QUANTITATIVE INVESTIGATIOXS OF AMINO ACIDS AND PEPTIDES. VI1
EQUILIBRIA BETWEEN AMINOACIDSAND FORMALDEHYDE' EDWARD H . FRIEDEN, MAX S. DUNN,
AND
CHARLES D. CORYELL
Department of Chemistry, University of California, Los Angeles, Calzfornia Received JuEy 18, 1941
It is evident from a consideration of previous studies on the complexes formed between amino acids and formaldehyde that the equilibrium constants and the interpretations of reaction mechanisms given by different workers are not in agreement. Because it seemed desirable to devise a new experimental approach to the problem, the polarimetric study of formaldehyde-amino acid relations described in this report was undertaken. The experimental method and its application to proline are described in the present paper. The results of the investigations of other amino acid systems are to be reported at a later time. The importance of systematizing the behavior of amino acids in the formol titration was emphasized in 1933 by Levy (3), who discussed the nature of the complexes and the available equilibrium data. By measuring the changes in pH as formaldehyde was added to solutions containing an amino acid and half of an equivalent of base, he was able to evaluate the equilibrium constants for the reactions. It was concluded that 2 moles of formaldehyde combine reversibly with 1 mole of monoamino acid anion, I n 1936 Wadsworth and Pangborn (7) investigated the changes in concentration of free formaldehyde, bound formaldehyde, and amino nitrogen with time. These authors concluded that the reactions occur in steps, resulting finally in the formation of a non-dissociable compound. The reversible association of amino acids with formaldehyde to form a molecular complex, the initial step postulated by Wadsworth and Pangborn, is probably the reaction involved in Levy's experiments. In view of these investigations it seems probable that the compounds postulated by earlier workers on the basis of analyses of substances isolated from mixtures of amino acids and formaldehyde are not those concerned in the formol titration. This work was aided by grants from the University of California and the Rockefeller Foundation. For the sixth communication in this series see Dunn, Butler, and Frieden: J. Phys. Chem. 46, 1123 (1941). 215
216
E. H. FRIEDEN, M. S . DUNN, AND C. D. CORYELL
The constants of the amino acid-formaldehyde equilibria were determined by Tomiyama (6) and Balson and Lawson ( l ) , who used Levy’s method. Tomiyama found evidence, later challenged by Levy, that only 1 mole of formaldehyde combined per mole of the amino acids glycine and alanine. On the other hand, Balson and Lawson presented data which were consistent with the conclusion that the reactions of 1, 2, and 3 moles of formaldehyde per mole of the amino acid lead to the establishment of three equilibria. The first two equilibrium constants determined by these authors are in close agreement with those calculated by Levy. The polarimetric method used in studying the amino acid-formaldehyde equilibria is relatively simple. It is based on the principle that the rotations of the complexes resulting from the combination of formaldehyde with optically active amino acid anions should differ from each other and from the rotation of the amino acid. By inspection of the curve relating rotation and formaldehyde concentration it should be possible to determine the number of complexes. From a detailed study of the changes in rotation occurring in amino acid solutions varying in formaldehyde concentration, it should be possible to determine the relative concentrations of the complexes and the equilibrium constants of the reactions. Uncertainties introduced in other experiments because of abnormalities of the hydrogen electrode in high concentrations of formaldehyde are excluded and the possibility of cationic amino acid reactions, which would seriously complicate the interpretation of previous investigations, is avoided by the authors’ polarimetric procedure. EXPERIMENTAL
Rotations were measured by means of a Schmidt and Haentasch polarimeter with an accuracy estimated to be f0.003”. The light source was a General Electric sodium-vapor lamp. A special polarimetric tube, constructed as shown in figure 1, was employed to avoid the necessity of preparing a large number of individual solutions. Water from a thermostat was circulated through the outer jacket of the polarimeter tube, A. The experimental solution was circulated through the side arms, D and D’, by the pump E, which was constructed from a 100-ml. beaker to which the outlets I and I’ were sealed. Rapid circulation of the solution was effected by the motor-driven, wide-bladed stirrer, F. Concentrated standard formaldehyde solution was introduced from a Normax semi-microburet, the tip of which is shown at H. The capacity of the system is about 150 ml., although i t w&s shown to operate efficiently on a volume of 75 ml. That the contents of the system were mixed rapidly was established in preliminary experiments. The polarimeter tube was constructed of Pyrex glass, the ends being ground plane parallel to a length of 40.00 f 0.02 cm. The end plates of optically flat plates were cemented to the polarimeter tube. A calibrated thermometer was inserted at C.
EQUILIBRIA BETWEEN AMINO ACIDS AND FORMALDEHYDE
217
Proline (c.P.;[a]:" = -85.00'; C = 0.500 g. per 50.0 ml.ofwater) was purchased from Amino Acid Manufactures. Merck's 39 per cent formalin, diluted to approximately 12 per cent concentration, was used in all experiments. The pH of a solution containing 10 ml. of the concentrated formalin in 25 ml. of freshly boiled distilled water was 6.21. The effect of the titratable acid of this solution (0.00025N ) was considered to be negligible in the present experiments. The formaldehyde solution was stored under nitrogen, was standardized iodimetrically ( 5 ) before and after each run, and was connected directly to the buret. The latter was filled by using nitrogen to pump the liquid. About 0.5 g. of proline was used in each run. To this was added the amount of base required to convert 99 per cent of the amino acid to the anionic form. The solution was diluted to 100.0 ml. and 75.0 ml. was pipetted into the pump. After circulating the solution for several minutes
FIG.1 to bring it to constant temperature, the rotation was measured. After each addition of formaldehyde the solution was circulated until mixing was complete as shown by the reappearance of a sharp image in the eyepiece of :he polarimeter. The solution was circulated until the rotation became constant. The results of the polarimetric titration of I(-)-proline with 3.718 M formaldehyde are given in table 1. In calculating the concentrations of proline and formaldehyde it was assumed that the volumes of the solutions of these substances are additive. (Davis (2) has shown that the concentration of formaldehyde in aqueous solutions is a linear function of the density over an extremely wide range.) The values of the molecular rotation, [MID,given in column 5 were calculated by means of the equation
[MI, =
ads.
X 2.5
M
218
E. K. FRIEDEX, M . S. DUNN, AND C. 1). CORYELL
TABLE 1 Results of the polarimetric titration of 1(-)-proline with 3.718 M formaldehyde FORMALDEHYDE
PROLINE OBBERVED ROTATION
Volume
Conoentration
Concentration
nil.
nolcs per liter
moles per liter
O.Oo0 0 205 0.410
0.680 1.Ooo 1.250 1.500 1.751 2.000 2.550 3.000 4.050 5.000 7.655 10.00
0.00 0.01011 0,02020 0.0334 0.0489 0.0610 0.0729 0.08-16 0.0965 0.1221 0.1432 0.1902 0.2322 0.3450 0 ,4365
0.02420 0.02410 0.02405 0.02395 0.02385 0,02380 0.02375 0.02365 0,02360 0.02338 0.02325 0.02295 0.02265 0.02195 0.02135
-1.082 -1.242 -1.351 -1.427 -1.471 -1.493 -1.503 -1.518 -1.516 -1,521 -1.526 -1.503 --1..500 -1.456 -1.414
FORMALDEHYDE, MOLES PER LITER
FIG.2
-112.0 -129.1 -140.3 -149.0 -154.2 -157.0 -158.5 -160.3 -160.7 -163.0 -164.0 -163.9 -165.6 -166.1 -166.1
EQUILIBRIA BETWEES AMIKO ACIDS AND FORMALDEHYDE
219
in which 31 is the molarity of proline. When the length of the polarimeter tube is 4 dcm., this equation is identical with the equation
[MI, = 0.01 [a],
x
M.W.
where J1.W. is the molecular weight and [a],is the specific rotation. The concentration of formaldehyde in the final solution was reduced to one obtaining earlier in the experiment by the addition of distilled water. The molecular rotations of the two solutions were found to be identical. I t was concluded from this evidence that the equilibrium reactions are reversible. In figure 2 molecular rotations are plotted as a function of formaldehyde concentration. The curve was drawn from a plot of theoretical rotations, calculated by using the constants derived below. The points shown as cirrles indicate molecular rotations calculated from the experimental observations. I t is obvious that proline and formaldehyde react to form a single complex and that, under the conditions of the experiment, the reaction is essentially complete a t 0.3 M formaldehyde concentration. Analysis of the curve reveals that one proline molecule combines with one formaldehyde molecule to form the complex. Using the symbols, A-- (amino acid anion), F (unbound formaldehyde), and AF- (complex), according to Levy’s notation, the equation for the reaction may be written
+
AF g AF(1) and Li, the association constant of the amino acid per mole of formaldehyde, becomes
The effect of change in ionic strength or of the medium on the equilibrium is expected to be slight because of the cancellation of charge effects. The constant, L1. may be evaluated from equation 2 transformed to logL1= log [AF-l - - log [F] [A-I When AF-/A- = 1, log [AF-]/[A-] = 0, and log L1 = - log [F]. Hence, if log [.\F-]/[A-] is plotted as a function of log [F],a straight line of unit slope will result if equation 1 expresses the conditions adequately. The value of -log [F] when log [AF-]/[A-] = 0 gives log L1directly. At any point on the curve, the relative concentrations of AF- and Acan be estimated from the observed rotation, assuming the latter to be the sum of the rotations of the ions and independent of the formaldehyde concentration, the latter being relatively low. Free formaldehyde can be
220
E. H. FRIEDEN, M. 9. DUNN, AND C. D. CORYELL
calculated from the total added formaldehyde and the estimated AF= concentration. If we let a1 = [ M I D of the proline anion (A-) and TABLE 2 Calculated values for the terms given i n ec itions 1 to 6
I C 1 129.1 140.3 149.0 154.2 157.0 158.5 160.3 163.0 164.0 165.6
0.440 1.021 1.948 3.060 4.09 4.90 6.27 10.20 13.00 22.35
0.01011 0.02020 0.0334 0.0489 0Io610 0.0729 0.0846 0.1221 0.1432 0.2322
m(I
IF1
[A-I
0.00271 0.00800 0.0174 0.0307 0.0416 0.0528 0.0637 0.1001 0.1205 0.2088
E l 1.4-1
-0.3565 0.0095 0.2899 0.4860
0.6115 0.6900 0.7972 1.0080 1.1140 1.3490
LOO
[Fl
-2.567 -2.097 -1.759 -1.513 -1.380 -1.278 -1.203 -0.999 -0.919 -0.680
l.4k
2,O
1.8
1.6
1.4
1.2
ID
0.8
- LOG [F] FIG.3 (MIDof the proline-formaldehyde complex anion [AI?] then, a t any point on the curve designated by the coordinates (a)and (ZF),
[A-]
+ [AF-] = 0.0242
(4)
and
[F] = ZF - [AF-]
(5)
Equation 4 is valid because the effect of dilution by formaldehyde is eliminated in calculating molecular rotations. The value of a1 is 112.0 (from table 1) and that of is 168.0(estimated from figure 2). The data, given in table 2 and plotted according to equation 2A, are expressed by the curve shown in figure 3. It may be seen that the points lie satisfactorily on a straight line of unit slope. The intercept at log [AF-]/[A-] = 0 is 2.025,which corresponds to a value of 105 for LI. The
EFFECT OF SUBSTITUENTS ON ACID STREKGTH OF BENZOIC ACID
221
latter value is in good agreement with the value of 112 found by Levy. The agreement with the value of 126 reported by Balson and Lawson is less satisfactory; apparently the value of 65 found by Tomiyama is in error. The reliability of the value of the present authors is estimated to be f 5 per cent. SUMMARY
A method is described amino acid-formaldehyde acids with formaldehyde. tion constant of the only formaldehyde.
for the determination of the constants of the equilibria by polarimetric titration of amino The value, 105 f 5, was found for the associacomplex formed between the proline anion and
REFERENCES LAWSON, A . : Biochem. J . SO, 1257 (1936). (2) DAVIS, W.A.: J. SOC.Chem. Ind. 18,502 (1897). (3) LEVY,M.:J. Biol. Chem. 99,767 (1932-33). (4) LEVY,M.,AND SILBERYAN, D . E . : J. Biol. Chem. 118, 723 (1937). (5) ROMIJN, G.Z.:Z. anal. Chem. 38.19 (1897). (6) TOMIYAMA, T.: J. Biol. Chem. 111, 51 (1935). (7) WADSWORTH,A , , AND PANGBORN, M. C . : J. Biol. Chem. 118.423 (1936). (1) BALSON, E. W . ,
A'
AND
HB 0.005M LiB 0.005 M LiCl 0.045M Quinhydrone
LiCl 1.00 M
H X 0.005iZI LiX 0.005A1 Au LiCl 0.045M Quinhydrone
