10- 



[A5p,Glu,Thr. SePj.Met. His] (85^) 



(a) 



[Cy3,A5p-NHj,GlijMHj,Met2]ly5(90^; 



[A5pNH2.AlQj,5ePj] 

 ill Typ (90%) 



100 300 350 •*» 



0.2N pH 3.1, 35° 



[Cy5.Glu-NH2,AlQ,VaI,5ep]Ly5(80X) [Cys.Asp NHj,Vül,Tlir;Pro,Lys]Phe ,,-. 



[A5p<fNHj),Glu2y(-NH2), 

 >H AlQ.VüljXeu.SenHis] 



(55%) 



15^ 



200 250 300 350 •«0 450 550 600 



I^GraduQlly increoaing pH i &<Q*J,(0.2N pH 31 —IN pH5.1), 35°- 



f;yaA5p,Ileu.Thn3ep]Arg(85%) fQ\ 



[Cy5,(A5p-NH2)j,Glu-NHj,Gly,Thp]Typ 

 (90%) 



^lü,5er]Typ 

 (90%) 



JL 



[ThiiSeriMet] 



50 150 200 250 300 350 400 450 



I- — 0.2N pH 31, 35° -l-Grad, incr pHï [Nal(0ZNpH3HNpH51),35'"H 



f\3p,AlQ,VQl,5er]l007o 



[Cys,Aspj(-NH2),Glui/(-NH2),Gly,Ala. (d) 

 ^1 Val,lleu2,Ppo,His]Typ (90%) 



[Val2,7ro,His]?he(95%) 



.j:^ 



Effluent ml 200 300 "400 700 600 ' 1100 1200 1300 



1- — 0,2n pH 31, 35°— KGrad incr pHx[Na'],{Q2NpH3i-2N pH5.1),35°H 



Fig. 3. The peptides in 20-hour chymotryptic hydrolysates of peptides O-Tryp 4, O-Tryp 

 9, O-Tryp 2, and O-Tryp 16 obtained from performic acid-oxidized ribonuclease. Chromato- 

 graphy of hydrolysates from approximately 5 micromoles of each peptide was carried out 

 on 1 50 X 0-9 cm. columns of Dowex 50-X2 in the sodium form. The effluent was collected in 

 2 ml. fractions. Aliquots (0-1 ml.) were removed for alkaline hydrolysis prior to analysis 

 by the ninhydrin method. The figures in parenthesis represent the yield of each peptide. 

 The sequence of the amino acids within the parentheses is undetermined. (From Hirs, 

 manuscript in preparation.) 



(a) O-Tryp. 4. Elution of the column was with 0-2n sodium citrate buffer at pH 3-1. 



(b) O-Tryp 9. Elution was with 0-2n sodium citrate buffer at pH 3-1 as far as 150 effluent 

 ml. At this point, the pH and molarity of the influent buffer were gradually increased by 

 the use of a mixing chamber of 610 ml. volume filled with the 0-2n buffer at pH 3-1 into 

 which N sodium citrate-acetate buffer at pH 5-1 was allowed to flow. 



(c) O-Tryp 2. The elution procedure followed that given in (b) except that the gradual 

 change of pH and molarity of the buffer was begun after 220 effluent ml. 



(d) O-Tryp 16. The elution procedure followed that given in (b) except that the gradual 

 change of pH and molarity of the buffer was begun after 400 effluent ml. After 610 effluent 

 ml., the reservoir on the mixing device was charged with 2n sodium citrate-acetate buffer 

 atpH5-l. 



