COMPOUND AMINO ACIDS.* A well known property possessed by amino acids is their ability to ...

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COMPOUND AMINO ACIDS.*
BY SADAICHI MIYAMOTO AND CARL L. A. SCHMIDT.
(From the Division of Biochemistry, University of California Medical School,
Berkeley.)
(Received for publication, March 10, 1930.)
INTRODUCTION.
A well known property possessed by amino acids is their ability
to combine with acids and with bases. The change in acidity
which takes place when such a reaction occurs is usually expressed
graphically as a titration curve. From such curves the acidic and
basic dissociation constants of amino acids can be calculated.
On examination of the constants of the naturally occurring amino
acids it is seen that they fall into three groups: (a) those having
predominantly acidic properties, (b) those having predominantly
basic properties, (c) those whose acidic and basic properties are
about equal.
It follows that when an amino acid with predominantly acidic
properties is added to an amino acid with predominantly basic
properties a change in the acidity of the solution should take place
to a measurable degree and this change should proceed in accord-
ance with the law of mass action. The result of this reaction should
be the formation of a salt which contains the two amino acids in
the molecule (RCOONHSR’). The chemical stability of such a
salt appears to be a function of the dissociation constants of the
two groups involved in the linkage. However, this type of com-
pound differs from the usual linkage (-COHN-) when two amino
acids unite to form a peptide. The stability of the peptide link-
age as pointed out by Levene and Simms (1) is also a function of
the dissociation constants of the two groups involved in the linkage.
With the exception of the recent publication of Han (2), who
* Aided by a grant from the Chemical Foundation, Incorporated and
the Research Board of the University of California.
327
This is an Open Access article under the CC BY license.
328 Compound Amino Acids
pointed out that histidine glutamate is probably formed in the
isolation of glutamic acid, no work dealing with compound amino
acids has apparently been carried out.
In the present investigation we have studied the formation of
compound amino acids by following the changes in acidity which
take place when an acidic amino acid such as glutamic acid is
added to a basic amino acid such as arginine.
x=Arginine + Aspartic acid
A=Histidine + Aspartic acid
O=Lysine + Aspartic acid
(All solutions 0.02 M)
2 3 4 5 6 7 8 9 10 11
PaH
FIG. 1. Titration curves of aspartic acid.
EXPERIMENTAL.
The technique employed in obtaining the titration curves was
similar to that described by Kirk and Schmidt (3). The amino
acids employed were either prepared in this laboratory or were
S. Miyamoto and C. L. A. Schmidt 329
commercial products. They were repeatedly recrystallized to
insure a high degree of purity. Histidine and lysine were obtained
as the dichlorides, arginine as the monochloride, and ornithine as
the mixed chlorides. Glutamic and aspartic acids were the free
acids. The hydrochlorides of the basic amino acids were neutral-
ized by the addition of an equivalent amount of 0.1 N NaOH. TO
x = Arginine + Glutamic acid
n=Histidine + Glutamic acid
o= Lysine + Glutamic acid
(All solutions 0.02M)
100
m so
4 80
-0
z 70
'g 60
2 50
a-
: 40
6 30
0
20
3 4 5 6 7 8 910 11
FIG. 2. Titration curves of glutamic acid.
5 cc. of the solution of basic amino acid in a 10 cc. flask varying
amounts of glutamic or aspartic acid were added, and, vice versa,
varying amounts of basic amino acid were added to a constant
amount of acidic amino acid and the volume brought to 10 cc. by
addition of carbon dioxide-free water, The solutions were 0.02 M
with respect to each of the amino acids. The hydrogen ion activ-
ity of each solution was determined electrometrically.
330 Compound Amino Acids
The titration curves are represented graphically in Figs. 1 to 3.
We have included a titration curve of ammonia with acetic acid
as an illustration of the titration curve of a weak acid with a weak
base and a curve of lysine with acetic acid. The titration curves
of arginine and lysine with glutamic acid are essentially the same,
x =Ammonia + Acetic acid
a-Lysine + Acetic acid
O=Ornithine + Aspartic acid
(All solutions O.O2M)
loo-
QO-
w
80-
-zi
D 70-
w
.
w60-
E 6-z
g-g ;50-
.-o c3 E40-
;Ex
O’E L 30-
-zKO
-2o- 0’
0
io-
A
0 I I I I ITi\
-2 3 4 5 6 7 8 9 10 11
FIG. 3. Titration curves of acetic and aspartic acids.
while a similar relation holds for arginine and lysine with aspartic
acid. The curves which histidine forms with aspartic and glu-
tamic acids respectively are similar but differ from the curves
first mentioned. The titration curve of ornithine with aspartic
acid is essentially the same as the titration curve of acetic acid and
ammonia.
S. Miyamoto and C. L. A. Schmidt 331
DISCUSSION.
In Figs. 1 to 3 the point of equivalence indicating complete salt
formation is shown by the abrupt change in the acidity of the solu-
tion on addition of a small amount of either amino acid in excess
of an equivalent. The histidine curves do not show the distinct
break in the curves. This is due to the fact that histidine is a
relatively weaker base than lysine or arginine.
Since the second dissociation constants of both the basic and
the acidic amino acids are very much smaller than the primary
constants, the assumption can be made that for the purpose of
forming compound amino acids the dicarboxylic amino acid acts
as R-COOH, and the diamino acid acts as R-NHZ.
The hydrogen ion concentration in the salt solution can be
calculated with the aid of an equation which is derived as follows:
It is assumed that both the acid and the base which form the
compound amino acid are weak electrolytes and obey the law of
mass action. The hydrolysis reaction can be written
Aii+H20*HA+ B0H
If 1 mol of salt is contained in V liters and X = the fraction
1-x
hydrolyzed, then at equilibrium v mols of salt will be practi-
1-x
tally completely dissociated; e.g., v = the concentration of
X
the ions i and i. V denotes the concentration of acid which is
assumed to be very slightly dissociated but relatively more so than
the base. Now
(i$ (Oii) = K,
Go (A) = K
(HA) 4
6, (0%
--(Ezj- = Kb
332 Compound Amino Acids
These when combined give
(HA) @OH) _ Ku _ K
(A, (A) Ka Ka H
Now
X X
(HA) = - and (BOH) = -
v V
Hence
= (OH) (1 - X) X2
, and K, = ___
b X (1 - X)2
Therefore
K, = (ii, L or & = K, (KH)), and pH = $ pK. pK,
(K&i’
K, and Kb are the acidic and basic dissociation constants re-
spectively of the acidic and basic amino acid, K, is the dissocia-
tion constant of water, and KIT is the hydrolysis constant of the
compound amino acid.
The ionization constant (K,) of a compound amino acid can be
calculated with the aid of the following equations:
RCOOH R’NHs
(HA) @OH)
Tl Tl
R600 + ii R’NH, + Oii
(A + I? (6 + OH)
?I
RCOO NHaR’
(BA)
(1) c;i, (OH) = K,
6 COG,
(2)
(BOH) = Rb
S. Miyamoto and C. L. A. Schmidt 333
(A) (6
(3) (HA) = Ka
(2 6) = K
(4)
AB ’
L+&=oH+A
(5)
03) Z acid = HA + i + BA
(7) 2 base = BOH + 6 + BA
(6)-O’) Z acid - Z base = (HA) + (a) - (BOH) - (6) (a)
(a)-(5) z: acid - I; base + (0;) - 6) = (HA) - (BOH)
or
(BOH) = Z base - Z acid - (OH) + (2) + HA (b)
from equation (5)
(ii, = i + oii - ii (0)
If equations (b) and (c) are substituted in equation (2)
.(A + OH - ii, (OH)
Kb =
(d)
2: base - z acid - (OH) + (ii, + (HA)
from equation (3)
(ii) = K, y (4
H
If this is substituted in equation (d)
(OH)
Kb =
HA - L: acid + L: base + H - OH
or
HA = (O6)2 - K, - Kb (Z base - Z acid + & - Ofi)
k)
&, - K, ‘4
H
334 Compound Amino Acids
If this is substituted in equation (e)
K, (OH)2 - K, - Kb (2 base - Z acid f A -
(A) = T - OH) (h)
H
K b - K ‘-’
i Oii
From equation (6)
BA = z; acid - (HA + A) 6)
Substituting equations (g) and (h) in equation (i), we get
(O& - K, - Kb (Z base - 2 acid + & - Ofi)
BA = aacid - 6)
Ka-KK,+
i H
If equation (h) is substituted in equation (c)
&OH-&+$ K (OH)2- K, - & (Z base - 2 acid + A - OH)
(k)
H
K b -KOH= +
i H i
Substituting equations (h), (j), and (k) in equation (4) and
setting
(OI$e - K, - Ka (2 base - I: acid + & - OfI) = s
K-K@!
b a+
H
we get
K, =
Z acid - [(l+p]
In Table I numerical values for K,, KE, and pH of the compound
amino acids studied are presented. K, and Kb refer respectively
5 i : k .? p ‘5 3 z rt ,
10-Z 1O-2 10-Z 10-l 10-2 10-Z 10-Z 10-Z lo-* 5 from ondon, ^ _
cids. T T DH KE -7 Experi. ( :a1ou- Med. nenta1 .- 3 x 4.55 x 10-6 6.00 6.37 4.85 1.8 X 5.00 x 10-s 4.50 5.00 x 10-c 5.88 6.34 4 x 6.17 1.12 x 10-c 5.76 1.7 x 1.62 X, 1O-6 6.45 6.65 3 x 1.78 X lo-* 4.81 5.13 1.5 x 1.78 X 1O-5 6.32 6.63 4 x 5.55 x 10-s 6.87 2 x 6.98 1 2.5 X 3.00 x 10-s 7.00 6.96 values do not differ more than 0.0hysical chemistry, New York and L i LI .- <- - ,,, _ . . _.
A p
TABLE I.* nts Relating to Compound Amino - Ka Kb I - 2 x IO-* 1.1 x 10-S 1.01 x 10-s 2 x IO-” 0.99 x lo-‘ 2 x lo-’ 4.46 x lo-6 2 x IO-’ 1.10 x 10-b 5.62 X IO+ 1.01 X 10-S 5.62 X lo-& 0.99 x 10-S 5.62 X lo+ 0.99 x 10-c 1.84 X KF 1.81 X lo+ 1.84 X 1O-6 - instead of ours the calculated pH I ktroduction to the principles of .- dy^-r.
Consta ................. ................. ................. ................. ................. ................. ................. ................. ................. en by Washburn Washb urn, E. W., --
. . . . . . . . . . . . . . . . . . . . . . . . . . . . n givs. ( _^ -
Arginine aspartate.. Histidine “ Lysine “ Ornithine “ Arginine glutamate Histidine “ Lysine “ Lysine acetate Ammonium acetate * With the equatioour calculated value318 (1915) .)
336 Compound Amino Acids
to the dissociation constant of the predominant group of each of
the amino acids in the compound. With the exception of the
basic dissociation constant of arginine which is an unpublished
value, the data have been taken from the tables of Kirk and
Schmidt (4). The data given under pH indicate a satisfactory
agreement between the calculated and experimentally determined
values of acidity in solutions of the compound amino acids. The
calculated ionization constant (K,) for ammonium acetate, with
molalities instead of activity values, varies but little over the
whole range of the curve and the average value checks with that
obtained from conductivity measurements. For the amino acids
data for the activity coefficients are not available. With molali-
ties for the purpose of calculating ionization constants, consider-
able variation is shown over the range of the titration curve.
This seemingly indicates that the activities of the amino acids
may vary considerably with varying concentration. The ioniza-
tion constants given in Table I are the averages of the values which
appeared to us as most probable.
In the present investigation only amino acids having pronounced
acid and basic dissociation constants have been used for the study
of compound amino acids since changes in the acidity of the
solution can be most readily followed. The salt formation can,
howcvcr, be extended to all amino acids having diffcrcnccs in
dissociation constants. The stability of salts formed from amino
acids having dissociation constants which are small is necessarily
not great.
Hardy’s (5) observation that many of the protein constituents
of tissues or tissue fluids do not exist as such but are bound up in
complexes is of importance in connection with this study. Com-
pound proteins which are formed by the interreaction of acidic and
basic proteins have been studied by a number of investigators.
Hunter (6) and Gay and Robertson (7) prepared a number of
compound proteins. Schmidt (8) demonstrated the formation
of compound proteins by following the changes in acidity which
take place when a predominantly acidic protein is added to a
predominantly basic protein.
The formation of compound amino acids and compound proteins
is probably not unlike the union of toxin and antitoxin and other
immune bodies. Thus Arrhenius and Madsen (9) have shown that