13] STRUCTURE & ENZYMATIC ACTIVITY OF RIBONUCLEASE 229 

 methods essentially the same as those employed by Sanger and his col- 

 laborators for insulin. ^^ In our studies, subtilisin digests of native ribonu- 

 clease were subjected to electrophoresis by the method of Michl^^ and the 

 five ninhydrin-positive bands, which also exhibited a positive reaction for 

 disulfide bonds,^^ were eluted. A typical ninhydrin pattern is given in Fig. 2. 

 One of these cystine-containing components proved to be too large and com- 

 plicated for direct study. The other four were oxidized with performic acid and 

 the resulting cysteic acid-containing peptides were separated and analysed for 

 amino acids. The results of these analyses are given in Table 5. A considera- 

 tion of these analyses, taken together with the available data on the amino 



Table 5 

 THE LOCATION OF DISULFIDE BRIDGES IN RIBONUCLEASE 



acid sequence and distribution along the polypeptide chain of ribonuclease, 

 permits the conclusion that the half-cystine residues of the protein are paired 

 as follows: No. I-No. VI, No. II-No. VIII, No. III-No. VII, and No. IV- 

 No. V (numbering the half-cystines from I to VIII, beginning at the A^- 

 terminal end of the protein). Drs Spackman, Stein and Moore^^ have 

 independently identified the first and fourth of these bridges as well and 

 are now in the process of examining the remaining two. Their studies are 

 of particular importance since complete agreement in the data from the two 

 laboratories will help to rule out the ever-present possibility that there might 

 have occurred some disulfide exchange or other rearrangement during the 

 course of experimental study. The assignment of the disulfide bridges to- 

 gether with the sequential information we now have enables us to construct 

 the schematic and provisional diagram shown in Fig. 3. In this figure the 

 heavily-encircled residues are arranged in an order which is known (or 

 nearly so — there exists an unresolved disagreement in the sequence adjacent 

 to half-cystine No. VI. This portion of the sequence should probably read, 



