Nov. 15, 1924 
Reaction Between Formaldehyde and Proteins 
473 
suggested the possibility of determin¬ 
ing the order of the reaction. Accord¬ 
ingly, an experiment was performed for 
the purpose of measuring the extent of 
transformation of the more soluble 
proteins into formed euglobulins after 
they had been subjected to the action 
of formaldehyde for definite periods of 
time. From the resulting figures the 
velocity constant was calculated by the 
use of the monomolecular formula, 
The values for K thus obtained were 
so irregular that it was concluded that 
the reaction was either not of the first 
order or that the simultaneous occur¬ 
rence of side or consecutive reactions 
was obscuring the results. In order to 
avoid complexities of this character, re¬ 
course was had to Ostwald’s ( 9 ) expe¬ 
dient of determining the order of a 
reaction. As employed in this study, 
this involved dilution of the original 
solution and the determination of the 
time required for the euglobulins in the 
diluted and undiluted solutions, respec¬ 
tively, to be increased by a definite 
percentage, or inversely, the time re¬ 
quired for the noneuglobulin proteins 
of each to undergo a given amount of 
transformation into euglobulins. 
As stated by Bigelow (2, p. 361), this 
method depends upon the fact that— 
if a reaction is of the first order, the time required to 
reach a given stage is not altered by altering the 
original concentration. * * * If it is of the sec¬ 
ond order the time required to reach a given stage 
is inversely proportional to the original concentra¬ 
tion. * * * If it is of the third order, the time 
required to reach a given stage is inversely propor¬ 
tional to the square of the original concentration. 
Thus, the time required for a given 
amount of work to be done in concen¬ 
trated solutions on the one hand and 
half concentrated on the other may be 
expressed by the following ratios: For 
first-order reactions as 1:1; for sec¬ 
ond-order reactions as 1:2; and for 
third-order reactions as 1 : 4. 
In formolized serum at least two 
proteins are converted into euglobulins, 
so at least two reactions must progress 
either simultaneously or consecutively. 
Therefore, since only one product, 
euglobulin, is formed and since the 
ratio between the time required for a 
given amount of work to be done in the 
concentrated and half-concentrated 
solutions is determined by comparing 
the rate of formation of this one pro¬ 
duct in the two solutions, the reactions 
involved in the transformation of the 
more soluble serum proteins into euglob¬ 
ulins are to be regarded as either side 
or consecutive reactions. Mellor (7, p . 
75) states that— 
a reaction may be really compounded of two or 
more side reactions of the same order and yet have 
the same formal integrated equation as a normal 
una-bi- * * * molecular reaction. 
Ostwald’s method is designed to over¬ 
come the disturbing influence of side or 
consecutive reactions. 
Provided that the reactions are side 
reactions and all belong to the same 
order, one of the above-named ratios 
should apply. On the other hand, if 
they belong to different orders and 
occur simultaneously, some interme¬ 
diate ratio should apply, while if they 
are of different orders and occur consec¬ 
utively, a change between the earlier 
and later ratios should take place. 
With these ideas in mind, the method of 
Ostwald was applied to the reaction 
which results in the formation of 
euglobulins in formolized serum. 
EXPERIMENT II 
To 190 c. c. of horse-blood serum of 
known protein content, and previously 
heated to 38° C., 10 c. c. of a formal¬ 
dehyde solution, which contained 3.74 
gm. of formaldehyde, were added. 
Simultaneously, to a second 190 c. c. 
of the same serum, 200 c. c. of a 0.9 
per cent NaCl solution and 10 c. c. of 
the same formaldehyde solution (all 
previously heated to 38° C.) were added. 
Both portions were held at 38° C.; 
10 c. c. samples of the first formolized 
serum and 20 c. c. of the second were 
withdrawn at frequent intervals, di¬ 
luted to 200 c. c., primarily to stop 
the reaction, and the euglobulins de¬ 
termined at once. All determinations 
were made in duplicate. From the 
results obtained at each interval the 
amount of the more soluble proteins 
which had not been converted into 
euglobulins was calculated by sub¬ 
tracting the euglobulins found at the 
given time from the proteins originally 
present. The results are shown in 
Figure 1. 
The time required for the more 
soluble proteins of the two serums to 
undergo a given degree of change was 
determined from Figure 1 by interpola¬ 
tion and is shown in Table II. 
6 For a full discussion of this method of determining the order of the reaction,known as the method of 
integration, any treatise of theoretical or physical chemistry may be consulted. In the equation for th© 
first order reaction, K =*a constant, known as the velocity constant; A =the original amount of the sub¬ 
stance present; and X=amount of A transformed in time, t; Zo 0 »=natural logarithm. 
