648
EDWIN L. SEXTON AND MAX S. D U "
SOLUBILITY OF CERTAIiY AMINO ACIDS I N AQVEOUS SOLUTIONS O F AMINO ACIDS AND PEPTIDES'.' EDWIN L. SEXTON8 AND MAX S. DUNK Department of Chemistry, University of California, Los Angeles, California Received October 8.9, 1946
It has been pointed out by Cohn (4)that small molecules of known structure should be investigated as a necessary first step toward an understanding of the interactions of ions and dipolar ions in biological systems. Simple systems of this type which have been studied by Cohn and coworkers (5, 6) include: asparagine with alanine, glycine, a-amino-n-butyric acid, diglycine, and lysylglutamic acid; cystine with glycine, alanine, a-amino-n-butyric acid, diglycine, and valine. The solubility relations of the analogous systems norvaline with glycine and glutamic acid with glycine, diglycine, and triglycine have been investigated in the present work. PREPARATION O F AMINO ACIDS AND PEPTIDES
Glycine: Commercial material was recrystallized three times from water and alcohol. Analysis: less than 0.004 per cent of chloride, P~OS, heavy metals, iron, or ammonia. N (Kjeldahl) : milliequivalents found, 2.266; milliequivalents calculated, 2.266. I( +)-Glutamic acid: Commercial monosodium glutamate was converted to I( +)-glutamic acid and the latter was recrystallized twice from water and twice from water and alcohol. Analysis (formol titration with the glass electrode): milliequivalents found, 2.378; milliequivalents calculated, 2.379. The specific rotation in 6 N hydrochloric acid given below agreed closely with the values obtained with other wmples of purified I(+)-glutamic acid prepared in the authors' laboratory.
dl-Norvaline: The synthetic material was recrystallized three times from water and alcohol. Analysis: less than 0.004 per cent chloride, P20,, heavy metals, iron, or ammonia. N (Kjeldahl): milliequivalents found, 6.110; milliequivalents calculated, 6.119. Diglycine: This material, prepared by the acid hydrolysis of diketopiperazine, was recrystallized twice from water and methanol. Analysis: less than 0.004 per cent of chloride, P206,heavy metals, iron, or ammonia. Formol titration 'Paper No. 37. For'Paper S o . 36 see Dunn e l al. (8). This work was aided by grants from the Gelatin Products Corporation, Merck and Company, Inc., and the University of California. 1From a thesis hubmitted by Edwin L . Sexton t o the Faculty of the Graduate School in partial fulfilrnent of the requirements for the degree of Master of Arts, June, 1942. 8Present address: Best Foods, Inc., Buffalo, New Pork.
649
SOLUBILITY RELATIONS IN AMIiiO ACID SYSTEMS
with the glass electrode: milliequivalents found, 2.165; milliequivalents calculated, 2.168. Tn'glycine: This material was prepared by the reaction of chloroacetyl chloride, diglycine, and sodium hydroxide. The intermediate product, chloroacetylglycylglycine, was recrystallized from water. Analysis (titration with the glass electrode) : milliequivalents found, 1.958; milliequivalents calculated, 2.057. A mixture of the purified intermediate and concentrated aqueous ammonia was allowed to stand 4 days a t room temperature, and the crystalline product was recrystallized twice from water and methanol and three times from water. Analysis: less than 0.004 per cent chloride, PzOS, heavy metals, iron, or ammonia. Formol titration with the glass electrode: milliequivalents found, 2.109; milliequivalents calculated, 2.112. EXPERIMENTAL PROCEDURE
An appropriate volume of the solution of the solvent amino acid in carbon dioxide-free distilled water, an excess of the solute amino acid, and ten pieces of 3-mm.glass rod were transferred to an oil sample bottle of 120-ml. capacity. Adequate precautions were taken to prevent leakage, and the bottle was placed in the rotator of a thermostated bath maintained a t 25.OOOC. & 0.01'.
2.51 Y G L w m
GLUTAYIC AClD
Milliequivalents found. . . . . . . . . . . . . . . . . . Milliequivalents present.. . . . . . . . . . . . . . . . Milliequivalents found (per cent of theoretical).. ..........................
1
2.158
I 0.970 M DicLYcmE 2.173 2.171
2.160 99.9
0.17Y M
lpmLycim
~
~
100.2
1.391 1.395 99.7
In order to determine the minimum time for equilibration, bottles were removed from the thermostat a t 24-hr. intervals, and the concentration of amino acids was measured. Although the values were constant in all cases after 24 hr., the glutamic acid systems were equilibrated for 72 hr. and the norvaline systems for 48 hr. Since the same results were found with the glutamic acid systems after 5 days and with the norvaline systems after 96 hr.,it was concluded that bacterial decomposition during these periods was negligible. It was shown in preliminary experiments that glutamic acid could be determined accurately in solutions of glycine and glycine peptides by electrometric titration with the sensitive glass-electrode apparatus described by Robertson (12). These data are given in table 1. Glycine in the solutions was determined essentially by Bergmann and Niemann's (1) trioxalatochromiate method. Although Block (2) determined glycine in the complex either by Van Slyke amino nitrogen or Kjeldahl analysis, only the latter procedure gave satisfactory results in the present experiments. The
TABLE 2 l ( + ) -Glutaniic acid-glycineuater ~(+)-GLUIAMlC ACID-GLYCINE SOLUIIGN GLYCINE SOLUTION
molorify
0.000 0.100 0.300 0.500 0.800 1,000 1.500 2.000 2.500 3.000
, 1 1
1.0605 1.0754 1.0890
4.20 4.32 4.39
, 1
2.180 2.821 3.475
0.1307 0.1475 0.1617
DIGLYCINE SOLUTION
PH
Density ( 2 5 T . )
I(+)-Glutamic acid
Diglycine
moles per 1000 g. water moles pn I000 g. w a f n
molnrily
0.9982 1.0046 1.0282 1.0539
O.Oo0 0.100 0.500 1 .Oo0
,
3.40 3.82 4.17 4.38
0.00 0.09681 0.5217 1.076
~
0.05912 0.06472 0.09973 0.1375
1.500 TABLE 4 I ( + ) -Glutamic acid-triglycine-water ~(f)-GLCIAUlC ACID-TRICLYCINE SGLOTIGN TPIGLYCINE SOLWTIGN
Density (Zj”C.1
~
PH
i
molori1y
0.000 0.0700 0.1400 0.2100 0.3000
GLYCINE SGLUTION
0.9982 1.0045 1.0077 1.0138 1,0191
I
, 0.000
1
l(+l-Glutamic acid moles per 1000 g. water
0.00 0,06773 0.1336 0.2003 0.2827
0.05912 0.06986 0.08125 0.08987 0.10025
3.40 3.78 3.96 4.02 4.10 TABLE 5 dl -Norvaline-alucine-water
dl-NOKVALlNE-GLYCINE SOLUIION
Density (25°C.)
1
PH
1
Glycine
1
dl-Norvaline
molca per I000 g. water moles per I000 p. wafer
molarily
0.500 1.000 1 ,800 2.300 3.000
Triglycine moles pw 1000 g. 1 6 I C I
1.0101 1.02P2 1.0357 1.0576 1.0701 1.0745
6.96 6.65 6.49 6.35 6.28 6.28 650
0.00 0.5101 1.033 1.878
0.7204 0.7178 0.7081 0.6946
SOLUBILITY RELATIOKS IN -4MIXO ACID STSTEMS
651
total-nitrogen values obtained by Kjeldahl analysis were reproducible to 1.O per cent. The same recovery factor, 81.2 per cent, was found in the analysis of 1 M solutions of glycine containing 0, 4.80, and 14.56 per cent of &norvaline. The concentration of the amino acids in the various solutions mas calculated from the densities of the solutions and the content of glutamic acid, glycine, and total nitrogen. The solubility data obtained are shown in tables 2-5. DISCUSSIOS
Advantage has been taken in the present studies of the equation utilized by Cohn (3) and coworkers in their investigations of dipolar ion interactions:
(P, -
+
F9)/2.303RT = log JV/-VO K,C = K,(DO/D)C
(Fa- p!) is the change in partial molal free energy of the solute amino acid due to electrostatic forces, AV is the mole fraction of the solute in a solution of dielecis the mole fractric constant D and concentration C of another dipolar ion, K , is a salting-out constant tion of the solute in water of dielectric constant Do, which increases with the length of the hydrocarbon side chain, and K , is a constant derived from electrostatic forces. In systems where K , is appreciable, K , - K , may be approximated from the value of the limiting slope of the curve resulting from a plot of N / S o against C. For systems in which K , is small in comparison to K,, the value K , - K , is given by the ordinate where D o / D equals unity for the straight line obtained by plotting log against Do/D. The value for K,/2 is given by the ordinate C where D o / D equals 0.5. The possibility of altered solubilities due to compound formation between dipolar ions may be neglected, since von Preylecki el al. (11) have shown by electrometric titration evidence that compound formation does not occur in the systems under investigation. Change in solubilities resulting from shifts in ionic equilibria of dipolar ions because of changes in pH d-ere not significant in experiments with the dl-norvaline-glycine system, which was investigated only at pH levels in close proximity to the isoelectric points of these amino acids. It was necessary, hon-ever, to correct the concentrations of dipolar ions in the glutamic acid-glycine and glutamic acid-glycine peptide systems for the concentrations of the ionic species present, since the solubility measurements were made over a range of pH values. The corrected concentrations of dipolar ions were calculated from the appropriate mass-action expressions of these equilibria, the activity coefficients given by Hoskins, Randall, and Schmidt (9) and 8mith and Smith (14), and the dissociation constants given by Schmidt and Miyamoto (13) and Xims and Smith (10). It was assumed that the experimental pH values mere a measure of log l / a ~ + . The observed p H values ranged from 5.96 to 6.25 for 0.04625-3.130 ill glycine and from 5.52 to 5.62 for 0.56804.7770 Ai' diglycine solutions. The p H of 0.065210.3000 .If triglycine solutions was 5.46. The values of the dielectric constants of the several systems were calculated ~
652
EDWVIS L. SEXTON AND M A X S. DLWN
from the values for the dielectric increment (6) given by Wyman and McMeekin (15) and Devoto (7). K , , K,, and K , - K , were calculated by Cohn's methods. A summary of these calculations is given in table 6. Since K , - K . is relatively small for the dl-norvaline-glycine system, it was not feasible to calculate the individual K , and K , values. A plot of log N I N , against concentration of TABLE 6 Dipolar ion system constants DIPOLAP ION SYSTEM
dl-r\'orvaline-glycine, . . . . . . . . . . . . . . . , . , -0.010 0.282 I(+)-Glutamic acid-glycine ..... . . . . . 0.802 l(+)-Glutamic acid-diglycine.. . . . . . . . ,
...I .
~
'
I
I
0.224 0.068 0.780 1 0.066
I
22.6 22.6 70.6
~
15 15 26
I 2 C O K . OF SOLVENT DlpOLAR ION MOLES PER LITER FIG.1. Plot of log X!S, against concentration of glycine and glycine peptides
glycine and glycine peptides is given in figurt: 1, and a plot of 1/C log Y/Wo against Do/D is shown in figure 2. It may be noted that the substitution of a carboxyl for a terminal methyl group has not only changed the sign of the limiting slope of the curve but has resulted in a 28.4-fold increase in the absolute value of the slope. These results would indicate that the change in free energy as measured by K , - K , is a function of the second pom-er of the dipole moment. In analogous systems containing
SOLUBILITY RELATIOXS I X AMINO ACID SYSTEVS
653
asparagine or cystine Cohn has found that the first, rather than the second power was involved. It was expected that studies would be made of the influence of different terminal groupings on dipolar ion interactions, but it seemed desirable t o present the present experimental data, since this work cannot be continued for an indeterminate time.
t '%\
I.4
GLYUNE GKYLGLYUNE A DIGKYLGLYUNE 0 0
2',o:
z
3
sac-
\ '\
\
\
"%.:
-\
0.2-
\
FIG. 2. Plot of 1/C log
.\'/TO againat Do/D
SCBIXIRY
Solubility relations hsve been determined for the systems dl-norvaline-glycine, 1( +)-glutamic acid-glycine, I ( )-glutamic acid-diglycine, and I ( +)-glutamic acid-triglycine. An interpretation has been made of dipolar interactions occurring in these systems in terms of the concentration of dipolar ions, dielectric and other constants n-hich were calculated from the experimental data, and an equation relating these factors. It has been shown that substitution of a carboxyl for a terminal methyl group caused a change in sign of the limiting slope and greatly increased the absolute value of the slope of the curve relating log iV/iYO and C. It was found, also, that the change in free energy as measured by K , - K . was a function of the second power of the dipole moment.
+
REFEREK'CES (1) BERGMANS, hI., A N D TIELIAXK, C . : J. Biol. Chem. 122,577 (1938). (2) BLOCK, R.J., ASD BOLLISG, D.: The Deferminafionof the Amino Acids, p. 5 1 . Burgess Publishing Company, Minneapolis (1940).
654
JOHN H. L. U'ATSOX
(3) COHN,E.J.: I n Cohn and Edsall's Proteins, Amino Acids and Peptides as Ions and Dipolar Ions, p . 217. Reinhold Publishing Corporation, New York (1943). (4) COHS, E.J.,h l C l 1 E E K I N , T.L., A N D B L A K C H a R D , h1.H.: Compt. rend. trav. lab. Cadsberg 22, 142 (1Y38). (5) COHN,E.J.,MCMEEKIN, T.L., AND BLAKCHARD, M.H.: J. Gen. Physiol. 21,651 (1938). (6) C O H S , E . J . , MCJIEEKIN, T.L., FERRY, J.D., A N D B L A N C H A R D , h1.H.:J. Phys. Chem. 43, 169 (1939). (7) DEVOTO, G . : Gam. chim. ital. 61, 697 (1931). (6) DUNS,M.S.,CAMIEN,hI.S., SHASKMAK, A N D BLOCK,H . : Archiv. Biochem., in press. (9) HGSKISS, w . h l . ,RANDALL, >I., .4KD S C H M I D T , C.L. J. Biol. Chem.88,215 (1930). (10) SIMS, L.F., ASD SMITH,P . K . : J . Biol. Chem. 101, 401 (1933). S.J. VOX, CICHONCKA, J., HGFER,E., A N D R A F G L O W S . 4 K , E.: Biochem. (11) PRZYLECKI, Z. 299, 230 (1938). (12) ROBERTSON, G . R . : Ind. Eng. Chem., Anal. E d . 3, 5 (1931). C.L.A., A K D hlrYa~roTo,S.:J. Biol. Chem. 99, 335 (1933). (13) SCHMIDT, (14) S M I T H , P . K . , . ~ N DSMITH, E.R.: J. Biol. Chem. 121, 607 (1937). T . L . : J. Am. Chem. SOC.66, 908 (1933). (15) Wuuax, J., AKD RICMEEK~S,
s.,
ELECTROX MICROSCOPE OBSERTATIOSS O F THE MORPHOLOGY OF SEVERAL GASES POLYMERIZED BY CHARGEDPARTICLE BOMBARDMEKT JOHN H. L. WATSON' Plant Research Department, Shawinigan Chemicals Ltd., Shawinigan Falls, Quebec, Canada Received January d , 1947
The polymerization of certain gases and vapors when bombarded by charged atomic particles is a nell-known phenomenon (1, 2). Interest in the microphysical structure of cuprene, as a polymer of acetylene formed in the presence of a cuprous oxide catalyst, led to electron microscope examination of cuprene formed by charged-particle bombardment (3). Extension of these observations for cuprene, and for polymerized hydrogen cyanide and cyanogen gases is given here.' The corona cuprene samples were received as brittle semi-transparent sheets, which JTere ground and mounted as a dry powder for microscope examination. This cuprene was formed in a semi-corona, cold discharge in a cylindrical glass tube with a central aluminum electrode. The polymer \vas taken from the deposit found on the inner wall surrounding the electrode. The alpha-rag cuprene sample used here is similar to that described elsewhere (3). The polymer was deposited upon the sides of a large glass flask 1 The author is indebted for the samples to D r . S.C . Lind, Dean of the Institute of Technology, University of Minnesota, and Dr. George Glockler of the Department of Chemistry and Chemical Engineering, Iowa State University. The "corona" cuprene samples were obtained from Dr. Glockler and the other samples from Dr. Lind.
