60 Essays in Biochemistry 



merits in relation to the use of these materials as plasma volume 

 extenders, the subtilis organism produced these glutamyl peptides on 

 a simple medium, Santon's medium, the organic constituents of which 

 are glutamic acid, glycerin, and citric acid. The organism grows as 

 a mat on the surface of shallow layers of this medium and produces 

 up to 1 gm. of peptide per liter. By incorporating into this medium 

 heavy nitrogen, either in the form of ammonia or of amino nitrogen 

 in the glutamic acid, it was possible to obtain pure peptide containing 

 4. 00% isotopic nitrogen excess, and this peptide on hydrolysis yielded 

 pure glutamic acid containing 3.99% isotopic nitrogen excess, indicat- 

 ing that this was the only amino acid present. We also confirmed 

 the fact that the glutamic acid residues in the peptide we obtained 

 were y linked. 3 



At this point in our studies the year of 1941 ground to a close leaving 

 behind it many new and pressing problems, among them the need 

 for a method of preserving blood or developing a substitute for plasma. 



It had become apparent to us that here was available an easily and 

 cheaply produced polypeptide composed of a single non-aromatic 

 amino acid linked in a physiologically acceptable peptide linkage and 

 possessing a free carboxyl group for each peptide linkage. There was 

 a reasonable possibility that this material might be non-antigenic, 

 because existing theories, based on considerable evidence, postulated 

 that only large peptides or proteins containing some aromatic amino 

 acids were antigenic. A particularly attractive and provocative feature 

 was that the presence of a large number of carboxyl groups would 

 assure maximal Donnan effects, so that if the molecule were large 

 enough to be non-diffusible from the blood stream it would have an 

 unusually high oncotic efficiency. 



Under the circumstances, it was evident that the possibility of the 

 use of this material as a plasma volume extender had to be explored. 

 It was very quickly demonstrated that the material as isolated from 

 B. subtilis filtrates by the methods of culture then employed was in 

 many respects an ideal plasma volume extender. In extensive tests 

 in small animals it was proved to be non-toxic and non-pyrogenic. 

 Its osmotic potency in vitro was found to be quite high, and this 

 osmotic potency was found to be additive to that of serum albumin 

 (Table l 4 ). The material was obtainable as a dry white powder 

 which was highly water-soluble as the sodium salt, and solutions of 

 the latter were stable to autoclaving. The material did not deteriorate 

 or develop any unfavorable properties on standing. The ease of prep- 

 aration from culture filtrate was quite remarkable. After removing 



