318 BIOCHEMICAL GENETICS 



more easily identified in a mixture of a smaller number of polypeptides released through 

 enzymatic digestion than in a mixture of a large number of amino acids produced by 

 acid hydrolysis. Enzymatic digestion with trypsin and chymotrypsin, followed by 

 two-dimensional paper chromatography, was used by Sanger and Thompson 1154 to 

 establish the amino-acid sequence of the glycyl chain of insulin. This system was 

 adapted to the study of hemoglobin peptides 633 with the incorporation of electrophoresis 

 into the procedure. The peptides of the trypsin digest were separated by paper 

 electrophoresis in one direction and further characterized by chromatography at right 

 angles to the direction of the electrophoresis. This procedure is called "finger- 

 printing." Detailed methodology, along with an improved method for trypsin diges- 

 tion, is described by Ingram. 632 The improved method reduces the time required 

 for trypsin hydrolysis from 40 to approximately 2 hours. The trypsin-resistant core 

 that remains after digestion of hemoglobin can be hydrolyzed by chymotrypsin. 617 

 If necessary, a drop of caprylic alcohol can be added to the solution without ill effects 

 to reduce foaming created by nitrogen stirring. A thick chromatography paper, 

 usually Whatman no. 3 or 3MM, is used as a supporting medium for electrophoretic 

 and chromatographic separations of the soluble peptides of enzymatic digests. Care 

 should be taken that the chromatography and electrophoresis are always run in the 

 same manner relative to the machine direction of the chromatography paper. Follow- 

 ing the method described by Ingram, 632 electrophoresis is performed first, using 

 pyridine : glacial acetic acid -.water (10:0.4:90) at pH 6.4, and ascending chromato- 

 graphy second, using n-butanol : glacial acetic acid: water (3:1:1). However, equally 

 good results are obtained by reversing the procedures; 688 the peptides are separated 

 by descending chromatography first, using n-butanol : acetic acid: water (4:1:5) and 

 electrophoresis second, using pyridine -.acetic acid: water (1 : 10:289) atj&H3.7 (figure45). 



The various peptides of human hemoglobins have arbitrarily been assigned numbers 

 for ease of reference. 632 An illustration of the chemical differences in no. 4 peptide of 

 A, S, and C hemoglobins has been presented elsewhere. 634 - 636 (It should be noted, 

 however, that the amino-acid sequences are incorrect in the references cited above; 

 the corrected sequences have recently been reported by Hunt and Ingram 618 and Hill 

 and Schwartz. 579 ) 



Comparative studies on peptides of hemoglobins can also be done with instruments 

 used for paper-strip electrophoresis. Resolution of the peptides is not so good as with 

 two-dimensional separation; however, the application of specific biochemical tests 

 on the separated peptides increases the sensitivity of the system. Such a system has 

 been used by Benzer et al. 79 to establish that there are 3 varieties of human hemo- 

 globin D, although the native hemoglobin of the 3 varieties are electrophoretically 

 and chromatographically similar. That chemical alterations occur in peptides 

 no. 23 and no. 26 for hemoglobins D a and D , respectively, illustrates that chemical 

 alterations in the hemoglobin molecule occur in places other than peptide no. 4, 

 which contains the chemical alterations of hemoglobins S and C. It has been found 

 that hemoglobin /also has a chemical alteration in tryptic peptide no. 23. 922 



