12] STRUCTURE OF RIBONUCLEASE 217 



Structure of the peptide, the presence of a large R group, such as leucine, 

 at the amino-end being inhibitory, whereas amino-terminal glycine reacts 

 very rapidly. In the cyclization of the phenylthiocarbamyl peptides, Edman's 

 original method has been used, the reaction being carried out in glacial 

 acetic acid saturated with dry hydrogen chloride at 100°. Attempts to employ 

 aqueous acid in the cyclization step have met with varied success. With 

 peptides containing several residues of serine or threonine not immediately 

 adjacent to the amino-terminal group, the use of dilute aqueous acid led to 

 partial hydrolysis of the peptide. Although the cyclization reaction in glacial 

 acetic acid does not always proceed as rapidly as it does with some peptides 

 in aqueous solution, significant partial hydrolysis in the non-aqueous system 

 has not yet been observed. The rate of the cyclization is dependent upon the 

 structure of the peptide, the presence of an aspartic, glutamic, or cysteic 

 acid residue at the amino-terminal position being markedly inhibitory. 



^ 02 



Cvsieic acid 

 (102) 



Asportic Qcid 

 (110) 



Analysis of original peptide 

 by ion exchange clirwnatogpaphy 



Alanine Valine 



. A°"' K 



(0.91) 



(101) 



(023) 

 l\ A 



Analysis of peptide remaining 

 after first degradation 



(1.11) 



A 



(0 88) 



.A__ 



(097) 



Analysis of peptide remaining 

 after second degradation 



(103) 



(017) 



A,. .. 



A 



(005) 



(100) 



Analysis of peptide remaining 

 after third degradation 



Effluent 



(013) 



(029) 



, y \ 



(OH) 



-0.2NNa* Citrate pH 3.24, 50°- 



280 320 360 

 — O2NpH4.10, 50°- 



Fig. 4. Edman degradation of Asp-NH2.Val.Ala.CySO3H.Lys (0-Tryp 5). The chromato- 

 grams show the amino acid composition of acid hydrolysates of the peptides present at 

 each stage of the procedure. The amino acid analyses are now performed on columns of 

 sulfonated polystyrene resins with the aid of automatic recording equipment.^^ 



In Fig. 4 is shown an example of how the Edman procedure has been 

 used for the determination of the sequence of the pentapeptide asparaginyl- 

 valylalanylcysteicyllysine (0-Tryp 5). The top curve in Fig. 4 represents a 

 quantitative amino acid analysis of the acidic and neutral amino acids in 

 a hydrolysate of the pentapeptide. A resin, Amberhte IR-120, similar to 

 Dowex 50-X8, was employed. Analyses such as these, in conjunction with 

 Edman degradations, are currently being carried out with automatic recording 



