TYPES OF UNION 19 



occur in the protein molecules, as they do in the molecules of the 

 polypeptids, is therefore highly probable and is furthermore 

 confirmed by the extreme paucity of free amino groups in the 

 native protein molecule. 



The content of free amino groups in the native protein molecule 

 may be determined either by the yield of nitrogen on treatment 

 with nitrous acid (43) (69) (38) (71) or by the method of titra- 

 tion with formaldehyde, originally proposed by Sorensen (66) 

 (67) (46) (38) which latter method also enables us to indirectly 

 estimate the free carboxyl groups. An examination of the vari- 

 ous proteins by either of these methods reveals the fact that the 

 content of free amino groups in the unhydrolysed protein mole- 

 cule is very small indeed. Thus Van Slyke and Birchard have 

 obtained the following results (71) : 



Percentage of Total Nitrogen Present in Free Amino Groups 



Hgemoglobin 6.0 



Casein 5.5 



Haemocyanin 4.3 



Gelatin 3.1 



Edestin 1.8 



GUadin 1.1 



Zein 0.0 



Heteroalbumose 8.1 



Protoalbumose 9.9 



On the other hand, edestin, after complete hydrolysis by hydro- 

 chloric acid, yields a volume of free nitrogen, on treatment with 

 nitrous acid, corresponding to no less than 79 per cent of its 

 total nitrogen content (69). The investigations of Kossel and 

 Gavrilov and of Van Slyke and Birchard have in fact shown 

 that the free amino nitrogen in the unaltered protein molecule 

 exactly corresponds in quantity with one-half the lysin nitrogen. 

 Hence zein, which contains no lysin (49) yields no free nitrogen 

 on treatment with nitrous acid. The period required for complete 

 interaction of proteins with nitrous acid (30 minutes) is longer 

 than that required by the a-amino groups (3 to 4 minutes), but 

 corresponds to that found for the co-amino group of lysin. From 

 these facts Van Slyke and Birchard infer that one of the two 

 amino groups of lysin, the co group, exists free in the protein 

 molecule, and that this group represents, within at most a frac- 

 tion of a per cent of the total protein nitrogen, the entire amount 



