ANTI-ENZYME IMMUNITY 197 



With the amino group it forms mono- and dihydroxymethyl deriva- 

 tives, R-NHCH2OH, R-NH(CH20H)2; with the amide group, 

 hydroxymethyl, R-CO.NHCH2OH, and, at elevated temperatures, 

 methylene diamide, CH2(R-CONH)2; with one imino group, 

 hydroxymethyl, R.i-N-CHoOH, and with two imino groups, meth- 

 ylene compound, (R2-N)2CH2; with the peptide linkage, hydroxy- 

 methyl groups, -C0.N(CH20H)-; with the guanidino group, a 

 drastically changed arginine derivative; with the hydroxy Qalcoholic) 

 group, acetals, R-O-CH2OH, and hemi-acetals, (R-COO)2CH2; with 

 the sidfhydryl (SH) group, thio analogs of acetals, R-S-CH2OH, and 

 hemiacetals, (R-S)2CH2. Action of formaldehyde on tryptophane, 

 tyrosine, phenylalanine, and histidine leads to the formation of addi- 

 tional rings. 



Fraenkel-Conrat and Olcott (1948) reported that under conditions 

 of pH and temperature which are used for tanning and for the prepara- 

 tion of toxoids and vaccines, formaldehyde introduces a methylene 

 bridge -CH2 between amines on the one hand and the reactive 

 groups of phenolic and imidazole rings on the other. The linkage is 

 resistant to acid hydrolysis. Condensations joining indoles, amines, and 

 formaldehyde under similar conditions may occur with the -NH 

 group of the indole ring. 



Very careful analytical determinations (Nitschmann and Hadorn, 

 1943) have shown that formaldehyde can partially (ca. 35 per cent or 

 more) be removed from formolized casein by prolonged washing at 

 room temperature. In combination with a protein, formaldehyde, there- 

 fore, exists in a form which is less firmly bound, or readily removable, 

 and in another form which cannot be readily removed. The point 

 was made that it is impossible, at present, to differentiate the chemical 

 groups involved in these two forms of binding. 



Velluz (1938) reported that in tetanus toxin formaldehyde combines 

 with tryptophane forming an irreversible heterocyclic three ring com- 

 pound, transforming the toxin into a new antigen. Pappenheimer 

 (1938) reported that diphtheria toxin treated with low concentrations 

 of formalin in alkaline solution forms an irreversible combination. The 

 number of acetylated free amino groups corresponded to the number 

 of £-amino groups of lysine present (5.3 per cent). In converting the 

 toxin into toxoid about 40 per cent of the total free amino nitrogen was 

 found still free. Eaton (1937) reported that 30 per cent of the amino 



