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A new haemoglobin variant: J-Rovigo alpha 53 (E-2) alanine → aspartic acid
Biochimica et Biophysica Acta, 342 (1974) 1-4 :(': Elsevier Scientific Pt,blishing Company, Amsterdam - Printed in The Netherlands
BBA 36666 A NEW H A E M O G L O B I N V A R I A N T : J-ROVIGO A L P H A 53 (E-2) A L A N I N E ASPARTIC A C I D
R. ALBERTI", G. M. MARIUZZI a, L. ARTIBAN[b, 12. BRUNI b and L. TENTORV aCattedra di Istochimica dell" Universith di Ferrara (Italy), Centro di Studi sulla Talassemia delr Ospedale Civile di Rov~,o (Italy), and blstituto Superiore di Sanita di Roma (Italy)
(Received September 14th, 1973)
SUMMARY A new haemoglobin has been found in Rovigo (Italy). This haemoglobin is an unstable variant of the alpha chain (alpha 53 (E 2) Ala -~- Asp), and is as fast moving as haemoglobin J. It represents a high percentage (35 49.5',,',) of the total haemoglobin. The amino acid composition of the new peptide is described.
In Rovigo (Italy) we have found in two Italian brothers and in their sons (five carriers in total) a new fast-moving haemoglobin variant of the alpha chain, alpha 53 (E 2) Ala - - Asp which has never been described before. MAT12RIALS AND METHODS Venous blood samples were collected in EDTA and standard haematological methods were employed. The red cells were counted ill a Coulter-counter and their osmotic fragility was determined according to Miale [I ]. Haemoglobin was extracted into both water-ether and water-carbon tetrachloride solutions. Haemoglobin typing was carried out by electrophoresis on a cellulose acetate strip in Tris-glycine buffer, pH 9.5. Separation was obtained on CM-cellulose column chromatography and relative amounts of Hb A, and Hb J were determined by starch-block electrophoresis. The haemoglobin instability of the abnormal fraction was detected using two techniques: the Carrel-Kay method (isopropanol treatment at pH 7.4) and the Jacob-Winterhalter method (incubation at 50 :'C, for 2 h) [2, 3]. The abnormal haemoglobin was further characterised by the separation of the globin chains on cellulose acetate [4] and by hybridisation with canine haemoglobin [5]. Globin was obtained by the Rossi Fanelli method [6]; the separation of the polypeptide chains and tryptic digestion was carried out as described by Clegg et al. [7, 8], and the fingerprinting was performed according to the Ingram method, modified by Baglioni [9]. The chromatographic spots were visualized with ninhydrin 0.2}~; (w/v) in acetone, and treated with specitic staining for histidine and tyrosine [10]. The peptides to be analyzed for their amino acid composition were directly eluted from the fingerAbbreviation: Hb. haemoglobin.
p r i n t i n g . T h e c l u t i o n was p e r f o r m e d with HCI 5.7 M. and h ) d r o l y s i s \sas carried out u n d e r v a c u u m in scaled vials for 18 h at 1(15 ' C a c c o r d i n g to S p a c k m a n ct al. [11]. T h e a m i n o acid c o m p o s i t i o n o f thc new p e p t i d c was d e t e r m i n c d . RESUI.TS T h e h a e m o g l o b i n was u n s t a b l e as was s h o w n fl'om the instability test uscd and the p r e c i p i t a t e o b t a i n e d was d a r k - r e d and extensive. E l e c t r o p h o r e s i s on h o r i z o n t a l cellogel a c e t a t e in T r i s - g l y c i n c buffer at alkaline p H r e v e a l e d the p r e s e n c e o f a f a s t - m o v i n g h a e m o g l o b i n v a r i a n t with a m o b i l i t y iden-
BBt,~36GC~
tt!
A
ANODE
13
:
' l
' i
l z
1 ,
1 ,
Fig. 1. Electrophoresis on (A) cellulose acetatc and (B) Tris-glycinc buffer, pH 9.5; Amido black stain. Both plates show: Top: Hb J-Rovigo: it will bc seen that this fraction is present in a more anodic position.than Hb A ; two fractions Az IA2 J and A2 normal) are visiblc. Bottom : normal haemoglobin.
tical to other haemoglobins J, and two Hb A2 fractions, thus indicating the presence of an alpha chain variant (Fig. IA and IB). Hybridisation with canine haemoglobin and analytical separation of the polypeptide chains on cellulose acetate showed that the variant was indeed in the alpha chain. The abnormal alpha chain was eluted before the normal alpha chain on CMcellulose column chromatography (Fig. 2).
E 0.2-
/3 ~
C:
0
N
-TRovigo
_A
0.1-
s'o
1;0
1;0
2;0
Effluent volume (rely
Fig. 2. CM-ce]lulose chromatography of the globin chain, alpha J-Rovigo is eluted before the normal alpha chain.
Fingerprinting of the tryptic hydrolysate of the purified abnormal alpha chain revealed the absence of the histidine-tyrosine positive spot alpha Tp VI (containing residues 41-56) and the appearance of a new peptide more negatively charged, giving a positive staining for histidine-tyrosine (Fig. 3). Table I shows the amino acid composition of the new peptide, which differs from the alpha Tp VI of haemoglobin A, by one additional residue of aspartic acid and the absence of the alanine residue at the alpha 53 position. This new haemoglobin variant which has never been described is defined as "Haemoglobin J Rovigo alpha2S3 A,. beta,".
C>
cKAT~
__ j . /
-Q+ Tp ~J Rovlgo
Fig. 3. T h e fingerprinting o f the tryptic hydrolysate of the a b n o r m a l alpha chain s h o w s the absence o f the n o r m a l alpha A T6 (residues 41-56) a n d the a p p e a r a n c e of a new peptide alpha ~ T6 m o r e negatively charged.
"FABLE I AMINO ACID COMPOSITION (MOI.AR RATIO) O1" NORMAL AND ABNORMAL ALPHA Tp VI. Amino acid
alpha ~Tp VI
I..,, s H is
0.90
I. IO
1.9{') 1.02 0.93 2. I0
2. I I 1.92 0.89 1.90
1.02 1.03 1.04
0.99 I. 17 I. I 0 0.IX) 1.04 1.02 0.83 2.12
Asp 'rhr
Ser Glu Pro GIy A la Val l.cu Tyr Phe
1.04 1.22 I. 14 0.80 1.94
alpha ~-R°~o Tp VI
T h e p r e s e n c e o f a s p a r t i c acid, r e p l a c i n g a l a n i n c in this v a r i a n t , repeats the findings o f o t h c r h a e m o g l o b i n s J, thus d e m o n s t r a t i n g that this s u b s t i t u t i o n can be in different p o s i t i o n s e i t h e r in the a l p h a o r in the beta chain [12].
Clinical and haenTatological findings A total o f 8 m e m b e r s o f the f a m i l y were investigated and five o f t h e m wcre carriers. N o n e s h o w e d a b n o r m a l findings on physical e x a m i n a t i o n . T h e h a e m a t o l o gical d a t a were n o r m a l " n o n e o f the h e t e r o z y g o t e s was a n a e m i c : the o s m o t i c fragility o f the red cells was n o r m a l : H b A2 was n o t c l e v a t e d : and H b J c o n s t i t u t e d 35-49.5 " , o f the total h a e m o g l o b i n . E v e n t h o u g h this h a e m o g l o b i n is m o l e c u l a r l y unstable, it d o e s n ' t c a u s e any clinical d i s t u r b a n c e s . REFERENCES I 2 3 4 5 6 7 8 9 10 I1 12
Miale, G. B. (1962) Lab. Med. Hemat., 3d Ed., p. 482, C.V. Mosby Cy., Saint Louis 1967 Carrel, R. W. and Kay, R. (1972) Br. J. of Haem. 23, 615-619 Jacob, H. S. and Winterhalter, K. H. (1970) J. Clin. Invest. 49, 2008-2016 Kornarmy, L. and Marshall, G. B. (1972) Am. J. Clin. Path. 58, 428-430 Huehns, E. R., Shooter, E. M. and Beaven, G. H. (1962) J. Mol. Biol. 4, 323 428 Rossi Fanelli, A., Antonini, E. and Caputo, A. (1958) Biochim. Biophys. Acta 30, 608-615 Clegg, J. B., Naughton, M. A. and Weatherall, D. J. (1965) Nature 2(]7, 945-947 Clegg, J. B., Naughton, M. A. and Weatherall, D. J. (1966) J. Mol. Biol. 19, 91 108 Baglioni, C. (1961) Biochim. Biophys. Acta 48, 392-396 Easly, C. W. (1965) Biochim. Biophys. Acta 107, 386-388 Spackmann, D. H., Stein, W. H. and Moore. S. (1958) Anal. Chem. 30, 119(I-I 195 Pootrakul, S., Wasi, P. and Nanakorn, S. (1967) Br. J. Haem. 13, 303-309
BBA 36666 A NEW H A E M O G L O B I N V A R I A N T : J-ROVIGO A L P H A 53 (E-2) A L A N I N E ASPARTIC A C I D
R. ALBERTI", G. M. MARIUZZI a, L. ARTIBAN[b, 12. BRUNI b and L. TENTORV aCattedra di Istochimica dell" Universith di Ferrara (Italy), Centro di Studi sulla Talassemia delr Ospedale Civile di Rov~,o (Italy), and blstituto Superiore di Sanita di Roma (Italy)
(Received September 14th, 1973)
SUMMARY A new haemoglobin has been found in Rovigo (Italy). This haemoglobin is an unstable variant of the alpha chain (alpha 53 (E 2) Ala -~- Asp), and is as fast moving as haemoglobin J. It represents a high percentage (35 49.5',,',) of the total haemoglobin. The amino acid composition of the new peptide is described.
In Rovigo (Italy) we have found in two Italian brothers and in their sons (five carriers in total) a new fast-moving haemoglobin variant of the alpha chain, alpha 53 (E 2) Ala - - Asp which has never been described before. MAT12RIALS AND METHODS Venous blood samples were collected in EDTA and standard haematological methods were employed. The red cells were counted ill a Coulter-counter and their osmotic fragility was determined according to Miale [I ]. Haemoglobin was extracted into both water-ether and water-carbon tetrachloride solutions. Haemoglobin typing was carried out by electrophoresis on a cellulose acetate strip in Tris-glycine buffer, pH 9.5. Separation was obtained on CM-cellulose column chromatography and relative amounts of Hb A, and Hb J were determined by starch-block electrophoresis. The haemoglobin instability of the abnormal fraction was detected using two techniques: the Carrel-Kay method (isopropanol treatment at pH 7.4) and the Jacob-Winterhalter method (incubation at 50 :'C, for 2 h) [2, 3]. The abnormal haemoglobin was further characterised by the separation of the globin chains on cellulose acetate [4] and by hybridisation with canine haemoglobin [5]. Globin was obtained by the Rossi Fanelli method [6]; the separation of the polypeptide chains and tryptic digestion was carried out as described by Clegg et al. [7, 8], and the fingerprinting was performed according to the Ingram method, modified by Baglioni [9]. The chromatographic spots were visualized with ninhydrin 0.2}~; (w/v) in acetone, and treated with specitic staining for histidine and tyrosine [10]. The peptides to be analyzed for their amino acid composition were directly eluted from the fingerAbbreviation: Hb. haemoglobin.
p r i n t i n g . T h e c l u t i o n was p e r f o r m e d with HCI 5.7 M. and h ) d r o l y s i s \sas carried out u n d e r v a c u u m in scaled vials for 18 h at 1(15 ' C a c c o r d i n g to S p a c k m a n ct al. [11]. T h e a m i n o acid c o m p o s i t i o n o f thc new p e p t i d c was d e t e r m i n c d . RESUI.TS T h e h a e m o g l o b i n was u n s t a b l e as was s h o w n fl'om the instability test uscd and the p r e c i p i t a t e o b t a i n e d was d a r k - r e d and extensive. E l e c t r o p h o r e s i s on h o r i z o n t a l cellogel a c e t a t e in T r i s - g l y c i n c buffer at alkaline p H r e v e a l e d the p r e s e n c e o f a f a s t - m o v i n g h a e m o g l o b i n v a r i a n t with a m o b i l i t y iden-
BBt,~36GC~
tt!
A
ANODE
13
:
' l
' i
l z
1 ,
1 ,
Fig. 1. Electrophoresis on (A) cellulose acetatc and (B) Tris-glycinc buffer, pH 9.5; Amido black stain. Both plates show: Top: Hb J-Rovigo: it will bc seen that this fraction is present in a more anodic position.than Hb A ; two fractions Az IA2 J and A2 normal) are visiblc. Bottom : normal haemoglobin.
tical to other haemoglobins J, and two Hb A2 fractions, thus indicating the presence of an alpha chain variant (Fig. IA and IB). Hybridisation with canine haemoglobin and analytical separation of the polypeptide chains on cellulose acetate showed that the variant was indeed in the alpha chain. The abnormal alpha chain was eluted before the normal alpha chain on CMcellulose column chromatography (Fig. 2).
E 0.2-
/3 ~
C:
0
N
-TRovigo
_A
0.1-
s'o
1;0
1;0
2;0
Effluent volume (rely
Fig. 2. CM-ce]lulose chromatography of the globin chain, alpha J-Rovigo is eluted before the normal alpha chain.
Fingerprinting of the tryptic hydrolysate of the purified abnormal alpha chain revealed the absence of the histidine-tyrosine positive spot alpha Tp VI (containing residues 41-56) and the appearance of a new peptide more negatively charged, giving a positive staining for histidine-tyrosine (Fig. 3). Table I shows the amino acid composition of the new peptide, which differs from the alpha Tp VI of haemoglobin A, by one additional residue of aspartic acid and the absence of the alanine residue at the alpha 53 position. This new haemoglobin variant which has never been described is defined as "Haemoglobin J Rovigo alpha2S3 A,. beta,".
C>
cKAT~
__ j . /
-Q+ Tp ~J Rovlgo
Fig. 3. T h e fingerprinting o f the tryptic hydrolysate of the a b n o r m a l alpha chain s h o w s the absence o f the n o r m a l alpha A T6 (residues 41-56) a n d the a p p e a r a n c e of a new peptide alpha ~ T6 m o r e negatively charged.
"FABLE I AMINO ACID COMPOSITION (MOI.AR RATIO) O1" NORMAL AND ABNORMAL ALPHA Tp VI. Amino acid
alpha ~Tp VI
I..,, s H is
0.90
I. IO
1.9{') 1.02 0.93 2. I0
2. I I 1.92 0.89 1.90
1.02 1.03 1.04
0.99 I. 17 I. I 0 0.IX) 1.04 1.02 0.83 2.12
Asp 'rhr
Ser Glu Pro GIy A la Val l.cu Tyr Phe
1.04 1.22 I. 14 0.80 1.94
alpha ~-R°~o Tp VI
T h e p r e s e n c e o f a s p a r t i c acid, r e p l a c i n g a l a n i n c in this v a r i a n t , repeats the findings o f o t h c r h a e m o g l o b i n s J, thus d e m o n s t r a t i n g that this s u b s t i t u t i o n can be in different p o s i t i o n s e i t h e r in the a l p h a o r in the beta chain [12].
Clinical and haenTatological findings A total o f 8 m e m b e r s o f the f a m i l y were investigated and five o f t h e m wcre carriers. N o n e s h o w e d a b n o r m a l findings on physical e x a m i n a t i o n . T h e h a e m a t o l o gical d a t a were n o r m a l " n o n e o f the h e t e r o z y g o t e s was a n a e m i c : the o s m o t i c fragility o f the red cells was n o r m a l : H b A2 was n o t c l e v a t e d : and H b J c o n s t i t u t e d 35-49.5 " , o f the total h a e m o g l o b i n . E v e n t h o u g h this h a e m o g l o b i n is m o l e c u l a r l y unstable, it d o e s n ' t c a u s e any clinical d i s t u r b a n c e s . REFERENCES I 2 3 4 5 6 7 8 9 10 I1 12
Miale, G. B. (1962) Lab. Med. Hemat., 3d Ed., p. 482, C.V. Mosby Cy., Saint Louis 1967 Carrel, R. W. and Kay, R. (1972) Br. J. of Haem. 23, 615-619 Jacob, H. S. and Winterhalter, K. H. (1970) J. Clin. Invest. 49, 2008-2016 Kornarmy, L. and Marshall, G. B. (1972) Am. J. Clin. Path. 58, 428-430 Huehns, E. R., Shooter, E. M. and Beaven, G. H. (1962) J. Mol. Biol. 4, 323 428 Rossi Fanelli, A., Antonini, E. and Caputo, A. (1958) Biochim. Biophys. Acta 30, 608-615 Clegg, J. B., Naughton, M. A. and Weatherall, D. J. (1965) Nature 2(]7, 945-947 Clegg, J. B., Naughton, M. A. and Weatherall, D. J. (1966) J. Mol. Biol. 19, 91 108 Baglioni, C. (1961) Biochim. Biophys. Acta 48, 392-396 Easly, C. W. (1965) Biochim. Biophys. Acta 107, 386-388 Spackmann, D. H., Stein, W. H. and Moore. S. (1958) Anal. Chem. 30, 119(I-I 195 Pootrakul, S., Wasi, P. and Nanakorn, S. (1967) Br. J. Haem. 13, 303-309