MAMMALIAN HEMOGLOBINS 317 



which produces amino-acid hydrazides and amino acids; the latter are derived only 

 from amino-acid residues possessing the free carboxyl group. Upon dinitropheny- 

 lation the hydrazide forms di-DNP-amino acid hydrazide and the amino acid forms 

 DNP-amino acid. The C-terminal, DNP-amino acids are then separated by fractional 

 extraction and can be characterized chromatographically and estimated colorimetri- 

 cally. In the analysis the dicarboxylic amino acids, aspartic and glutamic, may be 

 contaminants in the extract of the C-terminal amino acids. The application of the 

 method has been widely demonstrated, and a review of satisfactory results of analyses 

 of C-terminal residues of a variety of proteins is presented by Haruna and Akabori. 523 



The method as described is not useful for sequence analysis since all the peptide 

 bonds of the molecule are destroyed during the hydrazinolysis. Ohno 962 and Niu 

 and Fraenkel-Conrat 945 ' 946 have modified the hydrazinolysis method for sequential 

 analysis of the amino acids from the C-terminal end. The principle consists of partial, 

 rather than complete, hydrazinolysis of the protein followed by dinitrophenylation of 

 the peptides and extraction of the C-terminal, DNP-peptides from the other di-DNP- 

 peptide hydrazides. The C-terminal peptides are then completely hydrazinized and 

 the sequence inferred from the relative amounts of the hydrazinized amino acids present 

 in the mixture. The method is not highly accurate since some amino acids are partially 

 destroyed and the DNP-derivatives of dicarboxylic amino acid, that is, aspartic and 

 glutamic, are difficult to distinguish from the true C-terminal amino acid. 



Enzymatic methods for obtaining C-terminal analysis appear to be as satisfactory 

 as chemical methods. Use of carboxypeptidase-A and -B, 437 protaminase, 1370 and a 

 Streptomyces griseus proteinase 1157 have been reported. 



Another method (unpublished) can be used to identify the C-terminal peptide. 

 Prior to removing the sample of hemoglobin, lysine can be tagged through injection 

 of C 14 -labeled lysine. Autoradiography and specific chemical tests can be used to 

 identify the peptides that contain lysine and arginine, respectively. Peptides that possess 

 neither arginine nor lysine are presumably in the C-terminal position. 



Peptide analysis 



Knowledge of the chemistry of protein molecules expanded rapidly following 

 development of quantitative methods for analyzing the amino-acid content of proteins. 

 Amino-acid analysis fails, however, to disclose the exact nature of the chemical differ- 

 ences that exist between closely related hemoglobins, such as A and S, and a search was 

 made for another approach to enable investigators to elucidate any structural variations 

 hemoglobins may possess. Scheinberg et a/. 1164, 1165 reported that the differences 

 observed for the electrophoretic mobilities of A and S hemoglobins at neutral pH " 4 

 disappeared when these hemoglobins were subjected to electrophoresis in buffers at 

 low pH. Prom their studies, they deduced that the electrophoretic difference be- 

 tween A and S hemoglobins could be ascribed to a difference in the number of ionized 

 carboxyl groups each possesses. Ingram 633 reasoned that, if the difference 

 involved even a single charged group, such a specific chemical difference might be 



