The chemical effects of gene mutations in some 

 abnormal human haemoglobins 



J. A. HUNT AND V. M. INGRAM 



Medical Research Council Unit for Molecular Biology, 

 Cavendish Laboratory, University of Cambridge 



It has been known for some years that the inherited haemolytic anaemia, 

 known as sickle cell anaemia, is due to an alteration in the structure of the 

 haemoglobin molecule.^ This change is thought to be caused by a single 

 mutation of the gene responsible for haemoglobin synthesis.^ We can 

 now report that the effect of this mutation is to change only one amino 

 acid residue in polypeptide chains containing nearly 300 residues; one 

 of the glutamic acids in normal haemoglobin has been replaced by 

 valine. 



Earlier experiments indicated that the two haemoglobins differed electro- 

 phoretically,^'^ haemoglobin S — the sickle cell anaemia type — having 2-3 

 carboxyl groups fewer than haemoglobin A — the normal protein. Deoxy- 

 genated haemoglobin S has an abnormally low solubility* which causes the 

 sickling of the red blood cells characteristic of this anaemia. However, 

 determinations of amino acid composition,^-^ A^-terminaF-^-^ and C- 

 terminal amino acids^^* or sulphydryl groups^^'^^ did not show any signifi- 

 cant differences between them. More recently, ^^ degradation of the two 

 haemoglobins with trypsin showed that the alteration which produces haemo- 

 globin S resides in one very short section of the polypeptide chains. Whilst 

 most of the fragments in the two haemoglobin showed similar behaviour 

 on electrophoresis and chromatography, a pair of peptides, called No. 4, 

 were found in which these properties differed. The complete chemical struc- 

 ture of these two peptides has now been determined by amino acid analyses, 

 end group analyses, some Edman stepwise degradations and the results of 

 partial acid hydrolyses — the last are shown in Fig. 1 (a) — which led to the 

 formulation of the two sequences.^* They each consist of the nine amino 

 acids shown in Fig. 1 (b). It can be seen that they differ in only one amino acid ; 

 one of the glutamic acids of the normal No. 4 peptide is replaced by valine 

 in the sickle cell No. 4 peptide. To date further investigation of the other 

 peptides which had been obtained by trypsin digestion, has failed to reveal 

 any additional differences. The trypsin resistant cores of the two haemo- 

 globin molecules — about 30% of the molecule — have been digested with 



