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

 oxidized with performic acid, and the oxidation mixture was chromato- 

 graphed as before. 



The results from Zone C of the top chromatogram are shown at the 

 bottom of Fig. 5. The cysteic acid peptides formed by oxidation move well 

 ahead of the rest of the mixture originally present in Zone C, and which 

 has not been affected by oxidation. The fractions containing each of these 

 two fast-moving peaks were pooled and their amino acid composition was 

 determined quantitatively. The empirical formulae for the pair of cysteic 

 acid peptides is shown on the curve. In the same manner, a pair of cysteic 



s* 



Chromatography of an enzymatic hydrolysate 

 of ritonuclease on Dowex50-X2 

 Column: J8x30cm 



• Ninhydpin color 



'—^ Analyses for -5-5- bonds 



(modified meinod of BroTid una Kossell) 



-0.20 ■ 

 010 



Iffluent ml 80 160 240 320 400 480 560 540 720 800 880 960 1040 1120 

 •NQformatcOZKpHÔO.ÔS'—l-Gradualty increasing pMond [Nû*J(OZN,pH3O-2ONNûaceiate,pH50),35°-|-20N,pH7,5O°- 



0.5 



Effluent ml 



(CySOjH.Asp-NHj 

 Glu-MHj.MetSOîï 



(CySOjH.Asp.Ileu, 

 TlinSer.Arq) 



Kechpomatogropfiy of material in Zone C 

 after oxidation with performic acid 

 Column 09 x 30 cm., Dowex 50-X2 

 Ninhydnn colour 



40 



100 



120 140 150 180 ZOO 220 240 260 



Fig. 5. Determination of the position of the disulfide bonds in ribonuclease. Upper 

 curve: A tryptic and chymotryptic hydrolysate from 200 mg. of ribonuclease was chromato- 

 graphed. Lower curve: Fractions from Zone C of upper curve were pooled, desalted, 

 oxidized, and chromatographed. (From experiments of Spackman; cf. ^^.) 



acid peptides of the following composition was obtained from Zone B: 

 (Asp.NH2,Val,Ala,CyS03H,Lys)and(Asp.NH2,Thr,Asp.NH2,CyS03H,Glu. 

 NH2,Gly,Tyr). From the amino acid composition of these two pairs of pep- 

 tides alone, without the necessity for any sequence work, it could be con- 

 cluded that a disulfide bond links half-cystine residues I and 11, and another 

 links half-cystine residues IV and V. 



There is room for improvement in the results thus far, for the yields of 

 the peptides referred to are low, and sufficient quantities of the peptides 

 corresponding to the other two -S-S- bonds presumably present in the 

 molecule have not yet been isolated, although work in this direction is in 

 progress. 



With the generous permission of Dr Anfinsen, however, Dr Spackman's 

 results can be considered in conjunction with those obtained by Ryle and 

 Anfinsen^' on the same subject. By the use of quite different methods, they 

 have (see p. 229) obtained evidence for linkages between half-cystine resi- 

 dues II and VIII, and III and VII, as well as evidence bearing on the prob- 

 able correctness of the I- VI and IV-V linkages which we have found. Through 

 Dr Anfinsen's courtesy in allowing us to refer to his results, it is possible to 



