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.
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
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.
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.
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