Free Amino Groups of Haemoglobins 



and pig insulin have the same terminal amino-acids 9 but it is in 

 many ways a unique protein ; one of its exceptional properties 

 is non-antigenicity. 



FURTHER DETAILS OF THE STRUCTURE OF 

 HORSE HAEMOGLOBIN 



If we assume that these haemoglobin molecules are made up of several 

 open polypeptide chains, we presuppose that these chains are bound in 

 some manner to form the whole molecule. The method of binding 

 may in some instances be of a labile character, as it has been shown 

 that mild changes of conditions will reduce the molecular weight of 

 horse haemoglobin to half. However stronger cross-linkages between 

 the groups of three chains probably exist. 



Comparison of the numbers of e-amino groups of lysine which 

 reacted with FDNB, with the analytical figures for the total number of 

 lysine residues per molecule shows good agreement. This is in contrast 

 to results found for certain other proteins (Porter 10 ) and shows that in 

 the haemoglobins no interchain linkages occur through the lysine 

 side chains. 



The only known covalent linkage is the — S — S— bridge of cystine. 

 The content of this amino-acid in horse haemoglobin is low, not more 

 than three residues being present (see G. R. Tristram, this volume, p. 109), 

 and some of these are known to be in the reduced state, as — SH groups 

 become reactive on denaturation. It is clear therefore that should the 

 six polypeptide chains of horse haemoglobin be covalently bound into 

 two groups of three, there must exist another type of covalent cross- 

 linkage besides the — S — S— bridges of the cystine residues. 



A further important point which requires to be established in 

 elucidating the structure of horse haemoglobin, is whether the six 

 chains, which all have the same terminal amino-acid, are in fact 

 identical. Information has been obtained on this by an investigation 

 of the peptides produced by partial hydrolysis of DNP-globin. Partial 

 hydrolysis was carried out by dissolving the DNP-globin in 10 N. HO 

 and keeping the solution at 37°C for 4 hours. The terminal DNP- 

 peptides could then be fractionated on silica gel columns using suitable 

 solvents. It was possible to crystallize three DNP-valyl peptides 

 purified in this manner, and their structure was found to be as follows : 



/ DNP-valyl-leucine 



2 DNP-valyl-glutamyl-leucine 



3 The amide of 2. 



It is clear that at least two distinct types of chains must exist in the 

 molecule, as two different penultimate residues, leucine and glutamine, 



123 



