THE APPARENT DISSOCIATION CONSTANTS OF CERTAIN

AMINO ACIDS AND RELATED SUBSTANCES IN

WATER-ETHANOL MIXTURES*
BY T. IS. JUKESt AND CARL L. A. SCHMIDT
(Prom the Division of Biochemistry, University oJ California Medical
School, Berkeley)

(Received for publication, January 10, 1934)

Although numerous determinations of the apparent dissociation

constants of the amino acids in aqueous solutions have been re-
ported (1, 2), few data relating to the apparent dissociation con-
st,ants of these substances in other solvents are available. The
experiments reported in this paper deal with the latter aspects of
the subject.
We have used the expressions pK’, and pK’, for monoamino-
monocarboxylic acids, and PK’~, pK’,, and pK’, for amino acids
with t.hree buffer groups, in accordance with the nomenclature of
Van Slyke and Kirk (3) (cf. also Briinsted (4)). This nomencla-
ture is independent of the dissociation constant of the solvent.
The dissociation constants of organic acids when dissolved in
water-ethanol mixtures have been studied by Michaelis and Mizu-
tani (5), who used the hydrogen electrode. An examination of
twenty-seven carboxylic acid dissociation constants revealed in
each case a progressive increase in the pK’ values with increasing
concentration of alcohol. A similar study of eleven organic bases
by Mizutani (6) showed a less marked decrease in the paH values
of the half neutralized solutions. Hall (7) comments, “From their
results one may infer, neglecting the junction potential, that the
acidity constant of an uncharged acid is usually about lo3 times
smaller in ethyl alcohol than in water . . .”
* Aided by a grant from The Chemical Foundation, Inc., and the Re-
search Roard of the University of California..
We are indebted to the Cyrus M. Warren Fund of the American Academy
of Arts and Sciences for the loan of the type K potentiometer.
t National Research Council Fellow in Medicine.
359

This is an Open Access article under the CC BY license.

 pK’ of Amino Acids

Other workers who have applied the hydrogen electrode to alco-

holic solutions include Bishop, Kittredge, and Hildebrand (8) and
Treadwell and Schwarzenbach (9), while the results of Duboux
and Tsamados (10) and Halford (11) indicate that pK values in
water-alcohol mixtures measured electrometrically are in close
accordance with values obtained by other methods.
Several methods have been published for the estimation of
amino acids and peptides by titration in alcoholic solution (12-16).
These methods involve the addition of alcohol to the amino acid or
peptide solution and subsequent titration with sodium hydroxide
to the color change of an indicator, usually phenolphthalein. The
authors have generally assumed that the method was dependent
upon a marked decrease in the pK’, values. That this assumption
is not justified is shown by the work of Michaelis and Mizutani
(5) who point out that the pK’ of phenolphthalein is 8.6 in water
and 12.6 in 90 per cent ethanol, and hence that titrations of amino
acids and peptides in alcohol are dependent on a shift in the indi-
cator pK’ and not in the pK’ of the substance titrated.1 The
results of Kolthoff (18) and Hammett (19) point to a similar con-
clusion. Van Slyke and Kirk (3) apparently overlooked the effect
of alcohol on the indicator when they assumed that alcohol af-
fects the pK’2 value of an amino acid sufficiently to bring its buffer
group within the zone practicable for titration with alkali. Elec-
trometric measurements to be reported in this paper show that the
effect of ethanol is actually to increase the value of PK’~ slightly in
the case of a typical or-amino acid. In a calorimetric titration,
however, this effect is more than counterbalanced by the con-
siderable increase in the pK’ value of phenolphthalein. The
results of the present work are also in harmony with the generally
accepted idea that the amino acids exist chiefly as zwitter ions
(20) .z Edsall and Blanchard (22) have studied the activity ratio
* McCoy (17) pointed out 29 years ago that addition of alcohol affects the
color of alkaline phenolphthalein.
2 In this connection, it is pertinent to point out that certain published
statements should not be construed too literally (21). The awitter ion con-
cept involves two oppositely charged groups in the amino acid molecule;
i.e., a basic and an acidic group. Any excess numbers of either acidic or
basic groups are not to be regarded as twitter ions. This statement applies
also to proteins. In very few, if any, proteins is the number of basic groups
exactly equivalent to the number of acidic groups; usually there is an ex-
 T. H. Jukes and C. L. A. Schmidt

between the zwitter ion and the undissociated form of certain

amino acids by determining the dissociation constants of the ethyl
esters of the amino acids. They found that the zwitter ion form
overwhelmingly preponderates in water and that in 90 per cent
alcohol the zwitter ions still far outnumber the uncharged mole-
cules.3
The following factors must be considered in the determination
of apparent dissociation constants in ethanol-water mixtures:
(a) The junction potential; the results of Halford (11) indicate
that this is small in value. The high mole fraction, 0.56, of water
in the ethanol-water mixture used in the present investigation
would tend to make the junction potential small. (6) The effect
of the dissociation constant of the solvent on the ionization of the
solute; this has been discussed by Hammett (19): The present
results are interpreted on a comparative basis. The determina-
tions were carried out in so far as possible under identical conditions,
so that this factor does not affect the conclusions (cj. BrBnsted
(24)). It should be noted that the results of Lowenherz (25)
indicate that an ethanol-water mixture of the strength that was
used would have a dissociation constant of about one-tenth that
of water. (c) The influence of the dielectric constant of the sol-
vent upon the ionization of the solute; this has been used by some
authors to predict dissociation constants in non-aqueous solvents.
The effect of the dielectric constant of the solvent upon the ioniza-
tion of an electrolyte is more complex than is indicated by the
Nernst-Thompson rule. The subject has been discussed byBrad-
ley and Lewis (26). These authors, by reasoning from theoretical
standpoints, evolved equations for calculating the dissociation

cess of one or the other. These excess groups do not necessarily come
within the scope of zwitter ions. Since, in the protein molecule, the po-
sitions of the basic and acidic groups cannot be definitely placed at present,
the influence of these respective groups upon each other is still a matter
of conjecture. It is very improbable that if, for example, the protein
or peptide contains an excess number of carboxyl groups over the
number of basic groups, the former will be symmetrically placed with re-
spect to the basic group, and thus statistically come under the influence of
the latter group. It is more probable that the positions of the excess basic
or acidic groups are such that they can be regarded as being wholly free.
3 Czarnetzky and Schmidt (23) have shown that, in the solid state, amino
acids must be regarded as existing chiefly as zwitter ions.
 pK’ of Amino Acids

constants of salicylic and cyanacetic acids in ethanol-water mix-

tures. The calculated values were in excellent agreement with
those obtained from conductivity measurements. The appear-
ance of complicating factors, however, makes such predictions not
always so successful (27). The addition of ethanol to a concen-
tration of 72 per cent lowers the dielectric constant of water, which
at 25” is about 79, to a value in the neighborhood of 40 (28).
Amino acids increase the dielectric constant of water; in the con-
centrations worked with in the present investigation (0.02 to
0.05 M), the increase, however, is very small.
The various amino acids and related compounds used in this
investigation were products of known purity. They were re-
crystallized and in certain instances prepared in accordance with
standard procedures. The glycine ethyl ester was titrated as the
hydrochloride and cadaverine as the dihydrochloride. The amino
acids were isolated in the free state and titrated as such. Ethanol
was purified by distilling from a mixture containing a little sul-
furic acid, after which aldehydes and acids were removed by the
method of Dunlap (29).
For the measurement of paH, aliquots of the aqueous solution
of the particular substance were pipetted into test-tubes and vary-
ing amounts of aqueous standard solutions of hydrochloric acid or
of sodium hydroxide were added. Ethanol and water were then
added so that the final solution contained 3.6 cc. of ethanol and
1.4 cc. of water. This alcohol-water mixture will subsequently be
referred to as “72 per cent ethanol.” This particular mixture was
used, since it was found that it was the highest concentration of
alcohol in which the amino acid mixtures were still soluble. The
concentration of amino acids was 0.02 to 0.05 M. The paH values
were determined at 25” with the aid of a Clark cell and a Leeds
and Northrup type K potentiometer. A steady stream of hydro-
gen was not passed through the solutions as it was found to cause
evaporation of the alcoholic solution; hence the vessel was kept
closed after the air had been displaced by passing a current of
hydrogen through the cell for a period of about 20 seconds.
In the calculation of the pK’ values, the usual blank correction
was applied where necessary, i.e. in the more acid and alkaline
regions. The value for the blank was read from an empirically
constructed curve obtained by addition of acid or of base to the
ethanol-water mixture.
 TABLE I
A pparcnl Dissociation CorLslarLts

Vdues in ~lqueous Biblio- Values in 72 per cent I Siblio-

solution ethanol solution Braphic
Snbstancr graphic
~efermre refer-
NO. enre
PK’I pK’z pK’s pIi’, >I~‘* PIi’ NO.

Mtrnonminomollocar-
boxylic acids
Alanine. 2.36 9.72 (1) 3.55 0.02
Asparagine 2.17 8.86 (30, 31’ 3.09 9.10
Glycine 2.42 9.74 (1) 3.46 9.82
‘I . 2.54 9.81 3.52 9.99 (5)
Isoleucine 2.36 9.68 ,g 3.69 9.81
Valine. 2.32 9.62 (32) 3.60 9.73
cu-Amino-n-valeric 2.36 9.72 (21) 3.60 9.88
acid
+),- “ L‘ 4.02 10.40 (21) 5.35 0.39
Is- “ “ 4.21 10.69 (21) 5.74 0.47
Monoiminomonocar-
boxylic acid
Proline. 2.00 10.60 (33) 3.04 0.55
Monoaminodicar-
boxylic acids
Glutamic acid. 2.19 4.25 9.61 (34) 3.16 5.63 10.75
Aspartic “ 1.88 3.65 9.61 (2) 2.85 5.20 10.51
Dibasic monoacidic am
ino acids
Arginine 2.02 9.04 12.41 (35) 3.34 9.40 14.1
3.073
Ilistidine. 1.82 6.00 9.1’ (36) 3.00 5.85 9.45
T,ysine, 2.18 8.95 10.5; (35) 2.75 8.95 10.531
2.96 8.97 10.53*
3.23 9.00 10.531
3.56 8.95 10.49{
Organic bases
Ethylamine.. 10.82 (6) 0.20 (6)
Cadaverine 9.84 10.98 9.49 0.32
Glycine ethyl es-
ter............. 7.73 (37) 7.19
Carboxy acids
Acetic acid. 4.70 (5) 6.41 (5)
Monochloroacetic
acid........... 2.86 (5) 4.44 (5:
-
Unless otherwise indicated by the reference number the values given in
this table were obtained during the course of the present investigation.
* 60 per cent ethanol solution.
t 48 per cent ethanol solution.
$72 per cent ethanol solution.
$84 per cent ethanol solution.
363
364 pK’ of Amino Acids

The pK’ values obtained are given in Table I. They are com-
pared with the values which have been obtained in aqueous solu-
tions at the same temperature. Certain other data are also in-
cluded for comparison. Certain of the data are graphically
represented in Figs. 1 and 2 in order to bring out more clearly
the relation of the aqueous to the alcoholic titration curves.

Acid or Base Added

FIG. 1. Apparent dissociation curves at 25” of ethylamine (Curves l),
glycine ethyl ester (Curves 2), lysine (Curves 3), arginine (Curves 4),
glycine (Curves 5), and monochloroacetic acid (Curves 6). The solid lines
represent the titration curves in 72 per cent (by volume) of ethyl alcohol
and the dashed lines represent the titration curves in water. All curves
were calculated from the pK’ values given in Table I. The scale on the
horizontal axis is such that the length given by the arrow represents 1
equivalent of acid or of base added. The points on the curves represent
present experimental observations.

DISCUSSION

The pK’ values for the monoaminomonocarboxylic acids given

in Table I show that the effect of the alcohol is to increase the
value of pKll (aqueous solution) about 50 per cent. An increase
in the pK’2 value is also noted. It is, however, much smaller and
more irregular than the increase in the PK’~ values. On the basis
of these observations the statement of Van Slyke and Kirk (3),
“There are in each such amino acid two buffer groups, with pK’
 T. H. Jukes and C. L. A. Schmidt 365

values in the neighborhood of 2 and 9.5 . . . ,” may be amplified

as follows: these values are raised respectively to about 3.5 and
9.8 in 72 per cent ethanol.
With this statement as a guide, it was found possible to make
some interesting deductions relating to the naturally occurring
diacidic and dibasic amino acids. The effect of increasing the
distance in the molecule between the amino group and the carboxyl

13c

PaH
10

Acid or Base Added

lb. 2. Apparent dissociation curves at 25” of cadaverine (Curves l),
glutamic acid (Curves 2), asparagine (Curves 3), and acetic acid (Curves
4). The solid lines represent the titration curves in 72 per cent (by volume)
of ethyl alcohol and the dashed lines represent the titration curves in water.
All curves were calculated from the pK’ values given in Table I. The scale
on the horizontal axis is such that the length given by the arrow represents
1 equivalent of acid or of base added. The points on the curves represent
present experimental observations.

group in a monoaminomonocarboxylic acid will be considered

first. It has been shown by Schmidt, Appleman, and Kirk (38)
that the values for pK’1 and pKf2 are increased by increasing the
distance between the amino and the carboxyl groups. In terms
of the zwitter ion theory this effect, as would be expected, is to
decrease the acidity and to increase the basicity of the amino acid.
The effect of alcohol on the pK’z value of an a-monoaminomono-
 pK’ of Amino Acids

carboxylic acid as shown by the present data is an anomalous one,

for it has been shown by Mizutani (6) that the effect of the added
alcohol is to lower the pK’ value of an organic base. If it be as-
sumed that the proximity of the carboxyl group is responsible for
the anomaly, then the effect on PK’~ of increasing the distance
between the amino and the carboxyl groups should be relatively
less in 72 per cent alcohol than in water. To test this hypothesis,
the effect of alcohol on the pK’ values of cy-, y-, and &amino-?z-
valeric acids was determined. The data which are given in Table
I confirm the anticipation. When the distance between the amino
group and the carboxyl group was increased, although the pKlz
value was increased in the alcohol-water mixture, this increase
was less than a similar increase in the pK’, values when the amino
acid was dissolved in water. In the case of r-amino-n-valeric
acid the values of PK’~ in water and in 72 per cent alcohol are
identical. On the other hand, the proportionate increase in the
values of pK’1 in water and in 72 per cent alcohol with increasing
distance of the carboxyl from the amino group is approximately
the same. This is in harmony with evidence to be brought out
later that the effect of alcohol in increasing t,he value of the car-
boxy1 group is a fairly constant one and is very little affected by
varying the groups substituent to the carboxyl.
A dicarboxylic amino acid should show two pK’ values in the
neighborhood of paH 2 and 9.5 respectively, corresponding to the
carboxyl and amino groups attached to the or-carbon atom. The
third pK’ value should lie in the neighborhood of 4.0 because wit’h
the strengthening influence of the amino group decreased by its
distance from the carboxyl group, the second carboxyl group
should have a pK’ value approaching that of a simple aliphatic
acid such as acetic acid. In addition, the pK’ value of the second
carboxyl group of glutamic acid should be slightly greater than
that of the second carboxyl group of aspartic acid because the
strengthening influence of the amino group is further removed
from the carboxyl group in the case of glutamic acid. By block-
ing the second carboxyl group, e.g. by converting aspartic acid
into asparagine, a substance is produced with only two pK’ values4
4 Theoretically, a pK’ value corresponding to the -CONH, group should
be measurable. The dissociation of this group is probably too small to be
of significance.
 T. H. ,J&es and C. I,. A. Schmidt 337

both corresponding to those of an cr-monoaminomonocarboxyiic

acid. In the case of asparagine, the influence of alcohol on the pK’
va.lues should be the same as in the case of an amino acid such as
&nine.
An inspection of Table I reveals that the aforementioned antici-
pations are realized. The effect of alcohol in increasing the pK’
value of t,he amino group of the dicarboxylic amino acids is greater
than in the case of the amino group of a monocarboxylic amino
acid. The increased influence is probably due to the presence of
the second carboxyl group in the molecule, since the increase is
0.91 for aspartic acid and only 0.24 for asparagine.
Titration curves of the three hexone bases in alcoholic solution
were carried out. An examination of Ohe differences in the pK’
values of histidine in water and in 72 per cent alcohol shows that
the influence of alcohol is to increase the pK’, and PK’~ values,
while the pKIZ value corresponding to the second basic group is
lowered. On the other hand, in the case of arginine, all of the three
pK’ values are increased in alcoholic solution. Lysine was titrated
in four different concentrations of alcohol. The pK’ values were
obtained from the pG’ values according to Simms (39). Michaelis
and Mizutani (5, 6) found that there was an increase in the pK’
values of organic bases and a decrease in the pK’ values of carboxy
acids with increasing concentration of alcohol. In the case of
glycine, however, they found that only the pKfl value exhibited
this progressive change; the pK’, value showed a slight irregular
increase with a maximum in the neighborhood of 80 per cent alco-
hol. The data for lysine given in Table I show a somewhat similar
effect as a result of increasing the concentration of alcohol. If
the increase in the pK’1 values of lysine is plotted against increase
of alcohol concentration, a curve is obtained which lies very close
to a curve similarly plotted for glycine. The values for pK’,
of lysine exhibit a slight irregular increase with increasing con-
centration of alcohol; this behavior is similar to that of glycine.
The addition of alcohol does not influence the pK’, value of lysine.
Mizutani (6) showed that the pK’ values of aliphatic amines are
lowered by the addition of alcohol. The values given in Table I
show that alcohol lowers both pK’ values of cadaverine. The
effect stands in contrast to that found in the case of lysine.
It was anticipated that if the carboxyl group of glycine were
368 pK’ of Amino Acids

blocked as in the case of glycine ethyl ester, the pK’ value of the
amino group would not be increased but lowered by t,he addition
of alcohol, as in t.he case of an amine. The pK’ figures for glycine
ethyl ester bear out the predict,ion. A similar result has been
reported and similarly int,erpreted by Edsall and Blanchard (22)
who titrated the ethyl ester hydrochlorides of alanine and leucinc
in water and in 90 per cent alcohol.
From the foregoing the conclusion may be drawn that (a) the
zwitter ion hypothesis can be applied to amino acids when dis-
solved in alcohol-water mixtures; (b) the effect of ethanol is to in-
crease markedly the pK’ value of the carboxyl group; (c) the effect
of ethanol on the pK’ value of an amino group is dependent upon
the substituent groups in the molecule. A slight increase in pK’
is produced if the amino acid contains an a-carboxyl group. If the
carboxyl group is moved a sufficient distance from the amino
group, there may be a decrease in the pK’ value. Dicarboxylic
amino acids show a decided increase in the pK’ value.
The effects of the polyhydric alcohols, glycerol and mannitol,
upon the dissociation constants of glycine were examined. This
amino acid was titrated in aqueous solution, in glycerol solutions
containing 20 and 40 per cent by weight of glycerol dissolved in
water, and in mannitol solutions containing 2 and 4 per cent by
weight of mannitol dissolved in water. The usual necessary blank
titrations were carried out. The pK’, value of glycine at 25” was
2.37 in water, 2.49 in 20 per cent glycerol, 2.48 in 40 per cent
glycerol solution, 2.45 in 2 per cent mannitol, and 2.35 in 4 per
cent mannitol solution. The corresponding PK’~ values were, re-
spectively, 9.72,9.67,9.63,9.63, and 9.66. The effect of the polyhy-
dric alcohols upon the pK’ values of glycine is small. The be-
havior of glycine is in marked contrast to that of boric acid whose
dissociation constants are markedly increased by the additions of
mannitol (40). Loffler and Spiro (41) observed that the addition
of glycerol to an aqueous solution of glycine required an increase
in the amount of sodium hydroxide necessary to neutralize the
solution to phenolphthalein. The present data tend to indicate
that the effect of glycerol is upon the indicator rather than upon
the amino acid.5
5 It might be expected that the dielectric constant of the solvent would
influence the pK’ values of the dissolved amino acid. That there is no
 T. H. Jukes and C. L. A. Schmidt 369

The values given by Michaelis and Mizutani ($6) for the effect of
various concentrations of ethyl alcohol upon the pK’ values of
certain organic acids have been interpolated so as to obtain the
values corresponding to 72 per cent ethanol. The average increase
in the pK’ values of twenty-seven monobasic and dibasic organic
acids, expressed as a percentage of the pK’ value in aqueoussolu-
tion, is 46 f 3.5 per cent. A similar analysis of the changes in
pK’, values of fifteen amino acids caused by the addition of ethanol
was found to be 48 f 10 per cent. The correspondence between
the two sets of values is striking. No such correspondence is pos-
sible in terms of the classical theory of amino acid dissociation.
The depressant effect of alcohol upon the dissociation of a carboxyl
group is evidently changed but little by the nature of the groups
substituent to the carboxyl group.

SUMMARY

1. With the aid of the hydrogen electrode, the pK’ values of

certain amino acids, when dissolved in ethanol-water mixtures,
were determined. Similarly, the pK’ values of glycine in aqueous
mannitol and glycerol solutions were estimated.
2. Ethyl alcohol was found to decrease the dissociation of the
buffer groups which represent in terms of the zwitter ion theory
the carboxyl groups of the amino acids examined. This is in
harmony with the effect of ethanol upon the dissociation constants
of many other carboxylic acids.
3. The addition of ethanol was found to increase slightly the pK’
values corresponding to the a-amino groups of the amino acids

simple relationship is shown by the following measurements of the pK’

values for glycine in a number of solvents of widely differing dielectric con-
stants: water (dielectric constant = 79) pK’, = 2.37, PK’~ = 9.74;72 per
cent alcohol (dielectric constant = 40) pK’, = 3.46, PK’~ = 9.82; 76.4 per
cent by volume acetone (dielectric constant = 35) pK’i = 3.48, pKfz =
9.17; 20 per cent urea (dielectric constant = 88) pK’, = 2.97, pK’2 = 9.87.
The influence of the various solvents is chiefly on the pK’, values. The
pK’z values are relatively little influenced. This is in agreement with
the statement of Halford (34) that the dissociation of an unchargedacid,
(3.8. acetic acid, should be much more sensitive to changes in the dielcc-
tric constant of the medium than the dissociation of a charged “acid,”
c.q. ammonium ion.
370 pK’ of Amino Acids

studied. This effect has previously been overlooked in titrating

amino acids in ethanol solutions with phenolphthalein as indicator,
since the effect of the alcohol is primarily upon the indicator and
not upon the amino acid.
4. The effect of glycerol and mannitol upon the pK’ values of
glycine is slight.
5. The evidence presented harmonizes with the idea that the
amino acids exist in ethanol-water mixtures, as in aqueous solu-
tion, chiefly as zwitter ions.

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