288 Essays in Biochemistry 



the approach to determining the structure of a protein summarized in 

 this essay. Enzymatic hydrolysis with several different enzymes, fol- 

 lowed by fractionation of the peptides thus produced and amino acid 

 analysis of each before ultimate sequence determinations on a selected 

 few, is the procedure used in essence by Bell and his associates in their 

 studies on ACTH. It is also a simple and logical development of the 

 approach the organic chemist has employed for years in the structural 

 analysis of simpler compounds. The general use of quantitative tech- 

 niques will, it is felt, simplify the task with proteins and make for less 

 uncertainty in the final result. Obviously, a large number of routine 

 quantitative amino acid analyses are required, partly as a substitute 

 for, and always as a prelude to, detailed structural work. To render 

 this approach truly feasible, therefore, these amino acid analyses must 

 be made extremely simple and quick. It seems safe to say that none 

 of the existing procedures completely fills this need. There are two 

 obvious ways- of improving this situation. One is to render paper 

 chromatography truly quantitative and more fully mechanized. The 

 other is to render current column methods faster and more automatic, 

 a subject under continued study in our laboratory. 



The amount of information now available on the structure of pro- 

 teins is so limited that it is too early to expect any ironclad conclusions 

 to emerge. Nevertheless, a few suggestive facts are worth noting. 

 There seems to be some tendency for like types of amino acids to 

 cluster together along the peptide chain. For example, in insulin we 

 find the three sequences, -Glu.Glu-NH 2 -, -Glu-NHo.Leu.Glu.Asp- 

 NH 2 -, and -Asp-NH 2 .Glu-NH 2 -, thus placing acidic amino acids and 

 their amides near one another. In another part of the insulin molecule 

 we find, in the A chain, three out of six half-cystine residues in the 

 protein in the single sequence -Cys.Cys.Ala.Ser.Val.Cys-. There is also 

 a cluster of three aromatic amino acids together in the sequence 

 -Phe.Phe.Tyr-, and finally, the only two strongly basic amino acid 

 residues in the molecule are tucked off at one end of the B chain 

 separated by only seven amino acids. This clustering of like R groups 

 is even more obvious in the ACTH molecule. There is a sequence of 

 fourteen amino acid residues near the N-terminal sequence, of which 

 seven are either lysine or arginine. At one point the sequence 

 -Lys.Lys.Arg.Arg- occurs. At the opposite end of the chain there is 

 a cluster of five acidic amino acid residues out of a sequence of eight. 

 Although the information on ribonuclease is still meager, it is already 

 apparent that the basic amino acids also tend to cluster in this molecule. 

 Of the four arginine residues, two are separated from another basic 



