TYPES OF UNION IN THE PROTEIN MOLECULE 149 



In the synthetic polypeptides it may be inferred from the methods 

 of synthesis employed that the amino-acids are united with one another 

 through a nitrogen atom, and the fact that these polypeptides yield 

 amino-acids on treatment with the same hydrolyzing agents that 

 produce amino-acids from proteins, and the fact that polypeptides in 

 which this structure has been established by synthesis, occur among 

 the partial digestion-products of protein, combine to establish the 

 correctness of the view that in the proteins, as in the synthetic poly- 

 peptides, the union of amino-acids takes place through the neutraliza- 

 tion of amino-groups by carboxyl-groups. Furthermore the fact that 

 the proteins yield the Biuret-reaction also confirms this view of the 

 construction of the protein molecule. It has been found that only those 

 substances which contain two COHN groups or two CSHN 

 or two C(NH)HN groups, or under certain conditions, two 

 CHNH groups yield the biuret-test. That such groups as 



COHN c COHN 



I 



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

 the polypeptides, is therefore, established by a number of independent 

 lines of evidence, and the existence of a large number of such linkages 

 in the proteins is furthermore confirmed by the extreme paucity of 

 Free Amino-groups in the native protein molecule. 



The content of free amino-groups in native and derived proteins 

 may be estimated in either of two ways. The first depends upon the 

 liberation of nitrogen which occurs when free NH 2 groups react with 

 nitrous acid, in accordance with the equation: 



RNH 2 + HNO 2 = ROH + H 2 O + N 2 



one molecule of nitrogen being released for every free amino-group 

 originally present in the substance. The second method (Sorensen's 

 method of formol-titration) depends upon the fact that formaldehyde 

 reacts with amino-acids to form methylene derivatives in accordance 

 with the reaction: 



HOOCRNH 2 + HCOH = HOOCRN:CH 2 + H 2 O. 



If the solution be neutral to begin with, it will now be acid, owing to 

 the destruction of the basic NH 2 -group leaving the carboxyl-group 

 unopposed. The number of amino-groups destroyed by the formalde- 

 hyde, or rather the number of carboxyl-groups left unopposed, may be 

 estimated by the quantity of alkali required to restore the original 

 neutral reaction of the solution. 



An examination of the various proteins by either of these methods 

 reveals the fact that the content of free amino-groups in the unhydro- 



