150 J. A. HUNT AND V. M. INGRAM [9 



chymotrypsin and subjected to examination by fingerprinting^^ and chromato- 

 graphy ; no differences could be detected. 



This finding is in agreement with such differences between the two haemo- 

 globins as had already been established, when it is remembered that haemoglo- 

 bin (mol. wt. 66,700) has two identical half-molecules,^^ and that therefore 

 the No. 4 peptide occurs twice in the whole molecule. It still remains to 

 relate this change of amino acids to the solubility differences and to the 

 causation of the haemolytic anaemia. However, it is the kind of change 

 which one would expect. 



Human haemoglobin S is a clear case where the mutation of a nuclear 

 gene is inherited in a strictly Mendelian manner. Furthermore, it is well 

 established that this mutation alters the nature of the protein haemoglobin. 

 We can now show for the first time that the effect of such a mutation is a 

 chemical one and that it leads in this case at least to a substitution of only 

 one amino acid in the protein by another one. One may speculate that this 

 very small change in the polypeptide chain of the protein reflects a very 

 small change in the DNA chain of the gene, affecting perhaps not more 

 than one of the nucleotide base pairs of that chain. 



Another mutation of the same haemoglobin gene produces another ab- 

 normal protein, haemoglobin C.^^-^' This causes a severe anaemia only 

 when it occurs together with haemoglobin S. The solubility of haemoglobin 

 C is almost normal, ^^-^^ and so is its amino acid composition;^^ however, 

 it is easily distinguished electrophoretically, since it has even fewer net 

 negative charges per molecule than haemoglobin S which itself has two 

 fewer than the haemoglobin A molecule. 



Haemoglobin C has been degraded with trypsin and, as a second stage, 

 with chymotrypsin, using the methods described for haemoglobin S. The 

 mixtures of peptides obtained were compared with those from haemoglobin 

 A by the 'fingerprinting'^^ and other methods employed for haemoglobin S. 

 Again, it was found that all peptides behaved identically except the No. 4 

 peptide in tryptic haemoglobin A digests whose glutamic acid had changed 

 to valine in haemoglobin S. This same peptide was not found in tryptic 

 digests of haemoglobin C. Its place was taken by two new peptide spots, 

 one neutral and the other positively changed at pH 6-4.^^ Using the methods 

 referred to above, the amino acid sequence of these two peptides has been 

 determined and is shown in Fig. 1 (b). Clearly the same amino acid, glu- 

 tamic acid, of normal haemoglobin has changed also in the haemoglobin C 

 mutation; its place is taken by lysine. In each half molecule a negatively 

 charged side chain has been replaced by a positive group altering the net 

 charge of the protein by four units, twice as many as for haemoglobin S. 

 This interpretation agrees very well with the known electrophoretic series of 

 the haemoglobins which is A — S — C. The additional peptide found in tryp- 

 tic digests of haemoglobin C is due to the introduction of an additional 

 lysine peptide bond which is hydrolysed by the enzyme. 



