MECHANISMS OF GENE ACTION 281 



and a further separation is elicited by different rates of migration of the 

 peptides in particular solvents. The resulting two-dimensional separa- 

 tion consists of a distribution of spots, which can be visualized by 

 staining with ninhydrin; an example is shown in Figure 10.4. 



Hemoglobin contains 600 amino acids per molecule, arranged in four 

 polypeptide chains, two identical a-chains and two identical i3-chains. 

 For purposes of amino acid analysis, one need consider onlv the 300 

 amino acids of the basic half-molecule. Comparison of the fingerprints 

 of HbA and HbS indicated identity except for a single peptide, # 4. 

 Total amino acid analysis of this peptide from HbA showed that, of the 

 eight amino acids present, two were glutamic acid and one was valine, 

 while the corresponding peptide from HbS contained one glutamic acid 

 and two valines. The final step in analysis required determination of the 

 precise sequence of the eight amino acids in peptide # 4. The results 

 are shown in Table 10.2, together with the results of similar analyses 

 undertaken subsequently with other abnormal hemoglobins. The second 

 abnormal hemoglobin studied was HbC, in which a single amino acid, in 

 this case lysine, was substituted for the same glutamic acid in peptide 

 # 4. This result is particularly remarkable because genetic data indicate 



TABLE 10.2 

 Chemical Differences in Hemoglobins 



(From Hunt and Ingram, 1959, Biochemistry of Human Genetics, 

 Ciba Found. Symp., Little, Brown & Co., Boston, p. 114.) 



Hb Chain Peptide Amino Acid Sequence 



Val . His . Leu . Thr . Pro . GlO . Glu . Lys. . . 



Val . His . Leu . Thr . Pro . Val . Glu . Lys . . . 



Val . His . Leu . Thr . Pro . LyS . Glu . Ly^ . . . 



Val . His . Leu . Thr. Pro . Glu . Gly ; Lys . . . 



... Val . Asp . Val . Asp . GlO . Val . Gly . Gly .- 



Glu . Ala . Leu . Gly . Arg . . . 

 E /3 26 .. .Val. Asp. Val. Asp. Glu. Val. Gly. Gly. - 



Lys . Ala . Leu . Gly . Arg . . . 

 D0 26 ? 



L ^ ? ? 



D« a 23 ? 



I a 23 . ? 



Hopkins-2 a ? ? 



Q a ? ? 



Pa? ? 



Peptide #4 is a terminal peptide of the /3-chain, peptide #26 is inside the /3-chain. 

 . . indicates end of peptide. 



