REACTIONS OF FIXATIVES WITH PROTEINS. 2 59 



These facts suggest strongly that formaldehyde reacts with the 

 -NH2 groups of proteins. Lysine is largely involved. ^'^^' 177,203 



NH NH NH 



I I I! 



HC(CH2)4NH2 HC(CH2)3NH.CNH2 



r 



Lysine (left) and arginine {right), as components of proteins 



The side-group of arginine also reacts, but only above pH 8, 

 a degree of alkalinity unusual in microtechnical fixation. This 

 reaction is not fixative, for it does not stabilize the protein; indeed, 

 it increases susceptibility to swelling by acids (p. 64) and 

 shrinkage by high temperature. ^^^ 



When proteins are deaminized by the substitution of -OH 

 groups for -NHg, their capacity to bind formaldehyde is much 

 reduced. The work on this subject has been done largely with 

 collagen and casein, because the toughening of these substances 

 by formaldehyde is important in industry. The physical changes 

 associated with tanning by formaldehyde do not occur after 

 deamination. 



Formaldehyde could react with lysine by simple addition, with 

 formation of the side-group -(CH2)4NH.CH20H, or alternatively 

 the reaction could be a condensation to form -(CH2)4N=CH2 and 

 water. The former reaction would be a special case of the general 

 equation RH + CH20=RCH20H. The -OH in the additive com- 

 pound is reactive, and a methylene bridge, R — CH2 — R-"^, is thus 

 easily formed. Formaldehyde is commonly thought of as a reducer, 

 but it is to be noted that in this particular reaction it acts on the 

 contrary as an oxidizer. This was pointed out long ago by Kings- 

 bury,^^^ who did so much to help to place fixation on a scientific 

 basis. 



A methylene bridge between lysine side-groups on two previ- 

 ously separate protein chains at once suggests itself; but the 

 stoichiometric relation between lysine and formaldehyde is nearer 

 I : I than 2:1, and it is therefore probable that the linkage is 

 between the lysine of one protein chain and a different group con- 

 taining nitrogen in another protein. This could easily be glutamine. 

 A link between lysine and the nitrogen of the peptide (-NH.CO-) 

 link of the main protein chain is also possible. It will be remem- 

 bered, however, that the peptide link of polypeptides is scarcely 



