118 (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24)
E.
n.
FRIEDEX, M.
s.
DUKX AND
c.
D. CORYELL
HOPRINS, F. G.: Kature 126,328, 383 (1930). W.W.: Biochem. J. 16, 678 (1922). LEPESCHKIN, LEWIS,P. S.: Biochem. J. 20, 984 (1926). MIRSRY,A. E.: J. Gen. Physiol. 24, 709 (1941). MIRSRY,A. E., AND ANSON, M. L . : J. Gen. Physiol. 18, 307 (1934-35); 19, 427, 439 (1935-36). MIHSKY, A. E., AND PAULING, L.: Proc. Natl. h a d . Sci. U. S. 22, 439 (1936). SEURATK, H . : J. Phys. Chem. 40, 361 (1932). T. B.: Am. Chem. J. 14, 662 (1892). OSBORNE, OSBORNE, T. B., AND HARRIS,I. F.: Am. J. Physiol. 14, 151 (1905). PALMER, A. H.: J. Biol. Chem. 104, 359 (1934). S. M. : U. S. Public Health Reports 47, 241 (1932). ROSENTHAL, SCHMIDT, C. L. A.: The Chemistry of the Amino Acids and Proteins. Charles C. Thomas, Springfield, Illinois (1938). SPIEGEL-ADOLPH, &I.:I n J. Alexander's Colloid Chemistry, Vol. 2, p. 303. The Chemical Catalog Company, New York (1928). Wu, H., AND YANG,E.-F.: Chinese J. Physiol. 6,301 (1931). W u , H., AND YEN, D.: J. Biochem. 4, 345 (1924-25).
QUANTITATIVE ISVESTIGATIOX3 OF AMIXO ACIDS AND PEPTIDES. XIV
EQUILIBRIA BETWEEN A41v11K0 ACIDSAND FORMALDEHYDE: ARGINIKE AND LYSINE' EDWARD H. F R I E D E S , * MAX S. DUNK,
AXD
CHARLES D. CORYELL
Department of Chemistry, l'niversity of California, Lo8 Angeles, California Received January 2 , 1943
This paper presents the result,sof a polarimetric study of the equilibria between formaldehyde and I ( +)-arginine and formaldehyde and I(+)-lysine. Some of the results obtained with other amino acids have already been reported (1, 2, 3, 4). I. ARGIh-INE
A potentiometric study of the equilibria between arginine and formaldehyde has been reported by Levy (5). He found ( a ) that the equilibria were established slowly, erratic results being obtained unless the solutions were allowed to stand, and ( b ) that the data indicated that both arginine cation and arginine zwitter ion react with 2 moles of formaldehyde. There appeared to be no evi1 For the preceding paper in this series, see reference 4. This paper is part of a thesis submitted by Edward H . Frieden t o the Department of Chemistry, University of California, in partial fulfillment of the requirements for the degree of Dortor of Philosophy, October, 1942. This work was aided by grants from the University of California and from Merck and Company. * Present address: Department of Chemistry, University of Tesas, Austin, Texas.
INVESTIGATIONS OF AMINO ACIDS AND PEPTIDES. XIV
119
dence for the existence of the intermediate reaction (1 mole of formaldehyde per mole of arginine), Levy calculated the values 0.71 and 2.5 X IO6 for Ln and L21, respectively. (The symbols used follow the terminology introduced by Levy.) Polarimetric titrations were performed using solutions of I( +)-arginine monohydrochloride and I(+)-arginine free base. The amino acids used were the C.P. grade, obtained from Amino Acid Manufactures.
Experimental The apparatus used has been described previously (1, 2). Upon the addition of formaldehyde to a solution of I ( +)-arginine monohydrochloride, only slight
220
180
140
[MIHG 100
60
20
-20
40
80
120
160
200
240
280
320
360
400
TIME IN MINUTES
FIa. 1. Graph showing the change of rotation of mixtures of l(+?-arginine and formaldehyde with time. The concentration of arginine in all runs was 0.0523 M. The formaldehyde concentrations were a8 follows: 0,O.OOOM; X, 0.0103 M ; 0 , 0.0200M ; A , 0.0304 M ; v , 0.0378M ; o , 0.0506 M.
changes in rotation were observed. Within experimental error, the rotation was linear up to 2 M formaldehyde concentration. There was no indication of compound formation, although a considerable change in rotation was observed when the solution was allowed to stand in the tube for a period of 6 hr. This phenomenon was not investigated further. Upon the addition of formaldehyde to arginine free base, a rapid, apparently reversible reaction occurs until slightly more than 1 mole of formaldehyde has been added. Upon the addition of more formaldehyde, a definite time dependence of rotation is observed. This is consistent with the observations reported by Levy. In order to investigate the slow reactions observed, mixtures of formaldehyde
120
E.
n.
FRIEDEIZ, M.
20
BO
40
s.
DUNN AND
80
c.
IZO
IO0
D. CORYELL
160
140
180
TIME IN MINUTLS
FIG.2. Graph showing the change of rotation of mixtures of l(+)-arginine and formaldehyde with time. The concentration of arginine in all runs was 0.0526 M . The forrnaldehyde concentrations were as follows: 0,0.0623 M ; X, 0.1122M ; A, 0.2500 M ; 0,0.515M .
400
300
200
[MI% 100
0
2o
40
eo
eo
m
IZO
140
so
IAO
200
220
240
TIME IN MINUTES
FIG.3. Graph showing the change of rotation of mixtures of Z(+)-arginine and formaldehyde with time. The concentrations of arginine (2') and formaldehyde (F) were as follows :
0.0264 M, (F) = 0.0514 M
X,
(a*)
(Z*) = 0.1071 M , (F) = 0.206 M
0,
(Z*) = 0.1071 M , (F) = 0.0410 M
0 , (Z*) = A,
U, (Z*) = 0.10'71M , (F) = 0.0820 M
0.0268 M, (F) = 0.0308 M
121
INVESTIGATIONS OF AMINO ACIDS AND PEPTIDES. XIV
and arginine were prepared, placed in 2-dm. polarimeter tubes, and the rotation observed as a function of the time elapsed from the moment of mixing. Several runs were made at concentrations of 0.053 M arginine and 0.01 M to 0.53 M formaldehyde. Rate studies were also made a t both larger and smaller concentrations of arginine. The temperature of all runs was about 24OC. The observed rotations were converted to molecular rotations. The experimental results are shown in figures 1 and 2, in which molecular rotation is plotted against time elapsed from the moment of mixing. Another set of rate studies was made using different initial arginine concentrations. The rate curves are shown in figure 3.
Discussion It is apparent from figures 1, 2, and 3 that the reaction between arginine and formaldehyde occurs in two steps. An instantaneous initial reaction is followed TABLE 1 Initial and asymptotic rotation. the curves of figures 1 and 8 ARGININE
FOPUALDEHYDE
moles per lifer
moles per liter
0.0523 0.0523 0.0523 0.0523 0.0523 0.0523 0.0529 0.0529 0.0529 0.0529
O.Oo0 0.0101 0.om 0.0303 0.0404 0.0506 0.0630 0.1135 0.253 0.529
29.2 -3.0 -25.5 -45.0 -60.0 -65.0 -70 -84 - 102 -80 (?)
29.2 77.0 116 167 224 300 345 592 675 730
by a slower second reaction, the rate of the latter being apparently dependent upon either the concentration of formaldehyde or the concentration of the compound formed in the first step, or both. A valuable clue as to the stoichiometry of the two reactions involved can be obtained by plotting the initial and final rotations of figures 1 and 2 against the formaldehyde concentrations. The initial rotations (the rotations at aero time) are obtained by extrapolating the curves to their origin. The final rotations (the asymptotes of the curves of figures 1 and 2) were evaluated by plotting the rotations of a given set of data against the reciprocals of the elapsed time. The rotation a t 1/T = 0 is the asymptote. Table 1 gives the initial (Mo)and final (M,) rotations for each set of data available. From table 1 , curves relating M Oand M , to Z F can be drawn, as indicated in figures 4 and 5. From figure 4 it may be inferred that the compound which is rapidly formed from formaldehyde and arginine is to some extent dissociated. Figure 5 indicates that the product of the time-dependent reaction contains 2
122
E. H. FRIEDEP;. 31. S. D U S K AiLD C. D. CORYELL
moles of formaldehyde per mole of arginine, and that very little dissociation occurs. This is qualitative confirmation of Levy’s conclusions.
I
0.02
0.04
0.06
I
0.08
f
0.10
FORMALDEHYDE,
I
,
012
0.14
OM
,
0.11) 0.20
I
0.22
I
0.24
MOLES PER LITER
Fro. 4. Initial rotations of I(+)-arginine-formaldehyde mixtures plotted as a function of the formaldehyde concentration.
700
600
500
400
I4HG
300
200
100
FORMALDEHYDE. MOLES P E R LITER
FIG.5 . Asymptotic (final) rotations of mixtures of I(+)-arginine and formaldehyde plotted as a function of the total formaldehyde concentration.
The calculation of the initial and final rotations of the curves of figure 3 result in t,he values listed in table 2. Upon comparing the data of table 2 with those of table 1, it is observed that the initial and final rotations are primarily dependent upon the formaldehyde-
123
INVESTIG.4TIONS OF AMINO ACIDS AND PEPTIDES. XIV
arginine ratio, and are only slightly affected by a change in the actual concentration of either component. This is consistent with the assumption that the initial reaction involves the combination of 1 mole of formaldehyde and 1 mole of arginine to form a complex, since a more marked dependence of rotation upon absolute concentration is expected for any other possible reaction. TABLE 2 Initial and asymptotic rotations of the curves i n figure 3 ARGININE
FORYALDEEYDE
males per liter
molrr pcr likr
0.0268 0.0266 0.1071 0.1071 0.1071
0.0307 0.0512 0.0410 0.0820 0.2045
1
(Md
-72 -89 -27 -66 -82
320 535 112 220 540
If it is assumed that the rapid reaction is: Z*
+F
ZF*
the time-dependent reaction must follow the equation:
ZF*
+ F e ZF:
". . " The expression for the rate of the reaction is therefore: _.
.
.
.
*
d ( Z p ) - d(ZF:) dT dT
,.
- k(zF*)(F)
The dependence of the rate of the reactions of figures 1 , 2 , and 3 upon the free formaldehyde concentration can be ascertained by a comparison of the rate of reaction when (Z") = 0.0529 M and (ZF) = 0.1135 M with the rate when (Z") = 0.0529 iM and (ZF) = 0.253 (figure 2). From figures 4 and 5 it can be seen that the molecular rotations of ZF" and ZF: are -110" and 750°, respectively. Therefore, for any given set of data, the fraction of ZF" converted to ZF: during the time interval TI - 2'2 is: Fraction converted =
(MT2
- MT,)/860
(4)
in which M,, is the observed rotation at time 2'1 and M T zis the observed rotation at time Tz. For the two sets of data referred to, the initial rate of conversion of ZF* to ZF: is 1.545 X lo-' moles per liter per minute when ZF = 0.1135 hf, and 5.65 X IO-' moles per liter per minute when ZF = 0.253. The ratio of these rates is 3.66. If it is assumed that in each case the initial reaction has proceeded nearly to completion, the free formaldehyde concentrations are 0.0606 and 0.2001, which bear the ratio 3.29. Since the time intervals are not accurately known, the agreement is satisfactory, and it can be concluded that the rate of the time-dependent reaction of formaldehyde with arginine is first order with respect to formaldehyde.
124
E.
n.
FRIEDEN, M.
s.
DUKN AND
c.
D. CORYELL
The specific reaction-rate constant for the reaction of ZF* with formaldehyde was calculated using a set of data in which the ZF:Z* ratio is considerably greater than unity. If the ratio of total formaldehyde to total amino acid is designated by R, then the relative concentrations of ZF* and F shortly after mixing will be 1 and R - 1, respectively. Designating the fraction of amino acid in the form of the complex ZF* by x,equation 3 becomes:
- d!&T = k ( r ) ( R - 2 + 2)
(5)
TIME IN MINUTES
FIG. 6. Graphical test of equation 6 applied to arginine-formaldehyde rate data R = 4.79.
Transposing and integrating:
s 1 R-2
+
z(R +
I?+
2) =
-
s
- log (R
dl - l)] = kt
Plotting log (z R - 2)/s against T should give a straight line of slope -l/[k(R - 2)] and intercept - log ( R - 1). Figures 6 and 7 indicate the application of equation 6 for the two sets of data for which PZ = 4.79 and 2.15. The values of k calculated from the two slopes are 1.03 X IO-' and 1.14 X lo-', respectively. The latter is probably less reliable, since R - 2 is smaller in this instance than in the former case. The fact that both sets of data fit the predicted straight line so closely is further proof of the mechanism we have proposed. In his discussion of the reaction of arginine with formaldehyde, Levy noted that his attempts to study the time-dependent reaction proved unsuccessful. I t was thought worthwhile to study the rate potentiometrically to see if the
125
INVESTIOATIOXS OF AMINO ACIDS AND PEPTIDES. XIV
results were in conformity with the conclusions reached above. Accordingly, a fivefold excess of formaldehyde was added to a solution of half-neutralized arginine monohydrochloride, and the pH followed with time. The results are given in table 3.
0.1s
-
I
1
10
20
30
40
50
60
70
TIME IN MINUTES
FIG. 7. Graphical test of equation 6 applied t o arginine-formaldehyde rate data,
R = 2.15. TABLE 3 Rate of change of pH of half-neutralized 0.06p M l(+)-arginiw monohydrochloride and 0.870 M formaldehyde ELAPSEDIIYE
1
PH
0.5 1 .o 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5
1
ELAPSEDIIYE
1
PH
minulor
minulor
7.86 7.86 7.80 7.73 7.70 7.65 7.80 7.57 7.51 7.48 7.44
6.0 7.0 8.0
10.0 15.0 20.3
30.0 36.0 104
7.41 7.32 7.28 7.20 7.01 6.86 6.49
6.30
132
5.32 5.22
330
6.00
Before the addition of formaldehyde, the solution consisted of an equimolar mixture of the hydrochloride (C$ and the zwitter ion (Z*). The ZF:(Z*) ratio, after the addition of formaldehyde, was 9.82. From the polarimetric study it is known that with this amount of formaldehyde present, nearly all of
126
E.
n.
x. s.
FRIEDEN,
DUNN AXD
c.
D. CORYELL
the Zi has been converted to ZF=. The equilibrium is therefore governed by equation 7.
The time-dependent reaction is, as before: ZF*
+ F e ZF:
(2)
The relation of (H') t o (ZF*) can be shown to be given by equation 8:
( C z ) remains essentially constant. As before, (F) is replaced by R (ZF"), and (C')L1 is written as L:. Therefore:
In the following equations, R - 2 is designated by a and (ZF*) by equation 5
-
TC.
2
+
From
and
but from equation 8
Substituting into equation 5b:
--1 (pH
+ log L:) = kt
(10)
Solving for the pH: pH = -akt - log L:
(11)
Hence, if the pH be plotted against t , a straight line of dope -ak and intercept - log L: should be obtained. Figure 1 represents the data of table 3. It may be observed that the prediction of linearity is fulfilled for a considerable period of time, whereafter the curve flattens out. This flattening is to be expected if it i.i recalled that earlier experiments with arginine cation indicated the existence of a slow reaction of the cation with formaldehyde (page 2). It is obvious from equation 8 that if
INVESTIG.4TIONS OF AMINO ACIDS AND PEPTIDES. XIV
127
the cation, as well as the switter ion, reacts, the change in pH will be less than if the latter alone reacted. From the first part of figure 8, IC can be calculated to be 0.85 X lo-’, in fair agreement with the polarimetric data. The contrast between the slow reaction of arginine with formaldehyde and the rapid reaction of other amino acids led us to study the effect of formaldehyde upon the reactivity of arginine toward alkaline alcoholic @-naphtholand bromine (the Sakaguchi reaction). When the test is performed upon a dilute solution of arginine alone, a deep carmine color develops immediately, while formaldehyde alone gives a dark green color which soon changes to a light brown. When the test is immediately applied to a solution containing arginine and a large excess of formaldehyde, the carmine color develops as rapidly as before. If the arginine-formaldehyde mixture is allowed to stand for 20-30 min. before the test
TlMC IN MINUTES
FIG.8. Rate of change of pH of mixture of half-neutralized 0.0549 M arginine monohydrochloride and 0.270 .%fformaldehyde.
is applied, a dark green color appears, which gradually changes to a reddish brown; the latter solution is distinctly different from the color produced in any of the other tests. Since the Sakaguchi reaction is specific for the monosubstituted guanidino group [HzNC(=SH)NHR], it is clear that formaldehyde reacts slowly and irreversibly with the guanidino group of arginine. The structure of the product was not investigated further. 11. LYSINE
From studies of the effect of formaldehyde upon pKz and pKa of lysine, Levy ( 5 ) calculated equilibrium constants for the reaction of each amino group with 1 and 2 moles of formaldehyde. The polarimetric studies reported in this paper are not consistent with Levy’s results, although our experimental results and conclusions are incomplete. Both lysine zwitter ion and the anion can theoretically react with formalde-
128
E. H. FRIEDEN, M. S. DUX"
AND C. D. CORYELL
hyde. Accordingly, two polarimetric titration3 were performed. For the first, an equivalent quantity of base was added to a quantity of the hydrochloride. The pH of thr resultant solution was 9.8. From the PIC values of lysine it was calculated that at this pH 78.G per cent of thr amino acid is present as the aivitter ion, 13.8 per cent exists as the anion, and 10.6 per rent is present as the cation. This distribution, while unfavorable, is nearly the best possible under the circumstances. Two of the forms present (A- and Z") can react with formaldehyde. TABLE 4 Results of the ( p H = 9.8) polarzinetrzc tztratzon of 0.1459 d l l(+)-lyszne zwbtter ton wzth Y.820 M formaldehyde PORY.4WEBYDE
ml.
0.00 0.120 0.205 0.355 0.505 0.655 0.810 0.970 1.120 1.300 1.475 1.650 1.860 2.140 2.430 2.700 3.09 3.64 4.33 5.05 6.00 8.00 10.00 12.00 15.00
LYSINE
_____
moles per lifer
moles per life?
O.Oo0
0.1459 0.1456 0.1453 0.1447 0.1444 0.1438 0.1435 0.1430 0.1427 0.1421 0.1416 0.1410 0.1405 0.1400 0.1391 0.1384 0.1373 0.1360 0.1343 0.1323 0.1302 0.1257 0.1215 0.1176 0.1121
0.0092 0.0156 0.0290 0.0383 0.0494 0.0610 0.0729 0.0840 0.0943 0.1098 0.1220 0.1372 0.1571 0.1775 0.1960 0.2228 0.2600 0.315 0.351 0.410 0.528 0.638 0.741 0.883
1
OBSERVED ROFAIION
_______ 1.315 1.287 1.242 1.176 1.110 1.040 0.958 0.873 0.782 0.816 0.514 0.389 0.271 0.169 0.114 0.109 0.133 0.163 0.231 0.289 0.330 0.358 0.389 0.418 0.430
22.6 22.1 21.4 20.3 19.2 18.1 16.7 15.2 13.7 11.4 9.1 6.9 4.8 3.0 2.1 2.0 2.4 3.0 4.3 5.5 6.3 7.1 8.0 8.9 9.6
The titration was performed using 3.820 A 1 formalin, and the results are shown in table 4. Figure 9 shows the data of table 4 in convenient graphical form. Figure 9 indicates that lysine zwitter ion reacts with formaldehyde to form two compounds. The analogy between this reaction and that of the other amino acids with formaldehyde is not complete, however. The shape of the first part of the curve indicates that the simple equilibrium postulated previously does not hold in this instance. The second part of the curve, when ZF > 0.2 111,appears conventional enough, and was found to be subject to the treatment described for leucine (2). a2 and
INVESTIQATIONS OF AMINO ACIDS AND PEPTIDES. XIV
129
of ZF* and ZF:) were found to be -2.88' and 11.5', respectively. It was assumed that whatever reaction occurred in the region ZF = 0.0-0.2 M had used up one equivalent of formaldehyde. This assumption is justified, even though an appreciable amount of the amino acid is present as forms other than the zwitter ion. While the cation probably does not react with formaldehyde, the anion, present in approximately equal amount, is expected to react with twice as much formaldehyde as does the zwitter ion. The L Y ~(the molecular rotations
I
0.1
0.2
0.S
0.4
Ob
0.5
FORMALDLHVDE, MOLES
PER
0.7
0.8
LITER
FIQ.9. Change of rotation of 0.1459 M Z(+)-lysine zwitterion upon the addition of formaldehyde.
logarithmic treatment of the data resulted in the linear relationship depicted in figure 10. From the intercept a t log (ZF:)/(ZF*) = 0, Liz turns out to be 8.72. From the peculiar shape of the first part of figure 9, it is to be expected that the calculated log (ZF*)/(Z*) and log (F) terms would not fit the straight line anticipated for a simple association, and this is indeed found to be the case. It is possible that the presence of a fairly large amount of the anion complicates the picture by introducing one or more additional reactions. For the second titration, two equivalents of sodium hydroxide were added to Z( +)-lysine monohydrochloride, and the solution was titrated with 3.820 M formaldehyde. The experimental results are given in table 5 and graphed in figure 11.
130
E. H. FRIEDES, M. S. DUKX AKD C . D. CORYELL
Figure 11 indicates that we are dealing with two main equilibria, with some indication of further complex formation. The fact that the firqt sudden change
FIG.10. Logarithmic treatment of the (ZF:)/(ZF*) dehyde reaction.
-
(F) relation for lysine-formal-
TABLE 5 Results of the polarimetric titration oj 0.1918 M lysine anion with 9.880 iM jormaldehyde j
FOBYALDEHYDE
mi.
0,000 0.120 0,290 0.455 0.635 0.933 1.250 1.750 2.250 2.500 2. 750 3.000 3.260 3.750 4.25 5.25 7.00 9.00 12 00 18 00
,
LYSrNE
moles per lito
moles per lik!
0.000 0.0092 0.0221 0.0345 0.0480 0.0703 0.0935 0.1292 0.1646 0.1823 0.1992 0.2165 0.2340 0.2665 0.2995 0.3635 0.4700 0.584 0.743 1.013
0.1312 0.1310 0.1304 0.1300 0.1298 0.1290 0.1280 0.1267 0.1254 0.1250 0.1242 0.1239 0.1230 0.1220 0.1208 0.1186 0.1152 0.1112 0,1060 0.0965
1.270 1.268 1.228 1.198 1.170 1.115 1.020 0.776 0.433 0.170 -0.027 -0.200 -0.300 -0.441 -0.515 -0.516 -0.497 -0.423 -0.407 -0.316
24.2 24.2 23.5 23.1 22.6 21.6 19.9 15 3 8.6 3.4 -0.5 -4.0 -6.1 -9.1 -10.7 -10.9 -10.8 -9.5 -9.6 -8.2
in slope occurs at a total formaldehyde concentration which is about half that required to reach a minimum rotation suggests that the two equilibrium constants, 1513 and L&, are nearly equal. This is t o be expected for consecutive
INVESTIGATIONS OF AMINO ACIDS AND PEPTIDES. XIV
131
additions of formaldehyde to each of the free amino groups of anionic lysine. The total formaldehyde concentration at the minimum is slightly greater than twice that of lysine; hence only 2 moles of formaldehyde are concerned. The data of table 5 are not subject to analysis by the methods used for leucine, since the analytical method described depends upon the assumption that appreciable quantities of AF- and AFT do not coexist in solution. (It is estimated that in order for this method to be useful, the ratio of L1 to L: must be a t least 10 to 1.) It was found possible to obtain a tentatively acceptable value of the constant Ll*by calculating, for the first five data of table 5, values of (A-), (AF-), and F] corresponding to a series of estimates of 012. When oil was taken as 24.5' and
FORMALDEHYDE, MOLES PER LITER
FIQ.11. Change of rotation of 0.1312 M Z(+)-lysine anion upon the addition of formaldehyde.
a2 was assumed t o be 17', the straight line depicted in figure 12 was obtained upon plotting log (AF-)/(A-) against log (F). From this graph, Ll2 turns out to be 35.0. It must be pointed out, however, that a satisfactory mathematical approach to the problem presented by figure 11 has not yet been devised. I t may be pointed out that, according to figure 11, the e-amino group of lysine reacts with formaldehyde before the a-amino group reacts. This conclusion is based upon the fact that the addition of the first formaldehyde molecule changes the rotation considerably less than does the second addition. Presumably, the addition of formaldehyde to the amino group which is closest to the asymmetric center should affect the rotation more than a similar addition much further away. From the results of potentiometric studies of the lysine-formaldehyde eyuili-
132
E. H. FRIEDEN, M. S. DUNN AND @. D. CORYELL
bria, Levy (5) has calculated equilibrium constants for the four possible reactions. While we havc not been able to calculate all four of these constants, some comparisons are possible. Levy gives the values 89 and 240 for L12 and Lzz, respectively; hence, his value for L& would be 2.70. The discrepancy between this figure and the value of 8.72 calculated from figure 10 is considerable. It is difficult to see how the latter could be greatly in error. While we do not claim great accuracy for the value LI3 = 35.0 calculated from figure 12, it is to be noted that this value is approximately that to be expected for the association constant of formaldehyde with the amino group of a
I
-2 I LOG
-1.7
P
FIG,12. Logarithmic treatment of the (AF-)/(A-) hyde reaction.
- (F) relation for lysine-formalde-
six-carbon-atom amino acid. (Compare the values for leucine, valine, ete.) Levy’s figure of 252 is greater than would be expected. SUMMARY
1. The polarimetric study of amino acid-formaldehyde reactions has been extended to the basic amino acids I(+)-arginine and I(+)-lysine. It has been shown that the reaction of arginine and formaldehyde is an instantaneous, probably revemible combination of 1 mole of each of the reactants, followed by a slow irreversible combination of the produrt with another formaldehyde molecule. The latter reaction apparently involves the guanidino group of the amino acid. 2. The experimental data available from the reaction of lysine and formaldehyde could not be subjected to complete mathematical analysis. although
ACTIVATED NITROGENOUS CARBONS
133
they have been interpreted qualitatively. The polarimetric data appear to be inconsistent with the equilibrium constants derived by Levy. REFERENCES (1) FRIEDEN, E. H., D U N N M. , S., AND CORYELL, C. D . : J. Phys. Chem. 48,216 (1942). (2) FRIEDEN, E . H., DUNN,M, S., AND CORYELL, C. I),:J. Phys. Chem. 47.10 (1943). (3) FRIEDEN, E . H., DUNN,M. S., AND CORYELL, C. b.:J. Phys. Chem. 41, 20 (1943). (4) FRIEDEN, E . H., D U N N ,M. S., AND CORYELL, C. D . : J. Phys. Chem. 4 7 , s (1943). (5) LEVY,M.: J. Biol. Chem. 109, 365 (1935).
THE CATALYTIC ACTIVITY OF ACTIVATED NITROGENOUS CARBONS' PAUL F . BENTE AND JAMES H. WALTON Uepartment of Chemistry, University of Wisconsin, iMadison, Wisconsin
Received M a y 80, I$&
It has been shown by Larsen and Walton (10) that activated carbon prepared from ash-free gelatin is much more active in catalytically decomposing hydrogen peroxide than carbon derived from sugars. It was the object of the present investigation to prepare activated nitrogenous carbons from different sources and to study the catalytic activities of these carbons upon (1) the decomposition of hydrogen peroxide, (8) the oxidation of hydroquinone, and (3)the oxidation of potassium urate. PREPARATION OF CARBONS
The method of preparing the carbon was essentially that used by Larsen and Walton and consisted in charring the material at about 450°C., grinding to 100 mesh or more, heating in a vacuum a t 1OOO"C. for 45 min., and then activating the residue by heating in moist oxygen at 875°C. for 12 hr. Table 1 lists the sources of carbon and gives the ash and nitrogen content. The yield of activated c.arbon was 20 to 50 per cent of the char. The ash content was not considered sufficiently high to warrant any purification. Spectroscopic examinations of the ash indicated that traces of many metals were present. Iron was pkesent to an insignificant extent; this is interesting in view of the fact that the claim has been made that for certain reactions the catalytic activity of carbons is due to the presence of iron-carbon and iron-carbon-nitrogen complexes (14, 19). The per cent nitrogen present in the carbons as nitrogen of constitution was estimated by Kjeldahl analysis. Determinations of nitrogen by the Dumas 1 This investigation WIW financed by a grant from the Research Committee of the University of Wisconsin, Dean E. B. Fred, Chairman.
