62 Essays in Biochemistry 



able that a first attempt to improve the situation should be in the 

 direction of increasing the diameter of the molecule. This obviously 

 could be done by attaching to the free carboxyl groups further amino 

 acids, peptides, sugars, or any biological substance with a replaceable 

 hydrogen. Since the peptide itself had seemed to be pharmacologically 

 acceptable and physiologically promising, side chains of either glutamic 

 acid of glutamyl peptide were the first choice, as it was felt that they 

 would retain all the Donnan effects and contribute nothing new in the 

 way of possible disadvantageous pharmacological effects. 



After some exploration it was found that conversion of the peptide 

 to a polyazide could be accomplished, and that this polyazide conju- 

 gated readily in pyridine solution with side chains in the form of the 

 pyridine-soluble polymethyl ester of the peptide. After saponification 

 the polysodium salt of the conjugate was obtained. (These steps will 

 be described in more detail later.) The preparation of two such con- 

 jugates in small amounts was completed, and these were tested in 

 dogs. Fortunately for this purpose we were able to develop a simple, 

 rapid, and convenient method of microassay. It had been observed 

 that the peptide formed highly insoluble precipitate with some cationic 

 dyes. Dr. Joseph Victor, one of the group * who collaborated in bio- 

 logical experiments with the peptide, had developed this observation 

 into a quantitative method of peptide determination applicable to blood 

 and urine. This has since been elaborated to make it applicable to 

 the assay of peptide content of other tissues. 



Of a number of dyes tested, the results obtained with safranine 

 seemed to be the most reliable. With this material it was found that 

 at pH 5.98 in the presence of excess safranine the peptide was pre- 

 cipitated quantitatively if its molecular weight was 3000 or higher. 

 The presence of excess peptide in the mixtures interestingly enough 

 dissolved the precipitate. This is reminiscent of the behavior of 

 antigen-antibody precipitates with the peptide behaving in a manner 

 similar to a multivalent antigen. In the actual analysis precipitation 

 is complete in 15 or 20 minutes, and the decrease in the concentration 

 of dye remaining in the supernatant is proportional to the amount of 

 peptide present in the solutions being analyzed. Since safranine gives 

 no precipitate with any material in normal plasma or urine which has 

 been diluted 1:1, the analyses can be performed directly on these mate- 

 rials. Where tissue extracts are present, the extraneous safranine- 



* Drs. A. J. Patek, J. Victor, F. E. Kendall, A. Lowell, W. Bloom, and G. C. 

 Hennig at the Goldwater Memorial Hospital, Columbia Research Service, Welfare 

 Island, N. Y. 



