68 Essays in Biochemistry 



fairly stable in cold pyridine. This polyazide in pyridine solution is 

 a backbone ready for conjugation with side chains. 



Conjugation is effected by addition of pyridine solution of side chain 

 to pyridine solution of backbone azide in the presence of triethylamine ; 

 the reaction mixture is maintained at 0° for 12 hours with stirring 

 and at room temperature for another 24 hours and then precipitated 

 by addition of ether. The solid is dissolved in water and saponified 

 with alkali, and the sodium salt of the conjugate is separated from 

 unreacted peptide by fractional precipitation with alcohol. 



Since there is generally a large difference in order of magnitude of 

 molecular weights of the peptide and conjugate, fractionation is not 

 too difficult. The degree of fractionation is easily ascertained by an 

 end-group determination using Sanger's method. The pure conjugate 

 should give no reaction, as it has no terminal amino groups. 



It is obvious that molecular dimensions of the conjugate should be 

 subject to variation by alteration of the size of backbone and of side 

 chains, and of the ratio of side chain to backbone. Methods of varying 

 the size of side chain have been described above. The size of the 

 backbone can be controlled by alcohol fractionation of the sodium salt 

 of peptide to be used as backbone. The number of side chains on a 

 given backbone can be controlled by varying the relative concentra- 

 tions of side chain and polyazide groups in the conjugation mixture. 

 In this connection it was thought desirable to have available a method 

 for quantitative determination of azide concentration in the reaction 

 mixture. A rapid simple method was obtained by using the iron 

 hydroxamic method of Lipmann and Tuttle. 10 In this determination 

 it is important that the aliquot of pyridine solution of azide used 

 should be no larger than 0.1 ml., as the presence of too much pyridine 

 interferes with color formation, and that two drops of gum acacia solu- 

 tion be added before addition of the ferric chloride solution. This 

 latter helps stabilize the solution. 



A considerable number of these conjugates have been prepared, and 

 various aspects of their chemistry and biology have been investigated. 

 These include studies of their effects on formed elements of the blood 

 in connection with their use in the blood preservation, of their effects 

 on hemostasis, of their possible pathological effects on prolonged and 

 repeated injection in animals, etc. The results of these studies have 

 to date been negative in that no biologically unacceptable properties 

 of the peptide or conjugate have been observed. Physicochemical 

 studies have been carried out for purposes of characterization of the 

 various polymers produced. 



