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November 1988, Volume 38, Issue 11

Case Reports

HEMOGLOBIN E B-THALASSEMIA IN A PAKISTANI FAMILY

Aftab Ahmed  ( Max-Planck Institut fuer Biochemie, Abt. Protein chemie, 8033-Martinsried, West Germany. )
Atiya Abbasi  ( Max-Planck Institut fuer Biochemie, Abt. Protein chemie, 8033-Martinsried, West Germany. )
Gerhard Braunitzer  ( Max-Planck Institut fuer Biochemie, Abt. Protein chemie, 8033-Martinsried, West Germany. )
Zafar H. Zaidi  ( H.E.J. Research Institute of Chemistry, University of Karachi, Kaiachi-32, Pakistan. )

Hemoglobin E is a slow moving B chain variant of hemoglobin, first discovered by Itano<1/sup> Characterized by Hunt et al2 showed glutamic acid at B 26 to be replaced by lysine. It is a common variant of hemoglobin in the world and reported in high frequency from South-East Asia3-6  Cases of Hb E, in combination with thalassemia have been reported on the basis of electrophoretic pattern only. In this communication a case of Hb E with B thalassemia is reported on the basis of amino acid sequencing of the abnormal peptide.

METHODOLOGY

Blood sample of propositus was collected in EDTA. Hematological parameters were deter­mined by normal methods. Radiological exami­nation of the skeletal system was also carried out. Hemolysate was prepared by the classical method7. Electrophoretic separation of hemoglobin was carried out on cellulose acetate membrane8 and on 10% polyacrylamide gel at pH 83g9.
Hemoglobin components were separated by chromatography on DEAE-Sephacel column (l5x2.5cm) with 0.05M Tris/HC1 buffer pH 8.5. Sample was eluted with a linear gradient of 0-0.1 M NaC10
Reversed phase HPLC was used for the separation of globin chains. A column of Nucleo­sil-C4 was equilibrated with 0.1% aqueous tri­fluoroacetic acid (TFA). Sample was eluted with a linear gradient of acetonitrile from 35—60% in 60mm, at a flow rate of imi/min.
The abnormal (3 chain was oxidized and digested with trypsin (TosPheCH2C1-treated, Worthington) at pH 10.5 for lh, followed by pH 9.5 for 2h with enzyme to substrate ratio of 5:10011
Separation of tryptic peptides was carried out by reversed phase HPLC12 on a LiChrosorb RP2 column equilibrated with 0.OSM ammonium acetate. Peptides were eluted with a linear gradient of 0—40% acetonitrile in 60 min at a flow rate of imI/min.
Amino acid composition of the abnormal peptides was determined by an automatic amino acid analyzer Model LC 5000, (Biotronik GmbH, West Germany).
The amino acid sequence was determined in a liquid-phase sequencer Model 890B, (Beck­mann Instrument) according to the method of Edman and Begg13

RESULTS AND DISCUSSION

Hemoglobin E is the third most common variant of hemoglobin. Association of Hb E with (3-thalassemia produces severe clinical problems. In the present study hematological and bioche­mical examinations revealed the following: Hemo­globin 63g/dl, reticulocytes 11%, PCV 0.241/1, MCHC 26g/dl, bilirubin total 4.2mg/di and direct 2.6mg/dl, serum iron 220ig/dl, TIBC 320j.Lg/dl.
The morphology of the erythrocytes showed severe hypochromia, anisoschisto-and poikilocytosis, film suggestive of thalassemia.
Radiological examination of the skeletal system showed markedly generalized osteoporosis and blood dyscrasia.
The electrophoretic pattern of hemolysate on cellulose acetate membrane and polyacrylamide gel showed an elevated Hb F and a slow moving hemoglobin at the position of Hb A 2 (Figure 1).


Separation of hemoglobin components was achieved by chromatography on DEAE-Sephacel confirmed the results of electrophoresis  (Figure 2).


Reversed phase HPLC of hemolysate resulted in the separation of abnormal globin chain approx. 55% (Figure 3)

and showed the absence of BA chain.
Two abnormal peaks were observed in the fingerprint of peptides by RP-HPLC (Figure 4).


Amino acid analysis is presented in Table.

ACKNOWLEDGEMENTS

Authors are grateful to Fatmid Thalassernia Centre, Karachi, Pakistan for providing the blood sample and Ms. E. Mueller, Ms B. Schrank, R. Gautsch and Mr. C. Crombach for technical assistance. One of us (Ahmed, A) thanks the DAAD for providing a fellowship.

ACKNOWLEDGEMENTS

Authors are grateful to Fatmid Thalassernia Centre, Karachi, Pakistan for providing the blood sample and Ms. E. Mueller, Ms B. Schrank,
R. Gautsch and Mr. C. Crombach for technical assistance. One of us (Ahmed, A) thanks the DAAD for providing a fellowship.

REFERENCES

1. Itano, HA., Berggren, W.R. and Sturgeon, PJ. Identification of a fourth abnormal human haemoglobin. J. Am. Chem. Soc., 1954; 76: 2278.
2. Hunt, J.A. and Ingram, V.M, Abnormal human haemoglobins. VI. The chemical difference between     haemoglobins A and E. Biochem. Biophys.Acta, 1961;49: 520-536.
3. Lehman, H. and Singh, R. B. Haemoglobin E in Malaya. Nature (Lond), 1956; 178: 695.
4. Flatz, G., Pik, C. and Sringam, S. Haemoglobino­pathies in Thailand H. Incidence and distribution of elevations of haemoglobin A2 and haemo­globin F; a survey of 2790 people. Br. J. Haematol., 1965; 11: 227.
5. Saleem, M. Haemoglobinopathies. Pakistan Armed Forces Med. J., 1974; 25:9.
6. Farzana, F. Haemoglobinopathies. JPMA., 1975; 25:103.
7. Drabkins, D.L. Spectrophotometric studies; cry- stallographic and optical properties of haemoglobin of man in comparison with those of other species. 3. Biol. Chem., 1946; 164: 703.
8. Schneider, R.G. Development in Laboratory diagnosis, sickle cell disease diagnosis, manage-j ment, education and research. Abramson, H. Bertles, L.W. and Wethers, D.L, Ed. St. Louis, Mosby, 1973, p.230.
9. Maurer, H.R. Disc electrophoresis, Berlin, Walter de Gruyter, 1971; p. 44.
10. lUeinschmidt, T., Sladic-Simic, D. and Braunitzer, G. Column chromatography of human embryonic haemoglobins. Blut, 1982; 45 :283.
11. Hirs, C.H.W. The oxidation of ribonuclease with performic acid. I. Biol. Chem., 1956; 219 611.
12. Kratzin, H., Yang, C. Y., Krusche, J.U. and Hilschmann, N. Praparatative Auftrennung des tryptischen Hydrolysats eines Protein mit Hilfe­ der hochdruck-Fluussigkeitchromatographie. Die Primarstruktur einer monokionalen L-Kette vom K-Typ, Subgruppe I (Bence-Jones-Protein Wes). Hoppe Scylers Z. Physiol. Chem., 1980; 361:1591.
13. Edman, P. and Begg, G. A protein sequenator. Eur. J. Biochem., 1967; 1: 80.

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