The Free Amino Groups of Haemoglobins 



R. R. PORTER and F. SANGER 



A comparative study of certain features of the structures of a 

 number of mammalian haemoglobins has been carried out. x Inform- 

 ation as to the structure was obtained by identifying and estimating 

 the amino-acids which carry a free amino group.' 2 It was hoped 

 that such an investigation of the general structure of haemoglobins 

 from closely related and more distant species might suggest a 

 chemical basis for the immunological specificity of these proteins. 

 Their physical and chemical properties differ according to the species 

 of origin but only their serological behaviour shows a systematic 

 variation which follows their genealogical relationships. A graduated 

 variation in structure was in fact revealed and in horse haemoglobin 

 a preliminary study of the terminal peptides of the polypeptide chains 

 has given further information about the molecular pattern. 



THE DNP METHOD 



The principle of the method used for the identification and estimation 

 of free amino groups in proteins may be illustrated by the following 

 formulae : 



F NHt'Protein NH-protein 



/\ /\ 



NO, alcoholic 



+ 



\y / bicarbonate 



NO 2 solution NO 



NO, acid 



hydrolysis 



FDNB DNP-protein 



R 



I 

 NH— CH— COOH NHCH,CH,CH 2 CH 2 CH COOH 



/\ I 



NO, NO, NH, 



■+■ + amino-acids 



V 



NO, NO, 



a-DNP-amino-acid e-DNP-lysine 



The reagent used is 1:2: 4-fluorodinitrobenzene (FDNB), which 

 reacts quantitatively with the free amino groups of the proteins at 

 room temperature. On hydrolysis of the DNP-protein by strong acid 

 the yellow DNP-amino-acids, which are relatively stable, may be 

 separated from the other amino-acids and can be fractionated 

 successfully using partition chromatography on wet silica gel. 3 In this 

 manner they can be identified and estimated colorimetrically. 



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