212 C. H. W. HIRS, WILLIAM H. STEIN & STANFORD MOORE [12 

 tryptic hydrolysis at pH 7 and 25°, the peptides produced in the hydrolysis 

 were separated on a preparative scale on columns of Dowex 50-X2, and 

 their amino acid compositions were determined quantitatively on columns 

 of Dowex 50-X4.' The thirteen principal peptides, formed in yields of be- 

 tween 50 and 100 per cent, accounted for all of the 124 residues in the 

 peptide chain. To determine the order in which these peptides were joined 

 to one another, the products of chymotryptic and peptic hydrolyses were 

 similarly isolated and their amino acid composition determined. ^-^ From 

 the data so obtained, it was possible to deduce the arrangement shown in 

 Fig. 1 provided three assumptions were made. These were, first, that there 

 is no branching of the peptide chain, and that all of the residues are con- 

 nected to one another by peptide bonds between a-amino and a-carboxyl 

 groups ; second, that in peptides formed by the action of trypsin, the basic 

 amino acids, arginine or lysine, if present, occupy the carboxyl-terminal 

 position; and third, that in peptides formed by the action of chymotrypsin, 

 a tyrosine or phenylalanine residue, if present, occupies the carboxyl-terminal 

 position. 



The partial structural formula appears to represent a unique synthesis 

 of the results, in that, within the restrictions imposed by the assumptions 

 made in its derivation, no other arrangement of the peptides has been found 

 possible. Moreover, this formula accommodates all of the data secured thus 

 far. Every peptide that has been characterized appears in the scheme of 

 proteolytic cleavages shown in Fig. 1 . 



This partial structural formula already reveals much about the structure 

 of ribonuclease, but its main use has been to serve as a point of departure 

 for three relatively independent lines of research. These are : one, the deduc- 

 tion of the sequence of the amino acid residues along the chain ; two, the 

 determination of the location of the four disulfide links connecting the 

 eight half-cystine residues ; and, three, the localization of the 'active center 

 of ribonuclease, if indeed, one may hope that such a unique and relatively 

 small catalytic site can be found in this large molecule. The results of all 

 three lines of investigation undoubtedly bear on one another, but the pre- 

 sent discussion will be limited to the first two of them. 



The problem posed by the sequence of the amino acid residues in ribonu- 

 clease can perhaps best be considered in connection with Fig. 2, which 

 shows the sequences as they are known at the present. The location of 90 

 of the 1 24 residues in the chain has so far been established (cf. 9), and these 

 are underlined in the formula. Work is currently in progress to fill in the 

 remaining gaps in the sequence. 



In the sequence work, it was first necessary to prepare in good yield a 

 series of relatively small peptides which collectively represented all of the 

 residues in the chain of oxidized ribonuclease. The partial structural formula 

 was of great value in pointing the way. Because this formula showed the 

 manner in which the forty-odd peptides formed by enzymatic hydrolyses are 



