20
E. H. FRIEDEN, M. S. DUNN AND C. D. CORYELL
A general solution has been described for the four parameter equations denoting the change in rotation of leucine solutions upon the addition of formaldehyde. REFEREXCES (1) BALSON, E. W., AND LAWSON, A.: Biochem. J. 90, 1257 (1936). (2) Du”, M. S., AND WEINER,J. G.: J. Biol. Chem. 117,381 (1937). (3) FISCHER,E., AND MECHEL,L.: Ber. 49, 1355 (1916). (4) FRIEDEN, E. If., DUNN,M. S., AND CORYELL, C. D.: J. Phys. Chem. 46, 216 (1942). (5) LEVY,M.: J. Biol. Chem 99, 767 (1933).
QUANTITATIVE IKVESTIGATIONS OF AMINO ACIDS AND PEPTIDES. X I EQUILIBRIA BETWEEK AMINO ACIDSAKD FORMALDEHYDE : GLUTAMIC ACID’ EDWARD H. FRIEDENa, MAX S. DUNN,
AND
CHARLES D. CORYELL
Department of Chemistry, University of California, Los Angeles, Calzfornia Received September 11, 19@
The polarimetric study of the equilibria between amino acids and formaldehyde, applied by the authors to the reaction of formaldehyde with I ( -)-proline, d(-)-N-methylleucine, and I(-)-leucine (1, 2), has been extended in this paper to include the equilibria between formaldehyde and I( +)-glutamic acid. EXPERIMENTAL
The apparatus used for the polarimetric titrations has been described previously (1). I( +)-Glutamic acid3was obtained from Amino Acid Manufactures. A 0.1020 M solution of glutamic acid, containing enough sodium hydroxide to convert 99.5 per cent of the amino acid to the dianionic form, was titrated with 12.60 M formaldehyde. The filtered mercury arc (5461 A.) was used. The data obtained are given in table 1. The reaction was shown to be reversible by the addition of distilled water tu the solution, thus restoring the formaldehyde concentration to a value obtained earlier in the experiment. The rotation of the resulting solution was identical with that observed earlier under these same conditions. KO further change in For the preceding communication in this series, see Frieden, Dunn, and Coryell: J. Phys. Chem. 47, 10 (1943). This paper is part of a dissertation submitted by Edward H. Frieden to the Graduate School of the University of California in partial fulfillment of the requirements for the degree of Doctor of Philosophy, February, 1943. The authors were aided in this work by grants from the University of California and from Merck and Company. 2 Present Address: Department of Chemistry, University of Texas, Austin, Texas. 8 C.P., lot x 5 .
INVESTIOATIONS OF AMINO ACIDS AND PEPTIDES.
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rotation was observed when the solution was allowed to remain in the tube for 24 hr. The change of rotation of a solution of glutamic acid upon the addition of formaldehyde is described graphically in figure 1, in which the molecular rotation is plotted as a function of the concentration of formaldehyde. The solid curve represents the predicted relation, as calculated by the method to be described, while the experimental observations are indicated by the circles. The shape of figure 1 indicates that glutamic acid, like leucine, reacts with formaldehyde to Results of the polarimetric titration q
TABLE 1 .IO80 M I(+)-glutamic acid with 18.60 M formaldehvde
POPMAWEHYDE
GLUTAMIC ACID
OBSEBVED ROTATION
moles par1ilcr
s o l e s per liler
degrees
degrees
0.OOO
0.1020 0.1019 0.1018 0.1016 0.1016 0.1014 0.1014 0.1012 0.1010 0.1008 0.1006 0.1004 0.1000 0.0993 0,0990 0.0984 0.0971 0.0962 0.0944 0,0927 0.0887 0.0850
0.752 0.556 0.414 0.296 0.191 0.066 -0.023 -0.116 -0.243 -0.356 -0.429 -0.476 -0.521 -0.514 -0.478 -0.364 -0.248 -0,171 -0.028 0.076 0.242 0.336
18.4 13.7 10.2 7.6 4.7 1.6 -0.6 -2.9 -6.0 -8.9 -10.7 -11.9 -13.0 -12.9 -12.1 -9.3 -6.4 -4.5 -0.8 2.1 6.8 9.9
Volume
Concentration
ml.
0.00 0.060
0.110 0.155 0.200
0.250 0.300 0.350 0.450 0.550 0.650 0.800 1.000 1.250 1.500 2.000 2.50 3.00 4.00 5.00 7.50 10.00
0.0147 0.0270 0.0379 0.0489 0.0610 0,0737 0.0852 0.1093 0.1333 0.1570 0.1932 0.2405 0.299 0.358 0.473 0.584 0.695 0,910 1.117 1.600 2.045
form two complex compounds. The curve is governed by the four-parameter equation :
Equation 1was derived in the previous discussion of the reaction between leucine and formaldehyde (2). CYI,as,and a8are the molecular rotations of the compounds A-, AF-, and AFT, respectively, and L1 and LZare the mass-action constants for the equilibria involving the association of 1 mole of the amino acid with 1 and 2 moles of formaldehyde, in that order. The unknown quantities are a2,as,L1, and h.
22
E.
n.
FRIEDES,
hi.
s.
DUNN AND C. D. CORYELL
The use of the data of table 1 to derive numerical values for the unknowns is completely analogous to the solution presented for the leucine-formaldehyde reaction. Two approximations were found sufficient to place o i p a t -23.4' and a3a t 25.0'. With these values, the data of table 1 (ZFgreater than 0.3 M) can be utilized to calculate the log (AF,)/(SF-) - log (F) relation, These calculations result in figure 2. The straight line relating the two terms has a slope of unity, and confiims the original assumption as to the nature of the reactions concerned. The intercept of the curve on zero ordinate (log (AF;)/(AF-) equals zero) is -Q.OGO. and Li (defined as L ~ / L Iis) therefore 1.15. Knowing a1 (18.4') and aq,an approximate figure for h can be obtained from the first part of figure 1 ( Z F less than (0.2 M ) ) by calculating and plotting log
/6
/2 8 4 4
9
0
-9 -8
-16
: az /z 09 a b a~ 10 A t /4 / B PO FORP?ALD.F'nYDf, MOLES PfR 1ITER
2.2
FIG.1. The molecular rotation of dianionic I(+)-glutamic acid as a function of total formaldehyde concentration. The line is calculated from the four constants derived in the text.
(AF-)/(K) and log (F). The approximate value 80 obtained is used to correct the observed (M) terms for the rotational contribution of AFT, and from the corrected terms, the log (AF-)/(A-) - log (F) relation is recalculated. The graph of log ( A F - ) / ( K ) us. log (F) is reproduced in figure.3. Satisfactory agreement with the predicted slope is observed. The intercept is - 1.580, and L1 is therefore 38.0. From L1 and L:, Lz is calculated to be 43.7. Reference to figure 1, the solid line of which is obtained using the constants given above, shows that the fit of the predicted and experimental data is excellent except near the minimum. The constants given are thought to be within 5 per cent of the true values. Levy (3) has reported the values 22 and 24 for L1 and Lz, respectively. While the apparent discrepancy between these values and those calculated by the
INVESTIGATIONS OF AbfISO ACIDS AKD PEPTIDES.
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23
-
04 -
LOG
M
FIG.2. Logarithmic treatment of the equilibrium involving the addition of the second formaldehyde molecule.
LOG
(fl
FIG. 3. Logarithmic treatment of the equilibrium involving the addition of the first formaldehyde molecule.
present authors is large, it is significant that the ratio of the constants given by Levy is 1.09, Le., a value within the experimental error of the figure 1.15 reported in this paper. Levy’s L, value is not in harmony with figure 3. .
24
ALBRECHT, SCHNAKENBERG, DUNN AND MCCULLOUGH SUMMARY
A polarimetric study of the equilibria between formaldehyde and l(+)-glutamic acid is reported. The values 38.0 and 43.7 were found for the association constants of glutamic acid with 1 and with 2 moles of formaldehyde, respectively. REFERENCES (1) FRIEDEN, E. H., DUNN,M. S.,
CORYELL, C. D.: J. Phys. Chem. 46, 215 (1942). (2) FRIEDEN, E. H., DUNN,M. S., AND CORYELL, C. D.: J. Phys. Chem. 47, 10 (1943). (3) LEVY,M.: J. Biol. Chem. 99, 767 (1933). AND
QUANTITATIVE INVESTIGATIONS OF AMINO ACIDS A S D PEPTIDES. XI1 STRUCTURAL CHARACTERISTICS OF SOMEAMINOACIDS' GUSTAV ALBRECHTI, GEORGE W. SCHNAKENBERGS, MAX S. DUSN, AND JAMES D. RfcCULLOUGH
Department of Chemzstry, Universzty of Californza, Los Angeles, Calzfornza Received September 21, 1948
Knowledge of the structure of amino acids is of the greatest importance in attacking the problem of protein structure. Complete structures have been determined, however, only in the case of glycine (I), &alanine ( l l ) , and the closely related substance, diketopiperaeine (4). Because the difficulties of x-ray crystal analysis increase rapidly with the number of atoms in the molecule, it seems desirable to conduct preliminary investigations of the structural characteristics of other amino acids. A number of amino acids were surveyed in this manner by Bernal (2) in 1931. Five amino acids, not investigated previously, are treated in the present paper. Photomicrographs are shown for three of these amino acids and for four others the structures of which have been investigated by other authors. 1 The authors are indebted to Professors Linus Pauling and 0. L. Sponsler, in whose laboratories part of the x-ray measurements were made. Some of the data in this papei are taken from the thesis of G. Albrecht, which was presented to the Graduate School of the University of California in partial fulfilment of the requirements for the degree of Doctor of Philosophy, June, 1941. This work was aided by grants from the University of California and the Rockefeller Foundation. For the preceding paper in this series see Frieden, Dunn, and Coryell: J. Phys. Chem. 47, 20 (1943). 2 Present address: Department of Chemistry, Pomona College, Claremont, California. 3 Present address: Van Camp Sea Food Company, Terminal Island, California.
