Nov. 15, 1924 
Reaction Between Formaldehyde and Proteins 
481 
hyde. It is, of course, well known that 
the addition of formaldehyde to amino 
acids results in a reaction which may 
be represented by the equation: 
nh 2 -r-cooh+ch 2 o^± 
CII 2 N-R-COOH+H 20 
Thus, the basic group of the amino 
acids is attacked by formaldehyde and 
so loses its pronounced basic character, 
while the acid character of the amino 
acid is correspondingly increased. It 
seems plausible to attribute the pri¬ 
mary increase in acidity, which results 
from the addition of formaldehyde to 
serum, to reactions of that type; that 
is, in the first stage of the reaction in 
formolized serum, the formaldehyde 
may attack certain basic groups of the 
protein molecule as a result of which 
the protein molecule itself becomes acid 
in character. Such a reaction may be 
represented by the equation: 
NH 2 -R-C0H=N-R-C00H+CH 2 0£=> 
CH 2 N-R-C0II = N-R-C00H+H 2 0 
Following the initial increase in 
titrable acidity, a gradual decline was 
found to occur. This may be considered 
as the second stage of the reaction. It 
is well known that NH 2 — groups are 
not responsible for all the acid-com¬ 
bining capacity of the protein molecule, 
for all the NH 2 — groups of the mole¬ 
cule may be removed by the action of 
nitrous acid and the molecule will still 
combine with acid. Because of this 
and similar facts, the reserve acid-com¬ 
bining capacity of the protein molecule 
has been ascribed by Robertson (13, p. 
156 ) to the presence of enol, ■— C(OH) 
= N —, or similar linkages. To the 
nitrogen of this linkage is attributed 
the possession of two latent valencies, 
one positive and one negative. When 
the N is trivalent these two valencies 
neutralize each other; when pentava- 
lent, they are capable of neutralizing 
a negative and positive radical, re- 
spectivelv. 
H 
1 
— C(OH) = N — 
1 
Cl 
It is possible that the decrease in 
titrable acidity observed in formolized 
serums is due to reactions between such 
internal groups, on the one hand, and 
acid protein molecules, formed in the 
first stage of the reaction, on the other. 
The following formula represents such 
a possible combination: 
That a neutralization of the original 
and liberated — COOH groups of for¬ 
molized serums occurs seems assured. 
That the reactions described above 
offer a possible mechanism seems plau¬ 
sible. If this neutralization could be 
brought about by a combination of the 
acid groups with unattacked basic 
groups of the protein, then this neutral¬ 
ization could occur not only between 
adjacent basic and acid groups of the 
same molecule but between such groups 
of adjacent molecules. This would 
account for the finding that the reac¬ 
tion apparently belongs to the second 
order. We should thus have a polym¬ 
erization which would be expected to 
continue until all of the protein mole¬ 
cules which had been attacked by the 
formaldehyde had united with other 
affected or unaffected molecules. Thus 
the proteins would acquire the proper¬ 
ties of matter in mass and a network 
of the protein would extend throughout 
the serum, the visible result of which 
would be gratification. That gels 
possess a network is accepted, accord¬ 
ing to Lloyd (5), by many as a fact. 
Reiner and Marton (11) have recently 
attributed the formol-gelatification of 
serums to polymerization, though with¬ 
out advancing confirmatory evidence. 
This explanation of the mechanism 
of the changes which take place in the 
proteins of formolized serum is offered 
simply as a suggestion and with the full 
realization that the results presented 
herein are not sufficient to warrant at 
the present time a final conclusion. 
LITERATURE CITED 
(1) Banzhaf, E. J. 
1908. THE FURTHER SEPARATION OF ANTITOXIN 
FROM ITS ASSOCIATED PROTEINS IN HORSE SE¬ 
RUM. Proc. Soc. Exp. Biol, and Med. 6:8-9. 
(2) Bigelow, S. L. 
1914. THEORETICAL AND PHYSICAL CHEMISTRY. 
544p.,illus. New York. 
(3) Chick, II., and Martin, C. J. 
1910. ON THE “HEAT COAGULATION’' OF PROTEINS. 
Jour. Physiol. 40: 404-430, illus. 
(4) Henley, R. R. 
1923. CHANGES IN THE PROTEINS AND THE GELATI- 
FICATION. OF FORMALIZED BLOOD SERUM. Jour. 
Biol. Chem. 57: 139-151. 
(5) Lloyd, D. J. 
1922. NOTES ON SOME PROPERTIES OF DIALYSED 
gelatin. Biochem. Jour. 16: 530-540, illus. 
(6) Mann, G. 
1906. CHEMISTRY OF THE PROTEIDS. 606 p., illUS. 
London. 
(7) Mellor, J. W. 
1904. CHEMICAL STATICS AND DYNAMICS. 528 p., 
illus. London, New York, and Bombay. 
(8) Moll, L. 
1904. UEBER KtlNSTLICHE UNWANDLUNG VON 
albumin in globulin. Beitr. Chem. Physiol 
u. Path. 4: 563-577. 
H 
1 
CH 8 -N-R-C(OH) = N-R-COOH 
1 
OOC —R— N=(nO)C— R—N—CHj 
