17] ACTIVE FRAGMENT OF BOVINE SERUM ALBUMIN 293 



a strongly reducing medium such as neutral m sodium thioglycollate — the 

 solution turning into a gel on standing at room temperature. Apparently 

 when the internal disulphide bond is broken, the configuration of the mole- 

 cule is disrupted with loss of solubility and biological activity. Further 

 evidence of the importance of configuration was obtained. If a solution of 

 inhibitor is heated to 100° for 2 min. a visible precipitate forms and no 

 activity is left. If the inhibitor is dissolved in 3m urea at room temperature 

 then the activity is lost slowly — it is reduced to about half in 24 hours. It 

 seems clear that the combining site of the fragment is a relatively large 

 section of the peptide chain, and its abihty to combine with antibody is 

 dependant on the integrity of the native configuration. This is lost if the 

 disulphide bond is broken or if more labile bonds are broken by heat or 

 urea. At the same time the inhibitor is stable to ethanol at room temperature 

 and also to pH 2 and pH 10-5 — the latter observation suggesting that electro- 

 static bonds are not important in preserving the native configuration. 



When the immunological properties of this fragment were investigated it 

 became clear that only some, perhaps only one, of the combining sites of 

 the original antigen were present in the inhibitor. For example, with anti- 

 serum from rabbits after a short course of injections with bovine serum 

 albumin, the inhibitor would combine and subsequently prevent the preci- 

 pitation with serum albumin of about one-third of the antibody present. 

 This power to combine with only part of the antibodies could be demon- 

 strated clearly when the behaviour of whole antigen and inhibitor were 

 compared by precipitation in agar gel according to the technique of Ochter- 

 lony. In Fig. 1 it can be seen that a typical Y type of interaction occurs 

 showing that the inhibitor is combining with part of the antibody, the 

 remainder diffusing through until it meets the serum albumin front. This 

 presumably means that antibodies are present in the serum which are directed 

 to antigenic binding sites situated in the four-fifths of the molecule which 

 has been lost. 



It was interesting to observe that in serum, taken from animals which 

 had been given a prolonged series of injections of serum albumin, the in- 

 hibitor could combine with a much larger proportion of the antibodies 

 present and, in some cases, with all antibodies present giving complete 

 inhibition of precipitation. This is taken to mean that, as immunization 

 continues, the antibodies produced are directed against an increasing num- 

 ber of the potential antigenic sites on a protein. In the example quoted 

 where complete inhibition was achieved the molar ratio required for this 

 was 1-8 moles inhibitor to 1 mole serum albumin. This low value makes it 

 improbable that the inhibition is competitive and other evidence such as 

 the inability of excess serum albumin to reverse the inhibition supports this 

 view. This low ratio is also in contrast to the results of Landsteiner using 

 haptenes where a very large molar excess is required for inhibition of pre- 

 cipitation with the conjugated antigen. This difference may arise because 



