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HEMOGLOBIN, 1(5),
419-426 (1977)
HEMOGLOBIN ARLINGTON PARK A NEW HEMOGLOBIN VARIANT WITB TWO AMINO ACID SUBSTITUTIONS IN THE 6 CliAIN Junius C . Adams, 111 and Paul Heller
VA Westside Hospital and the Department of Medicine of the University of Illinois Abraham Lincoln School of Medicine, P.O. Box 8195, Chicago, IL 60680. Abstract Hemoglobin Arlington Park was detected accidentally as result of the use of a tiemolysate which contained hemoglobin with normal electrophoretic mobility as a control for peptide mapping. Peptide maps of this hemolysate revealed 3 new peptldes resulting from 2 amino acid substitutions to give a new variant with the composition (B6 GlwLys, 95 Lys-rClu). INTRODUCTION Hemoglobin Arlington Park was accidentally discovered in the course of the ongoing Veterans Administration Cooperative Study on the Sickle Cell Trait. A blood sample which contained a hemoglobin with a normal electrophoretic pattern was randomly selected to serve as a putatively normal control for peptide mapping.
Three abnormal peptides were present which corres-
ponded to those of the abnormal B chains of Hb C ( 8 6 GllrtLys) N-Baltimore (095 Lys-*clu).
Thus, Hb Arlington Park is a
previously undescribed 6 chain variant with two amino acid substitutions with opposite charges which result in normal electrophoretic mobility of this hemoglobin variant. 419 Copyright 0 1977 Iiy Marcel Ikklier. Inc. A l l Kighls Rewrveil. Ncilhcr this work nor any par1 may be reproduced or transmilled in any fiirni or by any nirahs. electronic or iiiechrnicrl. including photocopying. niicrofilming. and recording. or hy any informalitm storagc and r r l r i r v d syrlcla. willioul prrnitulon in writing from the publisher.
ADAMS AND tIELLER
420 MATERIAL AND METIIODS
The b l o o d s a m p l e , c o l l e c t e d i n Na2EDTA, o r i g i n a t e d from a 47y e a r o l d b l a c k m a l e who w a s known t o h a v e severe h y p e r t e n s i o n .
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When t h e d i s c o v e r y o f a n a b n o r m a l p e p t i d e map w a s made s e v e r a l d a y s l a t e r t h e p a t i e n t had succumbed t o a c e r e b r o - v a s c u l a r
accident.
Review o f h i s h o s p i t a l c h a r t d i d n o t d i s c l o s e a n y h e m a t o l o g i c abnormality.
The p a t i e n t had c a r d i o m e g a l y , b u t n o e v i d e n c e o f
c o n g e s t i v e or r e n a l f a i l u r e .
T h e r e a p p e a r e d t o b e no l i v i n g b l o o d
relatives. Tlie h e m o l y s a t e w a s p r e p a r e d by s t a n d a r d t e c h n i q u e s a n d t h e stroma e x t r a c t e d with toluene.
C e l l u l o s e acetate e l e c t r o p h o r e s i s
w a s c a r r i e d o u t i n t h e Beclanan M i c r o z o n e a p p a r a t u s u s i n g a t r i s -
EDTA-borate
b u f f e r , pH 8 . 6 (1). Acid a g a r e l e c t r o p h o r e s i s was
p e r f o r m e d u s i n g Bacto-Agar
( D i f c o L a b o r a t o r i e s ) a n d 0.04N c i t r a t e
b u f f e r , pH 6 . 2 ( 2 ) . The a a n d 8 c h a i n s o f g l o b i n , p r e p a r e d from t h e b l o o d o f t h e p r o b a n d and of i n d i v i d u a l s w i t h tlb AC a n d Hb AN-Baltimore,
were
s e p a r a t e d by a m o d i f i c a t i o n o f t h e p r o c e d u r e o f C l e g g e t a 1 ( 3 , 4 ) . Tlie v a r i a n t B-chains were a m i n o e t h y l a t e d ( 3 ) . d e s a l t e d on a
90x2.5 cm column o f B i o g e l P-2 e q u i l i b r a t e d w i t h f o r m i c a c i d , and freeze-dried. T r y p t i c p e p t i d e maps were p r e p a r e d a s p r e v i o u s l y d e s c r i b e d
( 4 ) and were s t a i n e d w i t h 0.021: b u f f e r e d n i n h y d r i n ( 3 ) and w i t h s p e c i f i c s t a i n s for h i s t i d i n e ( 5 ) a n d a r g i n i n e ( 6 ) .
Tryptic
p e p t i d e s were s e p a r a t e d by c h r o m a t o g r a p h y o n 0.9d.3 c m columns
IIEMOGLOBIN ARLINGTON PARK
421
o f Deckman PA-35 r e s i n a t 5OoC u s i n g a l i n e a r g r a d i e n t o f p y r i d i n e a c e t a t e b u f f e r s from O.ZN(pH 3.1) t o Z.ON@H 5.0) (7).
The
abnormal p e p t i d e s w e r e r e c h r o m a t o g r a p h e d on 0 . 9 ~ 6 0 c m columns of
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Aminex AG 5OW-2 r e s i n u s i n g t h e same c o n d i t i o n s ss f o r t h e i n i t i a l separation.
S u b t r a c t i v e Edman d e g r a d a t i o n s o f t h e abnormal p e p t i d e s
were c a r r i e d o u t a c c o r d i n g t o S h e a r e r e t a1 (8). abnormal BT-l0,ll
C l e a v a g e of t h e
a t t h e a s p a r t y l - p r o l y l bond by d i l u t e a c i d
h y d r o l y s s was performed by t h e method o f S c h r o e d e r e t a 1 ( 9 ) . modified a s previously described (4).
The two f r a g m e n t s were
s e p a r a t e d by column chromatography o n Aminex AG 50Wx2 a s d e s c r i b e d above.
The abnormal p e p t i d e s a n d p e p t i d e f r a g m e n t s w e r e h y d r o l y z e d
i n 5.7N HC1 i n vacuo f o r 22 h r a t l l O ° C and a n a l y z e d i n a Beckman 1 2 0 C amino a c i d a n a l y z e r .
RESULTS E l e c t r o p h o r e s i s on c e l l u l o s e a c e t a t e a t a l k a l i n e pH r e v e a l e d a hemoglobin p a t t e r n t h a t was i n d i s t i n g u i s h a b l e from t h a t o f Hh
accompanied by normal amounts o f Hb A 2 .
A
Citrate a g a r e l e c t r o -
p h o r e s i s also f a i l e d t o r e v e a l a n y a b n o r m a l i t y .
The chromatogram
of t h e p o l y p e p t i d e c h a i n s s e p a r a t e d o n carboxymethyl c e l l u l o s e
colunms a l s o had a normal p a t t e r n .
The t r y p t i c p e p t i d e map o f
t h e 8 c h a i n (Fig. l ) , however, was abnormal.
In a d d i t i o n t o t h e
n o r m a l 8 c h a i n p e p t i d e s , t h e r e were t h r e e new p e p t i d e s , d e s i g n a t e d X, Y and 2.
P e p t i d e X was h i s t i d i n e p o s i t i v e , p e p t i d e Y r e a c t e d
o n l y w i t h n i n h y d r i n , and p e p t i d e 2 was b o t h h i s t i d i n e a n d a r g i n i n e
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0
0
~
0 0
0:
0
-0
00
8"
0
0
0 0
0
0 0 0
0 0
0 0
-0
Tryptic peptjde maps of aminoethylated t 3 d r l i n g t o n Park (also A
C
showing the p e p t i d e s of 8 ), 8
N
and 8
Baltimore.
The dashed c i r c l e s
i n d i c a t e the p o s i t i o n s a t which p e p t i d e s a r e normally found and t h e arrows i n d i c a t e the new p e p t i d e s . 422
IlEElOCLOBIN ARLINCTON PARK
423
The p o s i t i o n and s t a i n i n g c h a r a c t e r i s t i c s of t h e s e
positive.
p e p t i d e s s u g g e s t e d t h a t t h e 8 c h a i n of Hb A r l i n g t o n P a r k had t h e combined amino a c i d s u b s t i t u t i o n s of Ilb C (10) and Ilb N-Baltimore
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(11).
Comparison of t h e p e p t i d e maps of t h e t h r e e abnormal
hemoglobins s u p p o r t e d t h i s s u g g e s t i o n ( F i g . 1 ) . Amino a c i d a n a l y s e s of t h e abnormal p e p t i d e s a f t e r t h e i r i s o l a t i o n by column chromatography a r e shown i n T a b l e 1.
The
amino a c i d c o m p o s i t i o n s of p e p t i d e X and of p e p t i d e Y were t h o s e o f BT-la
(i.e.
r e s i d u e s 81-6) and
8T-lb (1.e. r e s i d u e s 87-8) of
Hb C i n d i c a t i n g a s u b s t i t u t i o n of g l u t a m i c a c i d by l y s i n e a t p o s i t i o n 6.
The amino a c i d c o m p o s i t i o n of p e p t i d e Z was i d e n t i c a l
TABLE I AMINO A C I D COMPOSITION OF ABNORMAL PEPTIDES FROM 8 CHAINS OF Hb ARLINCTON PARK
PEPTIDE AMINO A C I D
LYS **SAE Cys
His Arg ASP Thr Ser Clu Pro GlY
-x 1.0
0.9
1.0
1.1
* **
0.9 1.0
-z
za -
0.7 1.9 1.0 2.8 1.9
0.8 2.1
Zb -
1.0
0.9
Ala
Val Leu Phe
-Y
PEPTIDE FRACHENTS*
1.1 3.1 0.9 1.1 1.2 1.0
3.1 1.8
2.0 1.8 1.1 2.9
1.o 1.1
0.8
1.1 1.0 0.8 3.0 1.0
1.0
P e p t i d e f r a g m e n t s o b t a i n e d by d i l u t e a c e t i c a c i d h y d r o l y s i s of peptide Z (see text). S-amino e t h y l -
424
ADAMS AND IIELLER
w i t h t h a t of BT-lO,ll
( i . e . r e s i d u e s 883-104) o f Hb N-Baltimore,
i n d i c a t i n g a second s u b s t i t u t i o n t h r o u g h which l y s i n e is r e p l a c e d by g l u t a m i c a c i d a t p o s i t i o n 95.
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The y i e l d s o f 8T-1,
BT-10 and BT-11 were a p p r o x i m a t e l y 65%
of t h o s e e x p e c t e d f o r t h e normal 8 - c h a i n . X,
The y i e l d s o f p e p t i d e s
Y and Z were a p p r o x i m a t l e y 35% o f t h o s e found in t h e abnormal
hemoglobins C and N.
Thus, t h e proband w a s h e t e r o z y g o u s f o r tlb
A r l i n g t o n P a r k , which c o n s t i t u t e d a b o u t 352 o f t h e t o t a l hemoglobin. L i m i t e d s e q u e n c e d a t a , o b t a i n e d f o r p e p t i d e s X. Y a n d ' Z by s u b t r a c t i v e Edman d e g r a d a t i o n s , c o n f i r m e d t h e s t r u c t d r a l i d e n t i t y o f t h e s e abnormal p e p t i d e s w i t h t h o s e o f
and gN.
Peptide Z
w a s a l s o t r e a t e d w i t h d i l u t e a c e t i c a c i d , which s p e c i f i c a l l y h y d r o l y z e s t h e a s p a r t y l - p r o l y l bond between B99 and 6100.
The
amino a c i d c o m p o s i t i o n s were i d e n t i c a l w i t h t h e t w o c o r r e s p o n d i n g f r a g m e n t s o f B T - l 0 , l l o f Hb N-Baltimore method ( T a b l e 1 ) . t h e C-terminal
o b t a i n e d by t h e same
A s e x p e c t e d , t h e f i r s t Edman d e g r a d a t i o n o f
fragment of t h i s p e p t i d e y i e l d e d p r o l i n t .
DISCUSSION The amino a c i d s u b s t i t u t i o n s i n Hb A r l i n g t o n P a r k presumably
arose by a second m u t a t i o n i n a gene c o d i n g f o r Hb C or Hb NBaltimore, or by a homologous c r o s s o v e r i n an i n d i v i d u a l h e t e r o zygous f o r t h e s e t w o hemoglobins, a l t e r n a t i v e mechanisms by which Hb C Harlem (86 G l w V a l , 73 Asp-cAsn) might h a v e a r i s e n ( 1 2 ) .
HEMOCLOBIN ARI.INGTON PARK
425
There are o t h e r 8 c h a i n v a r i a n t s w i t h t w o amino a c i d s u b s t i t u t i o n s i n t h e same p o l y p e p t i d e c h a i n : Hb C-Ziguinchor
58 Pro-tArg) (13) and Hb S - h a v i s
(66 G l u + V a l ,
(86 GlwVal,
8142 Ala-Val) (14).
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It is not s u r p r i s i n g t h a t i n a l l t h e s e d o u b l e m u t a t i o n s t h e 86
l o c u s is involved s i n c e HL S and C a r e t h e most common 8 c h a i n variants.
V a r i a n t s of t h e a c h a i n a r e rarer and, t h e r e f o r e , t h e
same s h o u l d be t r u e for d o u b l e m u t a t i o n s a t t h e a l o c u s .
Only
one such v a r i a n t h a s been r e p o r t e d , Hb J Singapore (a78 Asn*Asp.
79 Ala+Gly) ( 1 5 ) . and i t is p o s s i b l e t h a t i n t h i s abnormal hemog l o b i n t h e amino a c i d s u b s t i t u t i o n a t a 7 8 is n o t t h e r e s u l t of a m u t a t i o n a l e v e n t , b u t t h e consequence of a p o s t - t r a n s l a t i o n a l chemical r e a c t i o n between t h e n e i g h b o r i n g g l y c i n e and a s p a r a g i n e r e s i d u e s r e s u l t i n g i n deamidation of t h e l a t t e r ( 1 6 ) . ACKNOWLEDGEMENTS T h i s r e s e a r c h was s u p p o r t e d by VA Research Funds.
W e thank
L. W i l l s f o r e x c e l l e n t t e c h n i c a l a s s i s t a n c e .
REFERENCES 1.
Glynn, K.P..
Penner, J . A . ,
Ann. I n t e r n , Med. 2.
Robinson, A.R.,
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3.
Clegg, J . B . .
Biol.
9: 91,
50:
H a r r i s o n , A.P. and Z u e l z e r , W.W.:
745, 1957.
Naughton, M.A. 1966.
and Rucknagel, D.L.:
769, 1968.
Robson. M . ,
.I.Lab. C l i n . Med.
Smith, J . R .
and W e a t h e r a l l , D.J.:
J . Mol.
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4.
ADAMS AND IlELLER
hdams, J.C..
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43:
Tausk, K . and Heller, P.:
W i n t e r , W.P.,
Blood
2611, 1974.
5.
Mann, T. and Leone, E.:
6.
Yamada, S. and I t a n o , H.A.:
Biochern. J .
53:
1 4 0 , 1953.
Bfochim. Biophys. A c t a .
130:
538, 1966. 7.
J o n e s , R.T.:
i n Methods o f Biochemical A n a l y s i s , e d i t e d
by D. G l i c k , p . 205, I n t e r s c i e n c e , I n c . , N e w York, 1970.
8.
9.
S h e a r e r , W.T.,
Bradshaw, R.A.,
J. B i o l . Chem.
242:
C u r d , F.R.N.
5441, 1967.
S c h r o e d e r , W.A.,
S h e l t o n , J.R.,
and J o n e s , R.T.:
Biochemistry
10. H u n t , J . A .
207: 945, -
Naughton, H.A.
Nagel, R.L.
242: 248.
13. C o s s e n s , M . ,
1P59.
and Weatherall, D.J.;
Nature
and Ranney, 11.M.:
J. Biol.
1967.
Garel, M.C.,
and Rosa, J . :
A u v i n e t , J . , Basset, P., Gomes, P . F .
FEBS L e t t . 58: 1 4 9 , 1975.
14. Moo-Penn, W.F., Am.
C o h i c k , J.
1965.
12. Bookcliln, R.M., Chem.
Shelton, J.B.,
2: 992, 1963. N a t u r e 184: 640,
and Ingram. V.M:
11. C l e g g , J . B . ,
a n d P e t e r s , T.:
N a g e l , R.L. a n d Schmidt, R.M.:
A b s t r a c t 325,
SOC. Hematol., 1976, p . 162.
15. Blackwell, R.Q.,
Boon, W.H.,
L f u , C.S. a n d Weng, M . I . :
Biochim. B i o p h y s . Acta. 278, 482, 1972.
16. S h a t t o n , D.M.
and H a r t i c y , B.S.:
Nature
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1970.
HEMOGLOBIN, 1(5),
419-426 (1977)
HEMOGLOBIN ARLINGTON PARK A NEW HEMOGLOBIN VARIANT WITB TWO AMINO ACID SUBSTITUTIONS IN THE 6 CliAIN Junius C . Adams, 111 and Paul Heller
VA Westside Hospital and the Department of Medicine of the University of Illinois Abraham Lincoln School of Medicine, P.O. Box 8195, Chicago, IL 60680. Abstract Hemoglobin Arlington Park was detected accidentally as result of the use of a tiemolysate which contained hemoglobin with normal electrophoretic mobility as a control for peptide mapping. Peptide maps of this hemolysate revealed 3 new peptldes resulting from 2 amino acid substitutions to give a new variant with the composition (B6 GlwLys, 95 Lys-rClu). INTRODUCTION Hemoglobin Arlington Park was accidentally discovered in the course of the ongoing Veterans Administration Cooperative Study on the Sickle Cell Trait. A blood sample which contained a hemoglobin with a normal electrophoretic pattern was randomly selected to serve as a putatively normal control for peptide mapping.
Three abnormal peptides were present which corres-
ponded to those of the abnormal B chains of Hb C ( 8 6 GllrtLys) N-Baltimore (095 Lys-*clu).
Thus, Hb Arlington Park is a
previously undescribed 6 chain variant with two amino acid substitutions with opposite charges which result in normal electrophoretic mobility of this hemoglobin variant. 419 Copyright 0 1977 Iiy Marcel Ikklier. Inc. A l l Kighls Rewrveil. Ncilhcr this work nor any par1 may be reproduced or transmilled in any fiirni or by any nirahs. electronic or iiiechrnicrl. including photocopying. niicrofilming. and recording. or hy any informalitm storagc and r r l r i r v d syrlcla. willioul prrnitulon in writing from the publisher.
ADAMS AND tIELLER
420 MATERIAL AND METIIODS
The b l o o d s a m p l e , c o l l e c t e d i n Na2EDTA, o r i g i n a t e d from a 47y e a r o l d b l a c k m a l e who w a s known t o h a v e severe h y p e r t e n s i o n .
Hemoglobin Downloaded from informahealthcare.com by Chulalongkorn University on 01/10/15 For personal use only.
When t h e d i s c o v e r y o f a n a b n o r m a l p e p t i d e map w a s made s e v e r a l d a y s l a t e r t h e p a t i e n t had succumbed t o a c e r e b r o - v a s c u l a r
accident.
Review o f h i s h o s p i t a l c h a r t d i d n o t d i s c l o s e a n y h e m a t o l o g i c abnormality.
The p a t i e n t had c a r d i o m e g a l y , b u t n o e v i d e n c e o f
c o n g e s t i v e or r e n a l f a i l u r e .
T h e r e a p p e a r e d t o b e no l i v i n g b l o o d
relatives. Tlie h e m o l y s a t e w a s p r e p a r e d by s t a n d a r d t e c h n i q u e s a n d t h e stroma e x t r a c t e d with toluene.
C e l l u l o s e acetate e l e c t r o p h o r e s i s
w a s c a r r i e d o u t i n t h e Beclanan M i c r o z o n e a p p a r a t u s u s i n g a t r i s -
EDTA-borate
b u f f e r , pH 8 . 6 (1). Acid a g a r e l e c t r o p h o r e s i s was
p e r f o r m e d u s i n g Bacto-Agar
( D i f c o L a b o r a t o r i e s ) a n d 0.04N c i t r a t e
b u f f e r , pH 6 . 2 ( 2 ) . The a a n d 8 c h a i n s o f g l o b i n , p r e p a r e d from t h e b l o o d o f t h e p r o b a n d and of i n d i v i d u a l s w i t h tlb AC a n d Hb AN-Baltimore,
were
s e p a r a t e d by a m o d i f i c a t i o n o f t h e p r o c e d u r e o f C l e g g e t a 1 ( 3 , 4 ) . Tlie v a r i a n t B-chains were a m i n o e t h y l a t e d ( 3 ) . d e s a l t e d on a
90x2.5 cm column o f B i o g e l P-2 e q u i l i b r a t e d w i t h f o r m i c a c i d , and freeze-dried. T r y p t i c p e p t i d e maps were p r e p a r e d a s p r e v i o u s l y d e s c r i b e d
( 4 ) and were s t a i n e d w i t h 0.021: b u f f e r e d n i n h y d r i n ( 3 ) and w i t h s p e c i f i c s t a i n s for h i s t i d i n e ( 5 ) a n d a r g i n i n e ( 6 ) .
Tryptic
p e p t i d e s were s e p a r a t e d by c h r o m a t o g r a p h y o n 0.9d.3 c m columns
IIEMOGLOBIN ARLINGTON PARK
421
o f Deckman PA-35 r e s i n a t 5OoC u s i n g a l i n e a r g r a d i e n t o f p y r i d i n e a c e t a t e b u f f e r s from O.ZN(pH 3.1) t o Z.ON@H 5.0) (7).
The
abnormal p e p t i d e s w e r e r e c h r o m a t o g r a p h e d on 0 . 9 ~ 6 0 c m columns of
Hemoglobin Downloaded from informahealthcare.com by Chulalongkorn University on 01/10/15 For personal use only.
Aminex AG 5OW-2 r e s i n u s i n g t h e same c o n d i t i o n s ss f o r t h e i n i t i a l separation.
S u b t r a c t i v e Edman d e g r a d a t i o n s o f t h e abnormal p e p t i d e s
were c a r r i e d o u t a c c o r d i n g t o S h e a r e r e t a1 (8). abnormal BT-l0,ll
C l e a v a g e of t h e
a t t h e a s p a r t y l - p r o l y l bond by d i l u t e a c i d
h y d r o l y s s was performed by t h e method o f S c h r o e d e r e t a 1 ( 9 ) . modified a s previously described (4).
The two f r a g m e n t s were
s e p a r a t e d by column chromatography o n Aminex AG 50Wx2 a s d e s c r i b e d above.
The abnormal p e p t i d e s a n d p e p t i d e f r a g m e n t s w e r e h y d r o l y z e d
i n 5.7N HC1 i n vacuo f o r 22 h r a t l l O ° C and a n a l y z e d i n a Beckman 1 2 0 C amino a c i d a n a l y z e r .
RESULTS E l e c t r o p h o r e s i s on c e l l u l o s e a c e t a t e a t a l k a l i n e pH r e v e a l e d a hemoglobin p a t t e r n t h a t was i n d i s t i n g u i s h a b l e from t h a t o f Hh
accompanied by normal amounts o f Hb A 2 .
A
Citrate a g a r e l e c t r o -
p h o r e s i s also f a i l e d t o r e v e a l a n y a b n o r m a l i t y .
The chromatogram
of t h e p o l y p e p t i d e c h a i n s s e p a r a t e d o n carboxymethyl c e l l u l o s e
colunms a l s o had a normal p a t t e r n .
The t r y p t i c p e p t i d e map o f
t h e 8 c h a i n (Fig. l ) , however, was abnormal.
In a d d i t i o n t o t h e
n o r m a l 8 c h a i n p e p t i d e s , t h e r e were t h r e e new p e p t i d e s , d e s i g n a t e d X, Y and 2.
P e p t i d e X was h i s t i d i n e p o s i t i v e , p e p t i d e Y r e a c t e d
o n l y w i t h n i n h y d r i n , and p e p t i d e 2 was b o t h h i s t i d i n e a n d a r g i n i n e
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0
0
~
0 0
0:
0
-0
00
8"
0
0
0 0
0
0 0 0
0 0
0 0
-0
Tryptic peptjde maps of aminoethylated t 3 d r l i n g t o n Park (also A
C
showing the p e p t i d e s of 8 ), 8
N
and 8
Baltimore.
The dashed c i r c l e s
i n d i c a t e the p o s i t i o n s a t which p e p t i d e s a r e normally found and t h e arrows i n d i c a t e the new p e p t i d e s . 422
IlEElOCLOBIN ARLINCTON PARK
423
The p o s i t i o n and s t a i n i n g c h a r a c t e r i s t i c s of t h e s e
positive.
p e p t i d e s s u g g e s t e d t h a t t h e 8 c h a i n of Hb A r l i n g t o n P a r k had t h e combined amino a c i d s u b s t i t u t i o n s of Ilb C (10) and Ilb N-Baltimore
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(11).
Comparison of t h e p e p t i d e maps of t h e t h r e e abnormal
hemoglobins s u p p o r t e d t h i s s u g g e s t i o n ( F i g . 1 ) . Amino a c i d a n a l y s e s of t h e abnormal p e p t i d e s a f t e r t h e i r i s o l a t i o n by column chromatography a r e shown i n T a b l e 1.
The
amino a c i d c o m p o s i t i o n s of p e p t i d e X and of p e p t i d e Y were t h o s e o f BT-la
(i.e.
r e s i d u e s 81-6) and
8T-lb (1.e. r e s i d u e s 87-8) of
Hb C i n d i c a t i n g a s u b s t i t u t i o n of g l u t a m i c a c i d by l y s i n e a t p o s i t i o n 6.
The amino a c i d c o m p o s i t i o n of p e p t i d e Z was i d e n t i c a l
TABLE I AMINO A C I D COMPOSITION OF ABNORMAL PEPTIDES FROM 8 CHAINS OF Hb ARLINCTON PARK
PEPTIDE AMINO A C I D
LYS **SAE Cys
His Arg ASP Thr Ser Clu Pro GlY
-x 1.0
0.9
1.0
1.1
* **
0.9 1.0
-z
za -
0.7 1.9 1.0 2.8 1.9
0.8 2.1
Zb -
1.0
0.9
Ala
Val Leu Phe
-Y
PEPTIDE FRACHENTS*
1.1 3.1 0.9 1.1 1.2 1.0
3.1 1.8
2.0 1.8 1.1 2.9
1.o 1.1
0.8
1.1 1.0 0.8 3.0 1.0
1.0
P e p t i d e f r a g m e n t s o b t a i n e d by d i l u t e a c e t i c a c i d h y d r o l y s i s of peptide Z (see text). S-amino e t h y l -
424
ADAMS AND IIELLER
w i t h t h a t of BT-lO,ll
( i . e . r e s i d u e s 883-104) o f Hb N-Baltimore,
i n d i c a t i n g a second s u b s t i t u t i o n t h r o u g h which l y s i n e is r e p l a c e d by g l u t a m i c a c i d a t p o s i t i o n 95.
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The y i e l d s o f 8T-1,
BT-10 and BT-11 were a p p r o x i m a t e l y 65%
of t h o s e e x p e c t e d f o r t h e normal 8 - c h a i n . X,
The y i e l d s o f p e p t i d e s
Y and Z were a p p r o x i m a t l e y 35% o f t h o s e found in t h e abnormal
hemoglobins C and N.
Thus, t h e proband w a s h e t e r o z y g o u s f o r tlb
A r l i n g t o n P a r k , which c o n s t i t u t e d a b o u t 352 o f t h e t o t a l hemoglobin. L i m i t e d s e q u e n c e d a t a , o b t a i n e d f o r p e p t i d e s X. Y a n d ' Z by s u b t r a c t i v e Edman d e g r a d a t i o n s , c o n f i r m e d t h e s t r u c t d r a l i d e n t i t y o f t h e s e abnormal p e p t i d e s w i t h t h o s e o f
and gN.
Peptide Z
w a s a l s o t r e a t e d w i t h d i l u t e a c e t i c a c i d , which s p e c i f i c a l l y h y d r o l y z e s t h e a s p a r t y l - p r o l y l bond between B99 and 6100.
The
amino a c i d c o m p o s i t i o n s were i d e n t i c a l w i t h t h e t w o c o r r e s p o n d i n g f r a g m e n t s o f B T - l 0 , l l o f Hb N-Baltimore method ( T a b l e 1 ) . t h e C-terminal
o b t a i n e d by t h e same
A s e x p e c t e d , t h e f i r s t Edman d e g r a d a t i o n o f
fragment of t h i s p e p t i d e y i e l d e d p r o l i n t .
DISCUSSION The amino a c i d s u b s t i t u t i o n s i n Hb A r l i n g t o n P a r k presumably
arose by a second m u t a t i o n i n a gene c o d i n g f o r Hb C or Hb NBaltimore, or by a homologous c r o s s o v e r i n an i n d i v i d u a l h e t e r o zygous f o r t h e s e t w o hemoglobins, a l t e r n a t i v e mechanisms by which Hb C Harlem (86 G l w V a l , 73 Asp-cAsn) might h a v e a r i s e n ( 1 2 ) .
HEMOCLOBIN ARI.INGTON PARK
425
There are o t h e r 8 c h a i n v a r i a n t s w i t h t w o amino a c i d s u b s t i t u t i o n s i n t h e same p o l y p e p t i d e c h a i n : Hb C-Ziguinchor
58 Pro-tArg) (13) and Hb S - h a v i s
(66 G l u + V a l ,
(86 GlwVal,
8142 Ala-Val) (14).
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It is not s u r p r i s i n g t h a t i n a l l t h e s e d o u b l e m u t a t i o n s t h e 86
l o c u s is involved s i n c e HL S and C a r e t h e most common 8 c h a i n variants.
V a r i a n t s of t h e a c h a i n a r e rarer and, t h e r e f o r e , t h e
same s h o u l d be t r u e for d o u b l e m u t a t i o n s a t t h e a l o c u s .
Only
one such v a r i a n t h a s been r e p o r t e d , Hb J Singapore (a78 Asn*Asp.
79 Ala+Gly) ( 1 5 ) . and i t is p o s s i b l e t h a t i n t h i s abnormal hemog l o b i n t h e amino a c i d s u b s t i t u t i o n a t a 7 8 is n o t t h e r e s u l t of a m u t a t i o n a l e v e n t , b u t t h e consequence of a p o s t - t r a n s l a t i o n a l chemical r e a c t i o n between t h e n e i g h b o r i n g g l y c i n e and a s p a r a g i n e r e s i d u e s r e s u l t i n g i n deamidation of t h e l a t t e r ( 1 6 ) . ACKNOWLEDGEMENTS T h i s r e s e a r c h was s u p p o r t e d by VA Research Funds.
W e thank
L. W i l l s f o r e x c e l l e n t t e c h n i c a l a s s i s t a n c e .
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