64 Essays in Biochemistry 



Antigens were accordingly prepared in which the peptide was linked 

 to human globulin both via the azide method listed above and via a 

 diazo coupling reaction achieved by making a p-nitrobenzoyl derivative 

 of the peptide by reacting it with p-nitrobenzoyl chloride, reduction 

 of this to the p-amino derivative, and conversion of this to the dia- 

 zonium salt. Peptide was readily separated from protein peptide con- 

 jugates by alcohol fractionation. The conjugates were tested for 

 antigenicity in mice, guinea pigs, and rabbits, using both in vitro and 

 in vivo methods for the detection of antibodies. 



The results of the work along this line can be briefly summarized as 

 essentially negative in that extensive courses of immunization with 

 these antigens, as well as with living and dead bacterial vaccines 

 yielded at best questionable evidence of antibodies in rabbits and 

 guinea pigs, and very dubious protection in mice. The rabbit serum 

 thus produced gave a dubiously positive precipitin test with the peptide 

 used, and the guinea pigs did not become sensitive to the peptide as 

 evidenced by lack of any symptoms of an anaphylactic shock on injec- 

 tion of the peptide. The important point in relation to the present 

 subject of discussion is, however, the fact that under no circumstances 

 were the peptide itself, polymers of the peptide with itself, or vaccines 

 of the peptide-producing B. subtilis ever found to be able to stimulate 

 the production of antipeptide antibodies in animals, and the same 

 seems to be true in humans at the time of the present writing, although 

 human trials are not yet complete. 



In the meantime the turbulent pattern of war had created new and 

 more urgent problems, and the subsequent opportunities of peacetime 

 allowed the pursuit of more interesting ones so that shortly after com- 

 pletion of this immunological study our attention was diverted from 

 glutamyl peptide to other fields until 1950. The initiation of work 

 on glutamyl peptide was, however, destined to yield a rich reward 

 indeed. Following the war, interest in the peptide had persisted ;it 

 Camp Detrick, and investigations relating to its mechanism of bio- 

 synthesis had been conducted by Drs. Housewright, Thorne, and 

 Williams. 6 ' 7 ' 8 In an excellent series of studies they had been able to 

 obtain an enzyme preparation from cultures of B. subtilis which cata- 

 lyzed a transamidation reaction in which the y-glutamyl radical of 

 glutamine is transferred to n-glutamic acid and n-glutainyl-n-glutamyl 

 peptides. Tri- and probably higher peptides were formed by the same 

 system. During the course of these fundamental investigations these 

 workers had developed much information on conditions of formation 



