MAMMALIAN HEMOGLOBINS 31 o 



adult hemoglobins may have similar amino-acid compositions, total amino-acid 

 analyses fail to disclose the nature of the differences between closely related hemoglobins. 

 However, the analyses are necessary for estimating the number of peptides that can be 

 expected from enzymatic digestion for peptide analyses. 



Sulphydryl analysis 



Attempts have also been made to detect differences in the number of available 

 sulphydryl groups in hemoglobin by amperometric titration. 630, 637 As described 

 by Ingram, the number of available -SH groups is determined by measuring the num- 

 ber of silver atoms per molecule bound as Ag-S-protein. A weak ammoniacal silver 

 nitrate solution is added to the protein until Ag ions are no longer bound. The assump- 

 tions are made that the available -SH groups in hemoglobin will react with metal 

 ions such as Ag or Hg in a 1 : 1 ratio and that the metal ions will not react with non-thiol 

 groups in the protein. Comprehensive studies have revealed minor differences in the 

 number of thiol groups among hemoglobins of man, horse, ox, dog, cow, and 

 sheep, 637, 921 and comparative studies indicate a similar number of -SH groups for 

 human hemoglobins A, S, and C. Although earlier reports using silver ions suggest 

 that eight -SH groups are present, 597 recent studies using mercury ions report only 

 six -SH groups in human hemoglobins. 13 Further application of Hg titration in the 

 study of mammalian hemoglobins can be found in a recent report by Riggs. 1056 



Thus far, configurational differences in hemoglobins that could be produced 

 through disulfide linkage have not been observed. Nevertheless, formation of inter- 

 molecular disulfide linkages could produce hemoglobins of high molecular weight, 

 such as are encountered in some strains of mice. Comparative results of sulf hydryl 

 content of the native versus the denatured, heavy hemoglobin might be used to support 

 or reject the possibility of intermolecular disulfide bonding. Results obtained, how- 

 ever, should be interpreted with caution; Murayama 921 has reported that not all the 

 -SH groups in native hemoglobin may be titratable and that the end point determined 

 by amperometric methods is temperature dependent. 



End-group analysis 



Efforts to establish the amino acids that occupy the terminal position of the poly- 

 peptide chains of the globin moiety of hemoglobin have been more fruitful than 

 sulfhydryl studies. A free alpha-ammo (N-terminal) group remains at one end of a 

 polypeptide and a free carboxyl (C-terminal) group at the other end. These end 

 groups are free to undergo a number of organic reactions. 



N-terminal analysis. — Several methods for N-terminal analysis of peptides and 

 proteins have appeared in the literature. The method developed by Sanger 1153 is 

 based on the fact that 2-4-dinitrofluorobenzene will react with the free amino group 

 forming a bond which is more stable than the peptide linkage of the amino acids in the 

 protein. After addition of the dinitrophenyl group (DNP) the protein can be hydro- 

 lyzed and a fraction of the DNP remains attached to the N-terminal amino acid. 



