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Hemoglobin deer lodge: α2β22 His → Arg
Clin. Biochem. S, 46-50 (1972)
HEMOGLOBIN D E E R LODGE: a..,p._. ~ '"~ - ^"~ A. L A B O S S I E R E , ° F. V E L L A °, J. H I E B E R T , °° and P. G A L B R A I T H °°
*Department of Bioche~nistry, College of Medicine University of Saskatchewa~b Saskatoon a~d Mt. Decv Lodge Hospital. Win~ipeg 12, Manitoba (Received November 12, 1971)
SUMMARY
1. As part of an extensive search for abnormal hemoglobin v a r i a n t s in the population of the province of Manitoba, 4100 blood samples have been examined t h a t were obtained f r o m persons a t t e n d i n g Deer Lodge Hospital in Winnipeg. 2. One of these contained a m i x t u r e of the normal hemoglobins A1 and A, and an abnormal f r a c t i o n with an electrophoretic mobility, at alkaline pH, approximately intermediate between t h a t of A, and A... 3. Purification and characterization of the v a r i a n t f r a c t i o n showed t h a t it contained an arginine in place of a histidine residue in the position next to the N-terminal residue of the beta polypeptide chain. This new v a r i a n t has been designated hemoglobin Deer Lodge.
CASE REPORT
F.E.L., a 50-year-old male, was referred for evaluation of anemia in June 1971. Anemia had been recognized in 1965 when the patient was rejected as a blood donor, although before t h a t time he had donated blood on m a n y occasions. He experienced fatigue in the early m o r n i n g and t r a n s i e n t discomfort in the neck and low back. His diet was good and there was no history of blood loss. His f a t h e r allegedly anemic, was of Welsh-Dutch ancestry and his mother, of English ancestry but now deceased, had never been known to be anemic. His three brothers and two sisters, one son and a d a u g h t e r are alive and well and not anemic. Physical examination revealed no a b n o r m a l i t y a p a r t f r o m the presence of slight pallor and the presence of a palpable spleen tip. L a b o r a t o r y studies showed a volume of packed red cells of 36% with a hemoglobin of 12.6 g/100 ml. There were 3.9 million red cells per cubic mm. of blood and reticulocytes were enumerated at 120,000 per cubic mm. The white cell count was 4,800 per cubic m m of blood with 56% m a t u r e neutrophils, 2% eosinophils, 3% monocytes and 39% lymphocytes. Platelets numbered C o r r e s p o n d e n c e : Dr. F. V e l l a
HEMOGLOGIN DEER LODGE
47
185,000 per cubic mm and appeared adequate on the blood smear. E r y throcyte sedimentation rate was 32 mm in 1 h ( W e s t e r g r e n ) . Total s e r u m bilirubin, alkaline phosphatase and blood urea nitrogen were normal. Serum proteins and proteins electrophoresis were unremarkable. The red blood cells showed slight microcytosis and hypochromia and a sternal marr o w aspirate w a s normal except for the absence of hemosiderin. A sickle cell preparation with 2% sodium metabisulphite was negative a f t e r incubation at room t e m p e r a t u r e for 2 hours. The electrophoretic p a t t e r n of the hemoglobin of the patient's f a t h e r and daughter was indistinguishable from normal, while that of his son also contained the abnormal variant. RESULTS On electrophoresis of the patient's hemoglobin on filter paper (Tris - E.D.T.A. - - b o r a t e / b a r b i t u r a t e discontinuous b u f f e r system p H 8.8), t h e abnormal fraction had a mobility j u s t ahead of that of hemoglobin S and accounted for some 40 percent of the total pigment, while the As band w a s distinct and single and accounted for 2 percent of the total. A ferrihemoglobin solubility test gave normal results, while a 'one min alkali denaturation' test showed a value of less than 1 per cent. There was no evidence of an abnormal heat lability when a solution of hemoglobin w a s incubated for 3 h at 50 °. Electrophoresis on citrate-agar gels ( p H 6.2) showed results indistinguishable f r o m those of normal control hemoglobin samples (1). Purification of the abnormal fraction was achieved by repeated electrophoresis on filter-paper. Peptide maps were prepared f r o m t r y p t i c digests obtained from both normal and abnormal globins by methods t h a t have already been described (2). Maps f r o m the v a r i a n t globin showed the following characteristics: (a) absence of a spot (histidine-positive) in the position of ~Tp I (b) presence of a new spot (arginine-positive) situated between the spots for flTp VI and ~Tp X (~Tp Ia~"a) (c) presence of a new spot slightly more anodal than normal /3Tp I and staining only with ninhydrin (f~Tp Fb"b) (Fig. 1). These results suggested that a new trypsin-sensitive peptide linkage was present in the amino acid sequence represented by flTp I and t h a t it arose by substitution of a histidine by an arginine residue so t h a t on tryptic digestion two new peptides with different charge and chromatographic properties were produced. A f t e r t r e a t m e n t of the v a r i a n t hemoglobin with para-chloromercuribenzoate ( P C M B ) and electrophoresis in a T.E.B.-barbiturate discontinuous b u f f e r system containing PCMB p H 8.8 at room temperature, b y a n
48
LABOSSIERE et M.
0 o
Hb Deer Lodge a
ond
~ choint
0
,:2:)
0
0
0 0 OK) 0 0
0 0
0
o
0
o
o o
0 0 0
0 ':;-:'
0
0
0
--
x
+
FIG. 1. Tracing of peptide map of soluble tryptic peptides from globin of purified Hb Deer Lodge. (1) normal flTpI missing. (a) a new peptide f~TpI ~b"a, arginine-positive. (b) a new peptide flTpI "b"b. Electrophoresis (pH 6.4) in the horizontal and ascending chromatography in the vertical direction.
adaptation for filter-paper of the method of Rosemeyer and Huehns (3), the abnormal beta chains could easily be separated (4). Peptide maps prepared from the variant PCMB-globin showed : (a) absence of flTp I (b) presence of two new spots in the same positions and with the staining properties of flTp I'b"a and flTp I'b"b described above (c) three spots corresponding to the "core" peptides flTp X, flTp XI and flTp XII (which usually do not appear in maps of tryptic digests of globin not treated with PCMB) (Fig. 2). The spots flTp I*b"a and flTp I'~"b were cut, eluted and hydrolyzed in concentrated hydrochloric acid in sealed capillary tubes (105 °, 18 h) and shown to contain valine and arginine, and leucine, threonine, proline, glutamic acid and lysine respectively. Since the three "core" peptides exhibited identical properties in maps of PCMB-treated normal and abnormal beta chains, it was concluded that the variant globin contained only one amino acid substitution and that its molecular structure was a,fl2 ' h,.-..~ The variants of the beta chain which have been reported and which involve the N-terminal octapeptide (i.e. flTp I) are shown in Table 1. Three of these (S, C Harlem and C Georgetown) are associated with the sickling phenomenon. The failure to produce sickling and the normal results obtained with ferrihemoglobin solubility and with heat stability tests suggest that the variant here reported is unlikely to be associated with hemoglobin instability and its hematological consequences. The substitution of histidine by arginine has also been reported in the following three variants: Ztirich (residue 63 of fl chain) (15), F Malta
HEMOGLOGIN D E E R LODGE
0
Hb Deer Lodge i~
49
c holn$
0
0
0
0 0
8 :7-
C)
C)
0
--
x
+
O F/G. 2. Tracing of peptide map of tryptic peptides from PCMB treated ~ chains of purified Hb Deer Lodge. (1) normal ~TpI missing (2), (3) and (4), flTpX, ~TpX1 and flTpXII respectively. (a) a new peptide flTpI "t'"a, arginine-positive. (b) a new peptide ~TpI """b. Electrophoresis (pH 6.4) in the horizontal and ascending chromatography in the vertical direction.
TABLE 1 Tp I VARIANTS residue number
amino acid from to
1 2
val his
3 4 5 6
leu thr pro glu
7
8
-tyr arg ---val val
glu
lys ala ?deleted lys
lys
lys gly ?deleted --
variant -Tokuchi Deer Lodge ---S C Harlem (also 73 Asp--*Asn) C G Makassar Leiden C Georgetown (also ? 83 to 120) Siriraj G San Jose Leiden --
reference -5 present report ---6 7 8 9 10 11 12 13, 14 10 --
( r e s i d u e 117 o f 7 c h a i n ) (16), a n d A_. S p h a k i a ( r e s i d u e 2 o f $ c h a i n ) (17). S i n c e t h e fl a n d 8 c h a i n s o f h u m a n h e m o g l o b i n h a v e a n i d e n t i c a l N - t e r m i n a l o c t a p e p t i d e s e q u e n c e , h e m o g l o b i n s A~ a n d D e e r L o d g e a p p e a r t o h a v e arisen by similar mutations in the structural genes for these two chains.
50
LABOSSIERE et al.
ACKNOWLEDGMENTS
This work was supported by a grant from the Medical Research Council to F.V. We are grateful to Mrs. M. Hurley for technical assistance. REFERENCES 1. VELLA, F. Hemoglobin variants in Saskatchewan. Clin. Biochem. 1, 118-134 {1967). ~. VELLA, F., LORKIN, P. A., CARRELL, R. W. and LEHMANN, H. A new hemoglobin variant resembling hemoglobin E. Hemoglobin E Saskatoon: ~ - c.J,,~L~.~. Canad. J. Biochem. 45, 1385-1391 (1967). 3. ROSEMEYER, M. A. and HUEHNS, E. R. On the mechanism of dissociation of haemoglobin. J. Mol. Biol. 25, 253-273 (1967), /,. LABOSSIERE, A. and VELLA, F. Unpublished observations. 5. SHIBATA, S., IUCHI, I., MIYAJI, T. and TAKEDA, I. Hemoglobinopathy in Japan. Bull. ¥amaguchi Med. School. 10, 1-9 (1963). 6. INGRAM, V. M. Gene mutations in human haemoglobin: the chemical difference between normal and sickle-cell haemoglobin. Nature, 180, 326-328 {1957). 7. BOOKCHIN, R. M., NAGEL, R. L., RANNEY, H. M. and JACOBS, A. S. Hemoglobin C Harlem: a sickling variant containing amino acid substitutions in two residues of the ~-polypeptide chain. Biochem. Biophys. Res. Commun. 23, 122-127 (1966). ,~'. HUNT. J. A. and |NGRAM, V. M. A terminal peptide se:luence of human haemoglobin? Nature, 184, 640-641 (1959}. 9. BLACKWELL, R. Q., OEMIJATI, S., PRIBADI, W., WENG, M. I. and LIU, C. S. Hemoglobin G Makassar: /~" ,~,,~,~l:,. Biochem. Biophys. Acta 21~l, 396-401 (1970} l~J. DE JONG, W. W. W., WENT, L. N. and BERNINI, L. F. Haemoglobin Leiden: Deletion of /~6 or 7 glutamic acid. Nature, 220, 788-790 (1968). I1. GERALD, P. S., and RATH, C. E. Hemoglobin C Georgetown: First abnormal hemoglobin due to two different mutations in the same gene. J. Clin. Invest., 45, 1021 (1966). lz'. TUCHINDA, S., BEALE, D. and LEHMANN, H. A new haemoglobin in a Thai family. A case of haemoglobin Siriraj-/~ thalassemia. Brit. Med. J., 1, 1583-1585 (1965). 13. HILL, R. L., SWENSON, R. T. and SCHWARTZ, H. C. Characterization of a chemical abnormality in Hemoglobin G. J. Biol. Chem. 235, 3182-3187 (1960). 1/,. HILL, R. L., and SCHWARTZ, H. C. A chemical abnormality in Haemoglobin G. Nature, 184, 641-642 (1959). 15. MULLER, C. J. and KINGMA, S. Hemoglobin Zurich, a~A/J..,"3 '~. Biochim. Biophys. Acta. 50, 595 (1961). 16. CAUCHI, M. N., CLEGG, J. B. and WEATHERALL, J. D. Haemoglobin F Malta: a new foetal haemoglobin variant with a high incidence in Maltese infants. Nature 223, 311-313 (1969). 17. JONES, R. T., BRIMHALL, B., HUEHNS, E. R. and BARNICOT, N. A. Hemoglobin Sphakia: a delta-chain variant of hemoglobin A~ from Crete. Science, 151, 1406-1408 (1966).
HEMOGLOBIN D E E R LODGE: a..,p._. ~ '"~ - ^"~ A. L A B O S S I E R E , ° F. V E L L A °, J. H I E B E R T , °° and P. G A L B R A I T H °°
*Department of Bioche~nistry, College of Medicine University of Saskatchewa~b Saskatoon a~d Mt. Decv Lodge Hospital. Win~ipeg 12, Manitoba (Received November 12, 1971)
SUMMARY
1. As part of an extensive search for abnormal hemoglobin v a r i a n t s in the population of the province of Manitoba, 4100 blood samples have been examined t h a t were obtained f r o m persons a t t e n d i n g Deer Lodge Hospital in Winnipeg. 2. One of these contained a m i x t u r e of the normal hemoglobins A1 and A, and an abnormal f r a c t i o n with an electrophoretic mobility, at alkaline pH, approximately intermediate between t h a t of A, and A... 3. Purification and characterization of the v a r i a n t f r a c t i o n showed t h a t it contained an arginine in place of a histidine residue in the position next to the N-terminal residue of the beta polypeptide chain. This new v a r i a n t has been designated hemoglobin Deer Lodge.
CASE REPORT
F.E.L., a 50-year-old male, was referred for evaluation of anemia in June 1971. Anemia had been recognized in 1965 when the patient was rejected as a blood donor, although before t h a t time he had donated blood on m a n y occasions. He experienced fatigue in the early m o r n i n g and t r a n s i e n t discomfort in the neck and low back. His diet was good and there was no history of blood loss. His f a t h e r allegedly anemic, was of Welsh-Dutch ancestry and his mother, of English ancestry but now deceased, had never been known to be anemic. His three brothers and two sisters, one son and a d a u g h t e r are alive and well and not anemic. Physical examination revealed no a b n o r m a l i t y a p a r t f r o m the presence of slight pallor and the presence of a palpable spleen tip. L a b o r a t o r y studies showed a volume of packed red cells of 36% with a hemoglobin of 12.6 g/100 ml. There were 3.9 million red cells per cubic mm. of blood and reticulocytes were enumerated at 120,000 per cubic mm. The white cell count was 4,800 per cubic m m of blood with 56% m a t u r e neutrophils, 2% eosinophils, 3% monocytes and 39% lymphocytes. Platelets numbered C o r r e s p o n d e n c e : Dr. F. V e l l a
HEMOGLOGIN DEER LODGE
47
185,000 per cubic mm and appeared adequate on the blood smear. E r y throcyte sedimentation rate was 32 mm in 1 h ( W e s t e r g r e n ) . Total s e r u m bilirubin, alkaline phosphatase and blood urea nitrogen were normal. Serum proteins and proteins electrophoresis were unremarkable. The red blood cells showed slight microcytosis and hypochromia and a sternal marr o w aspirate w a s normal except for the absence of hemosiderin. A sickle cell preparation with 2% sodium metabisulphite was negative a f t e r incubation at room t e m p e r a t u r e for 2 hours. The electrophoretic p a t t e r n of the hemoglobin of the patient's f a t h e r and daughter was indistinguishable from normal, while that of his son also contained the abnormal variant. RESULTS On electrophoresis of the patient's hemoglobin on filter paper (Tris - E.D.T.A. - - b o r a t e / b a r b i t u r a t e discontinuous b u f f e r system p H 8.8), t h e abnormal fraction had a mobility j u s t ahead of that of hemoglobin S and accounted for some 40 percent of the total pigment, while the As band w a s distinct and single and accounted for 2 percent of the total. A ferrihemoglobin solubility test gave normal results, while a 'one min alkali denaturation' test showed a value of less than 1 per cent. There was no evidence of an abnormal heat lability when a solution of hemoglobin w a s incubated for 3 h at 50 °. Electrophoresis on citrate-agar gels ( p H 6.2) showed results indistinguishable f r o m those of normal control hemoglobin samples (1). Purification of the abnormal fraction was achieved by repeated electrophoresis on filter-paper. Peptide maps were prepared f r o m t r y p t i c digests obtained from both normal and abnormal globins by methods t h a t have already been described (2). Maps f r o m the v a r i a n t globin showed the following characteristics: (a) absence of a spot (histidine-positive) in the position of ~Tp I (b) presence of a new spot (arginine-positive) situated between the spots for flTp VI and ~Tp X (~Tp Ia~"a) (c) presence of a new spot slightly more anodal than normal /3Tp I and staining only with ninhydrin (f~Tp Fb"b) (Fig. 1). These results suggested that a new trypsin-sensitive peptide linkage was present in the amino acid sequence represented by flTp I and t h a t it arose by substitution of a histidine by an arginine residue so t h a t on tryptic digestion two new peptides with different charge and chromatographic properties were produced. A f t e r t r e a t m e n t of the v a r i a n t hemoglobin with para-chloromercuribenzoate ( P C M B ) and electrophoresis in a T.E.B.-barbiturate discontinuous b u f f e r system containing PCMB p H 8.8 at room temperature, b y a n
48
LABOSSIERE et M.
0 o
Hb Deer Lodge a
ond
~ choint
0
,:2:)
0
0
0 0 OK) 0 0
0 0
0
o
0
o
o o
0 0 0
0 ':;-:'
0
0
0
--
x
+
FIG. 1. Tracing of peptide map of soluble tryptic peptides from globin of purified Hb Deer Lodge. (1) normal flTpI missing. (a) a new peptide f~TpI ~b"a, arginine-positive. (b) a new peptide flTpI "b"b. Electrophoresis (pH 6.4) in the horizontal and ascending chromatography in the vertical direction.
adaptation for filter-paper of the method of Rosemeyer and Huehns (3), the abnormal beta chains could easily be separated (4). Peptide maps prepared from the variant PCMB-globin showed : (a) absence of flTp I (b) presence of two new spots in the same positions and with the staining properties of flTp I'b"a and flTp I'b"b described above (c) three spots corresponding to the "core" peptides flTp X, flTp XI and flTp XII (which usually do not appear in maps of tryptic digests of globin not treated with PCMB) (Fig. 2). The spots flTp I*b"a and flTp I'~"b were cut, eluted and hydrolyzed in concentrated hydrochloric acid in sealed capillary tubes (105 °, 18 h) and shown to contain valine and arginine, and leucine, threonine, proline, glutamic acid and lysine respectively. Since the three "core" peptides exhibited identical properties in maps of PCMB-treated normal and abnormal beta chains, it was concluded that the variant globin contained only one amino acid substitution and that its molecular structure was a,fl2 ' h,.-..~ The variants of the beta chain which have been reported and which involve the N-terminal octapeptide (i.e. flTp I) are shown in Table 1. Three of these (S, C Harlem and C Georgetown) are associated with the sickling phenomenon. The failure to produce sickling and the normal results obtained with ferrihemoglobin solubility and with heat stability tests suggest that the variant here reported is unlikely to be associated with hemoglobin instability and its hematological consequences. The substitution of histidine by arginine has also been reported in the following three variants: Ztirich (residue 63 of fl chain) (15), F Malta
HEMOGLOGIN D E E R LODGE
0
Hb Deer Lodge i~
49
c holn$
0
0
0
0 0
8 :7-
C)
C)
0
--
x
+
O F/G. 2. Tracing of peptide map of tryptic peptides from PCMB treated ~ chains of purified Hb Deer Lodge. (1) normal ~TpI missing (2), (3) and (4), flTpX, ~TpX1 and flTpXII respectively. (a) a new peptide flTpI "t'"a, arginine-positive. (b) a new peptide ~TpI """b. Electrophoresis (pH 6.4) in the horizontal and ascending chromatography in the vertical direction.
TABLE 1 Tp I VARIANTS residue number
amino acid from to
1 2
val his
3 4 5 6
leu thr pro glu
7
8
-tyr arg ---val val
glu
lys ala ?deleted lys
lys
lys gly ?deleted --
variant -Tokuchi Deer Lodge ---S C Harlem (also 73 Asp--*Asn) C G Makassar Leiden C Georgetown (also ? 83 to 120) Siriraj G San Jose Leiden --
reference -5 present report ---6 7 8 9 10 11 12 13, 14 10 --
( r e s i d u e 117 o f 7 c h a i n ) (16), a n d A_. S p h a k i a ( r e s i d u e 2 o f $ c h a i n ) (17). S i n c e t h e fl a n d 8 c h a i n s o f h u m a n h e m o g l o b i n h a v e a n i d e n t i c a l N - t e r m i n a l o c t a p e p t i d e s e q u e n c e , h e m o g l o b i n s A~ a n d D e e r L o d g e a p p e a r t o h a v e arisen by similar mutations in the structural genes for these two chains.
50
LABOSSIERE et al.
ACKNOWLEDGMENTS
This work was supported by a grant from the Medical Research Council to F.V. We are grateful to Mrs. M. Hurley for technical assistance. REFERENCES 1. VELLA, F. Hemoglobin variants in Saskatchewan. Clin. Biochem. 1, 118-134 {1967). ~. VELLA, F., LORKIN, P. A., CARRELL, R. W. and LEHMANN, H. A new hemoglobin variant resembling hemoglobin E. Hemoglobin E Saskatoon: ~ - c.J,,~L~.~. Canad. J. Biochem. 45, 1385-1391 (1967). 3. ROSEMEYER, M. A. and HUEHNS, E. R. On the mechanism of dissociation of haemoglobin. J. Mol. Biol. 25, 253-273 (1967), /,. LABOSSIERE, A. and VELLA, F. Unpublished observations. 5. SHIBATA, S., IUCHI, I., MIYAJI, T. and TAKEDA, I. Hemoglobinopathy in Japan. Bull. ¥amaguchi Med. School. 10, 1-9 (1963). 6. INGRAM, V. M. Gene mutations in human haemoglobin: the chemical difference between normal and sickle-cell haemoglobin. Nature, 180, 326-328 {1957). 7. BOOKCHIN, R. M., NAGEL, R. L., RANNEY, H. M. and JACOBS, A. S. Hemoglobin C Harlem: a sickling variant containing amino acid substitutions in two residues of the ~-polypeptide chain. Biochem. Biophys. Res. Commun. 23, 122-127 (1966). ,~'. HUNT. J. A. and |NGRAM, V. M. A terminal peptide se:luence of human haemoglobin? Nature, 184, 640-641 (1959}. 9. BLACKWELL, R. Q., OEMIJATI, S., PRIBADI, W., WENG, M. I. and LIU, C. S. Hemoglobin G Makassar: /~" ,~,,~,~l:,. Biochem. Biophys. Acta 21~l, 396-401 (1970} l~J. DE JONG, W. W. W., WENT, L. N. and BERNINI, L. F. Haemoglobin Leiden: Deletion of /~6 or 7 glutamic acid. Nature, 220, 788-790 (1968). I1. GERALD, P. S., and RATH, C. E. Hemoglobin C Georgetown: First abnormal hemoglobin due to two different mutations in the same gene. J. Clin. Invest., 45, 1021 (1966). lz'. TUCHINDA, S., BEALE, D. and LEHMANN, H. A new haemoglobin in a Thai family. A case of haemoglobin Siriraj-/~ thalassemia. Brit. Med. J., 1, 1583-1585 (1965). 13. HILL, R. L., SWENSON, R. T. and SCHWARTZ, H. C. Characterization of a chemical abnormality in Hemoglobin G. J. Biol. Chem. 235, 3182-3187 (1960). 1/,. HILL, R. L., and SCHWARTZ, H. C. A chemical abnormality in Haemoglobin G. Nature, 184, 641-642 (1959). 15. MULLER, C. J. and KINGMA, S. Hemoglobin Zurich, a~A/J..,"3 '~. Biochim. Biophys. Acta. 50, 595 (1961). 16. CAUCHI, M. N., CLEGG, J. B. and WEATHERALL, J. D. Haemoglobin F Malta: a new foetal haemoglobin variant with a high incidence in Maltese infants. Nature 223, 311-313 (1969). 17. JONES, R. T., BRIMHALL, B., HUEHNS, E. R. and BARNICOT, N. A. Hemoglobin Sphakia: a delta-chain variant of hemoglobin A~ from Crete. Science, 151, 1406-1408 (1966).