316 CHOH HAO LI [18 



identification of cysteine as the C-terminal residue after reductive cleavage 

 of the disulfide bridges* of the hormone protein. ^^ 



In earlier investigations,^^ difficulties were encountered in attempting to 

 cleave the disulfide bonds in the hormone by reductive procedures. Even 

 in the presence of 6m urea at pH 9-4, the disulfide bridges in prolactin are 

 only partially reduced.^'' For the complete reductive cleavage of the di- 

 sulfide bonds, the following procedure has been shown to be effective: 

 100 mg. of sheep or beef prolactin were dissolved in 10 ml. of 10m urea 

 containing 3% mercaptoethanol, and 4 ml. of cone. NH4OH were added 

 to bring the pH of the solution to 9-8. The whole solution was placed in 

 a cellophane bag and dialyzed against 125 ml. of the reducing mixture (i.e., 

 10m urea, 3% mercaptoethanol, pH 9-8) in a glass-stoppered cylinder. After 

 3 days, the contents of the bag were placed in a beaker containing 1 g. of 

 iodoacetamide; 10 minutes later, the whole solution was dialyzed against 

 cold running tap water. After 2 hours of dialysis, 200 mg. of iodoacetamide 

 were added to the bag to insure the complete alkylation of the — SH groups. 

 The reduced hormone (prolactin — SCH2CONH2) solution was thoroughly 

 dialyzed and lyophilized. If prolactin — SH was desired, the reduced solu- 

 tion was not reacted with iodoacetamide, but instead was dialyzed against 

 3% mercaptoethanol for 5 days, lyophilized and stored in vacuo. 



In electrophoretic experiments on starch, prolactin — SCH2CONH2 mig- 

 rated essentially as a single component with a mobility higher than that of 



© 



0.1 M-NOgCOj, 145 mg. 

 48 hrs., Prolactin 



, Native, 2 mg 

 , Reduced, 2 mg. 



10 20 30 



Segment Number (cm.) 



40 

 © 



Fig. 6. Zone electrophoresis on starch of prolactin 

 145 v, 48 hours. 



-S— CH2CONH2; 01 M-NaaCOa, 



the native hormone in O-lM-NagCOs (Fig. 6). In no case was there separa- 

 tion of reduced prolactins into more than one component after the reduc 

 tive cleavage of the disulfide bonds in the hormone protein. It was further 



* It should be recalled that both sheep and beef lactogenic hormones have 3 residues 

 of cystine per mole of hormone protein.^^ .59 



