K N I A AND G. S C H A P I R A
Instztut de Pathologie Mol$cula,re*, 24, rue du Faubourg Saint-Jacques, Parzs I4e (France) (Received N o v e m b e r 5th, 197 o)
SUMMARY
Two new allelomorphs of rabbit hemoglobin fiT5 peptide are described. One variant contains glutamine and asparagine while the other contains serine and histidine. Both variants can be separated on ion-exchange resin with a pyridine-acetic acid as well as a pyridine-formic acid gradient.
INTRODUCTION
VON EHRENSTEIN1 has reported that in the rabbit hemoglobin a-chain, a neutral amino acid can replace another neutral amino acid in six positions. He suggested that these multiplicities were due to translational variations. We demonstrated however that the ambiguity in position 29 (valine or leucine) in the peptide aT4 originated from an allelomorphism2, 3. Our results were consistent with those obtained by HUNTER AND MUNRO4. The question arises: is such a polymorphism also present in the r-chain? Vow EHRENSTEIN has reported fingerprint differences1. There are some discrepancies between the r-chain peptide amino acid compositions given by NAUGHTONAND DINTZlS~ and by BRAUNITZER et al. ~. Isoleucine is known to be inconstant in the r-chain 7. Cysteine appears to be inconstant**. METHODS
Hemoglobin was purified by starch electrophoresis in a barbital KCN buffer. Heine and globin were separated at --20 ° by the acetone-HC1 procedure, a- and rchains were separated according to the method of DINTZlS8. r-Chain was digested * I n s t i t u t d ' U n i v e r s i t 6 , Groupe U I 5 de l ' I n s t i t u t N a t i o n a l de la Sant~ et de la R e c h e r c h e M6dicale, L a b o r a t o i r e associ6 a u Centre N a t i o n a l de la R e c h e r c h e Scientifique. ** I n s e p a r a t i o n of a- a n d r - c h a i n s according to t h e m e t h o d o f DINTZlS 8, we noticed two slightly s e p a r a t e d r - c h a i n s ; a m i n o acid a n a l y s i s of initial a n d final p a r t s of t h e p e a k s h o w e d a difference in c y s t e i n e c o n t e n t ( u n p u b l i s h e d data).
B~och,m. Bzophys. Acta, 229 (1971) 712-715
RABBIT HEMOGLOBIN
7~t3
by trypsin at 37 ° and pH 8.6. The soluble peptides were separated by chromatography on a Technicon resin P 4 with a pyridine-formic acid gradient (o.I M, pH 3.5-2 M, pH 5) which proved to be capable of separating the two variants of the aT4 peptide of rabbit hemoglobin a-chain, whereas the pyridine-acetic acid gradient did not give the same resolution. We also used a Spinco PA 35 ion-exchange resin with a pyridine-acetic acid gradient (0.2 M, pH 3.1-2 M, pH 5) according to the method of JONES9. Further purification of peptides was carried out on a Biorad AG I-X2 ionexchange resin with a gradient a-picoline-N-ethylmorpholine-pyridine-acetic acid of decreasing pH's by the method of SCHROEDER et al. 1°. In one case it was found necessary to rechromatograph one particular peak on a Biorad AG 5oW-X2 ionexchange resin with a pyridine-acetic acid gradient. Complete hydrolysis of peptides was performed with 6 M HC1 for 22, 36 and 72 h at IiO ° under vacuum. Analyses of the amino acid composition of the hydrolysate was achieved with a Beckman Spinco 12o C amino acid analyzer.
Fig. i. r-chain peptide map. Top figure, homozygous rabbits A; middle figure, homozygous rabbits B; bottom figure, heterozygous rabbits AB.
RESULTS We first studied the chromatograms of the r-chain from fifteen rabbits chosen at random. Whatever the resin or the developer, we found three types of chromatograms. Fig. I represents the three types obtained with a Beckman PA 35 resin and a pyridine-acetic acid developer. Peak A is present on chromatogram A (top figure), but not on chromatogram B (middle figure). Peak B is present on chromatogram B, but not on chromatogram A. Both peaks, although smaller, are present on chromatogram AB (bottom figure). Peaks A and B could represent two variants of the same peptide. They were purified, and the amino acid composition determined (Table I). The two corresponding peptides differ by two positions: A contains Glx and Asx, B contains His and Ser. Biochim. Biophys. Acta, 229 (197I) 7t2-715
714
j. DELAUNAY et al.
TABLE I AMINOACID COMPOSITIONOF THE VARIANTSA AND B Amino acid
A
B
Lys His NH3 Arg Asx Thr Ser Glx Pro Gly Ala Cyg Val Met Ile Leu Tyr Phe
o.93
o.93 o.96
2.94
2.13
2.7o 1.91 0.82 1.o8 2.o6
4.o8 0.97 0.93 1.o9 1.94
1.07 0.76
o 98 0.97
0.74
0.94
2.43
2.32
OF
THE PEPTIDE fiT5
I t is concluded t h a t these two peptides, fiT 5 (Asn, Gin) a n d fiT 5 (Ser, His), represent two v a r i a n t s of t h e p e p t i d e fiT5. O n l y t h e p e r m u t a t i o n between Set a n d Asn is possible on the basis of a single nucleotide change in t h e codon. T h u s His m u s t necessarily p e r m u t e with Glu or Gln. On the same basis however, only the p e r m u t a t i o n w i t h Gln is possible (Table II). The p e r m u t a t i o n s seem therefore to b e : Asn ~ Ser a n d Gln ~ His. T h e fact t h a t we never o b s e r v e d a n y different e l e c t r o p h o r e t i c m o b i l i t y of t h e whole hemoglobin on s t a r c h seems also to eliminate t h e presence of A s p a n d Glu in v a r i a n t A. H i s t i d i n e is a n e u t r a l residue at p H 9.3 a n d t h u s does n o t change t h e electrophoretic m o b i l i t y on starch. On the o t h e r hand, histidine b e h a v e s as a basic residue at low p H . This p r o b a b l y explains w h y the p e p t i d e flT5B a n d flT5A s e p a r a t e easily w i t h a p y r i d i n e - a c e t i c acid developer which is known, in m a n y cases, to give no s e p a r a t i o n of p e p t i d e s with identical charges. TABLE II P O S S I B L E P E R M U T A T I O N S B E T W E E N T H E R E S I D U E S D I F F E R I N G IN T H E VARIANTS A
PEPTmE fiT5
CAU
HiSCAC
GAU Asp(~AC
AAU ASnAAC
GAA GlUGAG
CAA GlncAG
+
+
--
+
--
+
--
UCU UCC
UCA SerucQ AGU AGC
Bzochzm. Biophys. Acta, 229 (I971) 712-715
AND B
OF
715
RABBIT HEMOGLOBIN
One of the ambiguities likely fits with the discrepancy in position 5o between the sequence given by BRAUNITZER et al.8: Phe~t Phe42 Glu4~ Ser44 Phe45 Gly46 Asp47 Leu4s Ser49 Ser~o Alast Ash52 Alas3 Va154 Met55 Asn~e Asn57 Pro~8 Lysso
and the one established according to DINTZIS8: Phe41 Phe42 Glx43 Ser~4 Phe4~ Gly4e Asx,~ Leu48 Ser49 Asxso Alas1 Asx52 Alas2 Va154 Met55 Leu58 Asx57 Pross Lyss9
We did not find the other discrepancy observed by these authors in position 56 (Asn or Leu, respectively). But neither BRAUNITZER et al. nor DINTZIS mentioned any His in the peptide fiT5. Six rabbits were found with peptide A (Asn, Gln), six with peptide B (Ser, His) and only three with both peptides. In rabbits AB, the two peptides were present in equal amounts. After purification we obtained twice as much peptide B (IiO nmoles) from rabbit B fl-chain as from rabbit AB fl-chain (50 nmoles). These values are consistent with the possibility of two allelic forms of the fl-chain structural gene. We have not done any breeding experiment; however, these results make allelomorphism quite likely. The apparent inconsistency between the equal frequency of both alleles A and B in the population and the low proportion of heterozygous rabbits AB may be due to the small number of rabbits studied. ACKNOWLEDGMENTS
We wish to thank Dr. D. Labie for helpful discussion. REFERENCES I G. VON EHRENSTEIN, Cold Spring Harbor Symp. Quant. Biol., 31 (1966) 705 . 2 G. SCHAPIRA, J. C. DREYFUS AND N. MALEKNIA, Biochem. Biophys. Res. Commun., 32 (1968) 558 • 3 G. SCHAPIRA, M. BENRUBI, N. MALEKNIAAND L. REIBEL, Biochim. B*ophys. Acta, 188 (1969) 216. 4 T. HUNTER AND A. MUNRO, Nature, 223 (197 o) 127o. 5 M. A. NAUGHTON AND H. M. DINTZlS, Proc. Natl. Acad. Sci. U.S., 48 (1962) 1822. 6 G. BRAUNITZER, S. B. BEST, F. ULRICH AND B. SCHRANK,Z. f. Physiol. Chem., 347 (1966) 207. 7 M. RAmNOVlTZ, M. L. FREEDMAN, J. M. FISHER AND C. R. MAXWELL, Cold Spring Harbor Symp. Quant. Biol., 34 (1969) 567. 8 H. M. DINTZlS, Proc. Natl. Acad. Sci. U.S., 47 (1961) 247. 9 R. T. JONES, Cold Spring Harbor Syrup. ~uant. Biol., 29 (1964) 297. IO W. A. SCHROEDER, R. T. JONES, J. CORNICK AND K. MCCALLA, Anal. Chem., 34 (I962) 1571. II R.V. ECK AND M. O. DAYHOFF,Atlas of Protein Sequence and Structure, National Biochemical Research Foundation, Silverspring, Maryland, 1966.
Biochim. Bzophys. Acta, 229 (1971) 712-715