STUDIES ON THE INTERACTION BETWEEN HEAVY

METAL SALT AND PROTEIN.

By

KEIZO KODAMA.

(From the BiochemicalLaboratory, Institute of Medical Chemistry,Tokyo Imperal

University,Tokyo, Director Prof., S. Kakiurhi.)

(Received for pnblication, .March3, 1923.)

I. INTRODUCTION.

The action of the salts of heavy metal as precipitant or as

coagulant of protein has been the topic of innumerable researches
in the protein chemistry. This is due partly to the attempt of
most of the old investigators to gain a clear conception as to the
mysterious nature of the protein body by isolating and analysing
the compounds of the protein with the heavy metal salts, and
partly to the theoretical interest associated with the colloidal
behaviour of protein. There is really an abundance of the reports
enumerated as evidence that the protein combines with the salts
of heavy metal in definite proportions. These experiments, however,
were carried out under different conditions and the results were so
divergent that the existence of any stoichiometrical relation can
hardly be recognized.
On the other hand, a group of the colloid chemists advanced
the adsorption theory to account for the non-concordance encountered
by analytical chemists. Since the interest of the investigators of
colloidal phenomena was chiefly focussed on the alterations of the
aggregating state, the special behaviour of protein, such as the
precipitability at the low and high concentration of heavy metal
salt and the solubility at the intermediate concentration has much
attracted their curiosity. They attempted to explain those phenomena
in the same manner as in the case of the suspenoids, especially
in those cases where the precipitation occurred at the low con-
505
506 K. Kodama:

centrations of the salts. As the explanation accepted by the

majority of colloid chemists is based on the assumption of an
adsorption of ions by colloidal particles or of a reaction occurring
between two colloids such as protein and hydroxide of heavy
metal, it seemed them natural that any definite proportion of
combining ratio between protein and heavy metal salts could not
result.
But unfortunately all the previous workers failed to take into
consideration one of the main variables, i.e. the hydrogen ion
concentration of the protein solutions.
In this connection, Robertson (1908) concluded very
reasonably as follows:-
"In the majority of investigations upon this subject no care
has been taken to maintain a neutral or otherwise constant reaction
in the medium from which the protein heavy metal compound is
thrown out, each observer has prepared these compounds under
different conditions and no clear distinction has been made between
the precipitation of the protein by the heavy metal salt and its
coagulation. It is not matter either for surprise or bewilderment,
therefore, that different observers have obtained very conflicting
results in determining the heavy metal content of these compounds.
In the light of our present knowledge the majority of these results
are perceived to be meaningless, since they were obtained with
compounds, or mixtures of different compounds prepared under
inadequately controlled conditions."
Quite recently J. Loeb (1922) has also raised the conclusive
evidence that the measurements of the hydrogen ion concentration
are paramount for understanding the chemical and physical
behaviour of proteins, and it was demonstrated in an ingenous
manner that the silver ion like other heavy metal ions would not
combine with protein at the acid side of its isoelectrical point.
In this experiment on albumin the writer attempted to come
near the solution of the problems concerning the interaction between
protein and heavy metal salts and came to the conclusion that
the hydrogen ion concentration is the predominant factor in not
 Heavy Metal Salt and Protein. 507

only the precipitation but also in the sa-called peptisation of the

once formed precipitate of heavy metal proteinate by escess of the
salt. And further the proof was furnished that the protein is not
determining factor in its combination with heavy metal salt as
Loeb Claims an the basis of bis experiment with gelatin.

II. THE PRECIPIT

ATIONOF PROTEINAT THE Low CONCENTRATION
OF HEAVYMETAL SALT.
Panli, (1904) first assumed that the weil dialysed protein
solution could not be precipitatecl by tue low concentration of
heavy metal salt; whereas slightly alkalised, it became readily
precipitated. And for this phenomenon he gave the following
plausible explanation.
" Schreibt man die bei niedrigen Konzentration auftretende
Fallungen den einfachen Metallionen zu, dann sind freilich
Widersprüche nicht zu vermeiden. So bleibe es unerklart, warum
der Schwellenwert fur das einwertige Silber, zweiwertige Kupfer
und dreiwertige FercHon zusammenfallt. Vergleicht man den
Schwellenwert des Silber und Wasserstoffions, so zeigt sich bei
gleicher Ladung trotz der bedentend grosseren Wanderungsgesch-
windigkeit des H-ions das Silbersalz der vollig dissozierten Saizssiure
weit uberlegen u. dgl. Alle Schwierigkeiten verschwinden unter der
von Biltz, Neisser und Friedmann und Landsteiner begrun-
deten Annahme, dass das kolloidal geloste Metallhydrosycl
dieser stark hydrolytisch zersetztes Scliwermetailverbindungen die
Eiweissfallung ermittelt etc."
According to this saterLent the view hell by Pauli is far
from convincing for the failure of generalis:ttion of the valency rufe,
which is called Schultze-Linder-Picton's law and prevails in the
domain of suspensions colloid as well as in some Gasas of emulsions
colloid, should not Leeessarily correlate that metul ion has no effect
as precipitatiog agent of protein. lndeed, iu the Gases of suspensoid
and emulsoid such as globulin, the stability of the colloidal sol vanishes
at its isoelectrical point and precipitation or cuagulation occmss. While
in the Gase of albuwin the process to bring it tu its isoeleetrical
 508 K. Kodama:

point can not induce its precipitation, because albumin is soluble

even at its isoalectrical point. Hence, factor other than the

neutralisation of its charge must play a more important role in the

chemismus involved in the precipitation. And what would be

expected in this respect is the solubility of the compound of the

protein with heavy metal salt. Since the solubility of any

compound has little to do with the valency of the united ion, but

rather with the special behaviour of electron carried by it, though

on its relation to the solubility of the resulting compound nothing

can be said definitely in the present state of our knowledge, it

might well be assumed that the theory advanced by Pauli is not

the only possible explanation with regard to the precipitation of

protein with heavy metal salt.

To verify the above assumption the following experiment was

uundertaken.

The egg albumin used in this experiment was prepared by

recrystallising three times from ammonsulphate solution and by

dialysing for 2 weeks against water, followed by the further

purification for freeing from the combining salt by the electro-

endosmotic arrangement.

The protein thus prepared showed the electrical conductivity

0.6 •~10-6 Mho and the reaction corresponding Pa 5,4. It was

not precipitable simply by adding a dilute heavy salt solution,

unless alkalised to some extent. This can be seen from the

following experiment. To each of test tubes containing 5 cc. of

sodium hydroxide solution of various concentration 1 cc. of the

albumin solution was added and well mixed. 2 cc. of the salt

solution were then put in each test tube and the mixture was

slightly shaken. As will be seen in the following table the

precipitation occurred only when sodium hydroxide in an amount

of more than 1/400 mol was added.
 Heavy Metal Salt and Protein . 509

TABLE I.

When an albumin solution is made more alkaline than its

isoelectric point, it is charged negatively owing to the following
chemismus.

If to such a solution the heavy metal salt is added the

cationic metal ion will combine with anionic protein ion, and when
solution is not too dilute, the resultant compound, being less soluble
than either protein at isoelectric point or its alkaline salt, will be
thrown out of solution. When, however, the amount of sodium
hydroxide exceeds a certain value, the heavy metal ion is carried
away as insoluble metal hydroxide before it enters into combination
with protein ion and precipitation of protein, therefore, no more
occurs.
Moreover, if the albumin was added to the mixture consisting
of heavy metal salt and sodium hydroxide in the same proportion
as in the above experiment, it also was not precipitated. The
metal hydroxide produced retains no power to precipitate protein.
This might be ascribed partly to the fact that metal hydroxide is
less effective owing to its rare dissociability and partly to the
510 K. Kodama:

mutual repelling action of the similarly charged particles between

protein ions and metal hydroxide, as metal hydroxide may be
charged negatively in an alkaline medium. In this connection
reference may be made to the investigations of Powis (1915) on
the preparation of colloidal ferric hydroxide either in the positively
charged or negatively charged condition. Hitherto ferric hydroxide
and other metal hydroxide has been regarded as typically a
positively charged colloid. P o w i s has shown, however, that the
sign of the charge is the matter of environment during preparation.
Colloidal solutions of ferric hydroxide are usually prepared by
dialysing a solution, say, ferric chloride, the resulting colloid is
positively charged, which is attributed by Powis to the fact that
under these conditions the he-ion is preferentially adsorbed by the
colloid particles. Under the conditions, in which the colloid is
formed in the presence of relatively large amount of OH ion, this
ion is adsorbed in greater quantities and confers its charge to the
colloid particle.
Since Pauli claims that the positive metal hydroxide may be
responsible for the precipitation of protein the latter must be
negatively charged, that is, it must exist at the alkaline side of
its isoelectrical point. This condition, ho,\ever, does not favour
the existense of the positive metal hydroxide. If Pauli's view be
correct, therefore, it must be expected that the precipitation is
limited to occur over narrow range which permits the simultaneous
existence of negatively charged protein ion and positively charged
metal hydroxide. This is really not the case, because the precipi-
tation can occur even at the acid side of the isoelectrical point of
albumin, as will be seen from the next experiment. The above
simple consideration now leads to the conclusion that the view
held by Pauli is presumably untenable for the precipitation of
protein by heavy metal salt at its low concentration.

III. THE INFLUENCEOF THE HYDROGENION CONCENTRATION

ON
THE PRECIPITATION
OF ALBUMINBY HEAVYMETALSALT.
As proved by the foregoing experiment the protein must be
 Heavy Metal Salt and Protein. 511

alkalised to some extent to be readily precipitated by heavy metal

salt. This fact suggests itself that the reaction of medium plays an
important role in the precipitation of protein by heavy metal salt.
As already mentioned Loeb has done much ingenious work in this
connection, which might be regarded as epoch-making in the
physical chemistry of protein. According to him protein exists in
three states, defined by their hydrogen ion concentration, namely:
(a) as non-ionogenic or isoelectric protein, (b) as metal proteinate,
and (c) as proteinacid salt. He demonstrated, using M/64 of
silbernitrate that the gelatin solution which has a PH value
greater than 4.7 becomes opaque and then black when exposed to
light, while all the solutions of PH smaller than 4.7 remain
transparent and he concluded that the cation Ag is only in
chemical combination with gelatin when the PH is greater than
4.7, while at PH 4.7 or below gelatin is not able to combine
with Ag ionogenically.
This view, however, is not shared by Bancroft (1921) who
considered that Loeb was working at such an extreme dilution
that the specific effects of all ions but hydrogen and hydroxyl ions
are practically negligible.
To do justice to Bancroft it is necessary to find out the
relation between the concentration of heavy metal salt and the
liminal hydrogen ion concentration necessary for the precipitation
of protein.
Experiment: To each 1 cc. of albumin solution (3%) in a
number of test tubes were added first varying quantities of NaOH,
then certain volumes of distilled water and finally varying amounts
of a heavy metal salt. The volume of distilled water is always
so chosen that the final volume of the solution becomes 6 cc.
After the mixture had been well mixed appearance of turbidity
was examined, which might be inferred as evidence that the
albumin entered into combination with heavy metal salt. Hydrogen
ion concentration was then determined colorimetrically in all cases
of the salts except of the zinc salt. A potentiometrical determi-
nation was inadequate, because nobler metals like Au, Ag and Cu
512 K. Kodarna:

etc. have a lesser tendency to ionise, so that when they are

brought into contact with hydrogen molecule, they confer their
charge on hydrogen, converting the latter into ionised state. As
indicator for colorimetrical determination methylred and parani-
trophenol were used. They showed no appreciable error due to
the presence of protein and salt, so far as the concentrations in
this experiment are concerned. But with a gold chlorid solution
above a certain concentration they do not serve well owing to
the original yellow colour of the salt and to the alternation of the
nuance of the indicator to a considerable extent.
The results are summarised in the following Table II and
illustrated in Figure 1.

TABLE II.

It will be seen from this table that each salt enters into
combination with protein at different hydrogen ion concentration.
This apparent fact does not favour the view held by Loeb that
the heavy metal ion can be only combined at the alkaline side of
 Heavy Metal Salt and Protein. 513

the isoelectrical point of protein. Since the isoelectrical point of

egg albumin was proved to be PH=5.7 by Kakiuchi and
Koganei (1922) it must' be expected, if allowance be made for
Loeb's idea, that the precipitation should occur at PH 5.7. But
this is not the case in the above experiment. It must be concluded,

Fig. 1. The liminal H-ion concentration for the precipitation of protein

by heavy metal salt.

therefore, that the isoelectrical point of protein is of minor significance

in the reaction between heavy metal salt and protein. According
to the results above obtained gold chloride seems to Le the most

powerful precipitant. Other salts are arranged in the order

effectiveness as: HgCl2>AgNO5, CuSO4,>ZnSO1.
This different behaviour of each heavy metal salt can l e
accounted for by assuming that the molecular adsrbability or
affinity of each heavy metal ion towards protein is different, so
that the resulting metal proteinate reveals the different solubility.
In this respect, reference may be made to the valuable work of
514 K. Kodama:

work of Michaelis and Rona (1922), that the charcoal can

adsorb silberion and copperion as well as hydrogen ion and ferric
ion and mercuric ion extraordinarily well.
Taking this interesting fact into consideration the writer
furnishes the following explanation for the chemismus involved in
precipitation.
1) If egg albumin exists at the alkaline side of its isoelec-
trical point it combines readily with heavy metal salt, for example
with AgNO3 in the following manner.

2) If egg albumin exists at the isoelectrical point or rather

at its acid side it can combine with heavy metal salt only when
the metalion is relatively good molecular adsorbable in comparison
with hydrogenion and its concentration increases to a certain
extent. The more concentrated the heavy metal salt, the more
hydrogenion must exist to hinder protein from its combination with
heavy metal salt by the law of mass action. This fact stands in
fair agreement with experimental results, Alkali metal and
erdalkali metal ions, though present abundantly, can not displace
hydrogen ion at all, so that the precipitation is never induced.
In this case the nature of the isoelectrical point may be precisely
appreciated. Since zinc ion is less molecularly adsorbable it is
greatly influenced by hydrogen ion so that it combines only with
the protein, which exists at the alkaline side of its isoelectrical
point. The liminal PH, therefore, in the case of zinc salt lies at
5.7 which K a k i u c h i and his coworkers claim to be an isoelec-
trical point of crystallised egg albumin, militating against the
current view that it lies at PH 4.7.
The chemismus can be schematically represented as follows:-
 Heavy Metal Salt and Protein . 515

If such a chemismus actually takes place, then it must be

expected that the reaction of the environment must turn out to be
more acidic owing to the free nitric acid. That this is really the
case can be shown by the followingexperiment.
To 5cc. of several heavy metal salts, each contained in a
test tube and previously brought to PH= 5 .2, 1 cc. of 3% egg
albumin (Pu=5.4) was added. After content had been well
mixed Pg of the resulting solution was measured colorimetrically .
The results are indicated in the followingtable.

TABLEIII.
Changeof PH owingto the interactionbetweenheavy metal salt anal
protein.
Concentration
of protein0.5%.

At this point, it must be recollected that the order of the

effectiveness of each heavy metal salt in acidifying the medium
stands quite in agreement with that of the precipitating efficiency
of each salt. This fact leads to the important conclusion that the
heavy metal salt is the more effective as precipitant, the more it
displaces the hydrogen ion.
Furthermore, the followingexperiment furnishes a support for
this conclusion.
To each 1 cc. of egg albumin solution (PH=5.4) in a
number of test tubes were added 2 cc. of potassium chloride
solution of varying concentrationand 3 cc. of M/100 HgC12 (PH=
5.3). The hydrogen ion concentration was determined colorime-
trically before and after the addition of HgCl2. As the control
distilled water of the same acidity as albumin solution was used.
The results are shown in Table IV.
 516 K. Kodama:

TABLE IV.
Relation between the change of PH and the precipitation of egg albumin.
End-concentrationof albumin =0.5%.
End-concentration of HgCl2=M/200.

Now it is apparent that the increasing turbidity was always

accompanied by a corresponding decrease of Pa value. The more
concentrated the potassium chloride, the less ionised is the mercuric
chloride, so that the concentration of potassium chloride is reached
where no turbidity of protein appears, owing to the sufficient
diminution of active Hg-ion.
The diminution of Hg-ion may be caused by the formation
of complex salt with the composition of K2HgCl4. Such a com-
pound may undergo hydrolysis to a certain extent giving a rise in
the amount of hy-droxil ion. That this is really the case can be
seen in the above experiment, the PH of Control solution showing
an increase on the addition of potassium chloride. It was long
known in practical medicine that the HgCl2 acts as disinfecting
agent more agreeably when it is mixed with sodium chloride.
This fact can be explained by assuming that the so small amount
of Hg-ion as is contained in HgC1 NaCl mixture is unable to
cause the precipitation of protein, but is sufficient to bring bacteria
to death.
 Heavy Metal Salt and Protein. 517

IV. THE DISSOLUTION

OF THE PRECIPITATEAT HIGHER
CONCENTRATIONof HEAVYMETALSALT.
The chemisnnis in the dissolution of the precipitate by the
excess of the salt has remained for a long time a puzzle to
investigators along this line.
Galleotti (1904) applied the phase rule, which was originated
by Roozeboom for 2 phasic heterogeneous system, thus intending to
attack this problem from the thermodynamical view point. But
the unapplicability of this rule for the system o" protein and heavy
metal salt was detected by Robertson (1918), so that it is no
more worth while to survey his work.
The majority of other investigators on this subject hold
almost the same view that the dissolution of the once formed
precipitate is to be ascribed to the formation of complex compound.
In this respect Pauli (1912) affords the following words.
"Versetzt man eiue Eiweisslosung etwa (,lurch Zink-od
Kupfersalz wit einen Uberschuss von Schwermetallsalz, so geht sie
wieder ins Lusung. Die Erkhirung dieses Phenomenon ist noch
nicht ganz sicher. Fiir Eieralbumin iunerhalb 0.5.-4.n Zinksulfat,
somit in Konzentrationen, die relativ wenig hydrolytisch dissozieren
umd es ist deshalb rahrscheinlich, doss die Aufhebung der Flockung
gemass einer Annahme Hardys wit der Bildung einen neuen
Komplexen Zinkhydroxydprotein + Zinksulfat zusammenhiingt, so
wie etwa Halogensilber Niederschkige im ulerschnss von Halo-
gensalz in Lusung gehen kounen."
The analogical explanation was also given by Zsygmondy
(1920) in the following manner.
"Im Cbersehuss des Fallungsmittels ist der Niederschlag
zuweilen lvslich. Diese Wiederloslichkeit luann verglichen werden
wit der Bildung von Komplexen ebenso wie Init der gewiihnlichen
Peptisation etc. Fasst man den Vorgang kolloidehemisch auf, so
liisst es sich etwa in folgender Weise zur Darstellung bringen :
Die gefallte Niederschliige nehmen Zinkionen auf gerade so wie
das Gel der Zinnsiiure Stannationen adsorbiert and die Ionen
518 K. Kodama:

erteilen den Eiweissteilen positive electrische Ladung. Die Folge

davon wird eine weitgeheude Zerteil ung des Niedersehlags sein:

Das peptisierte Kolloid l at daher positive Ladung."

As far as the writer knows, there is no convincingexperiment,
which affordsdirect evidenceto this idea. It seems to be deduced
from the observation of Lottermoser concerning in the dissolu-
tion of the silver haloid by excess of silversalt. But such a
analogy is of course far from satisfying.
As already discussed in the previousexperiment the hydrogen
ion concentrationshows the surpassing influence for the precipita-
tion of albumin by heavy metal salt. If there exists the hydrogen
ion concentrationin such an amount that the adsorption of hydrogen
ions exceeds the adsorption of heavy metal ions, the precipitation
will no more result. Hence if one adds a slight amount of acid
to the solution, in which the precipitate of metal proteinate has
been just thrown out, the precipitate will again promptly disappear.
This is especially the case with zineproteinate.
It must be here recollected that the heavy metal salts such
as CuSO4,ZnSO4and Pb(CH_1COO),etc., which can bring about
dissolution of protein precipitate by excess of their amount, are
likely to be hydrolysed and their solution acquires pronounced
acid reaction. It will, therefore,be expectedthat the further addition
of such heavy metal salt always correlates with the increase of
acidity of the solution, so that the combination between albumin
and heavy metal salt may not take place. This conception might
be acknowledged by the next experiment.
To 1cc. of albumin in a test tube were added first 1cc. of
M/1000 NaOH, then certain volumes of distilled water and finally
varying amount of M/100 CuSO, solution. The volume of water
added was always so chosen as to make the total volume up
to 6 cc.
 Heavy Metal Salt and Protein. 519

TABLE V.

The change of PH by increasing corcentration of salt and its relation to the

precipitation of albumin.
End-concentration of protein 0.5%.

The same experiment was repeated with ZnSO,, the result of

which is given in the following table.

TABLE VI.

N. B. Pa is determined electrometrically.

Hitherto it has been usually believed that such a dissolution

does not take place with silver salt. But under certain conditions
it is observable. The following experiment which was performed
520 K. Kodama :

in the same manner as in the case with CuSO4 may serve as

evidence.

TABLE VI[

It is apparent from this result that the dissolution of the

precipitate by excess of salt was always accompanied by the
increase of acidity of the solution.
Since such a process of dissolution can be also produced by a
mere addition of dilute acid to the same acidity, which prevails in
the solution of protein when heavy metal salt begins to cause
redissolution of the precipitate, it might well be assumed that the
essential feature of the process may be the increase of hydrogen
ion concentration.
However, there remains yet the possibility of the complex
salt formation.
Now, if such a chemisnius really prevails in the phenomenon
involved in dissolution it must be expected that the concentration
of heavy metal salt will decrease in such a case. Whether the
decrease really occurs or not can be shown by measuring the
concentration of salt. In this respect, the following experiments
were undertaken.
To each 7 cc. of egg albumin solution in a series of Erlen-
meyer flasks were added varying amounts of N;100 NaOH, a
 Heavy Metal Salt and Protein. 521

certain quantity of distilled water, so as to make the total volume

up to 16 cc. and finally 1 cc. of M/10 AgNO3 After well mixing
the concentration of Ag and H-ion and the refractivity of the
solution were measured.
As the silver electrode the platinum plate N\as used which was
coated with electrolysed silver. The determination of Ag ion was
found against tenth normal silber electrode, using Leeds and
Northrops potentiometer and sensible galvanometer, which reacts
well to 0,5 millivolt fluctuation.
Refractivity was measured on filtrate from precipitate with the
object of estimating the quantity of albumin remaining uncoagu-
lated. The results are summed. up in the following Table.

TABLEVIII.
End-concentration
of albumin=0.3%
End-concentration
of AgNO3=1/171 mol.

The next experiment only differed from the above in keeping

the concentration of NaOH constant and varying the quantity of
AgNO3.
 22 K, Kodama:

TABLE IX.
End-concentration of albumin =0.5%
End-concentrationof NaOH=1/1700 mol.

This turbidity is clue to the AgOH, because the refractivity gave no inform-

ation on the precipitation of albumin.

N. B. 1. The refractivity due to NaOH or salt is negligible,

2. ƒÎ0 means the pot-nlial of the control solution containing distilled water

instead of albumin. As the presence of sodium hydroxide causes the

precipitation of silver oxide the potentials obtained here 'Vary accord

ing to the amount of the latter.

A similar experiment was repeated with zicc salt. As the

electrode 10% zinc amalgam was used, prepared by melting 10

gm. of zinc powder (Kahlbaum) into 90 gm. of pure mercury.

The determination was carried out against saturated caromel

electrode.

TABLE X.

End-concentration of albumin=0.382%.
 Heavy Metal Salt and Protein. 523

It will be noticed from the above experiments that when the

peptisation took place the diminution of the concentration of heavy
metal salt could not be followed at all. This fact gives valuable
support to the idea that the essential features of the dissolution by
excess of salt lie in the increase of the acidity. It seems to me,
therefore, that the process which is called peptisation by many
authors can be easily elucidated by the fact above mentioned.
Further the results show how erroneous is the statement that
the flocculation of protein by heavy metal salt is an irreversible
process. Provided the conditions are really reverse l the coagula-
tion, certainly in the eases referred to, can be reversed too.

CONCLUSIONS.

In these experiments the interaction between crystalline egg

albumin and heavy metal salts such as AgNO3, ZnSO4, CuSO4,
HgCl, and AuC;; was studied, and the following conclusions were
obtained.
1. Heavy metal ion but not its hydroxide seems to be
responsible for the precipitation of protein by the heavy metal
salts at its low concentration.
2. Hydrogen ion concentration plays a leading role in the
precipitation of protein by heavy metal salt, its increase to a
certain extent preventing the combination of protein with heavy
metal ion. The isoelectrical point of protein, however, does not
play here so important a part, as J. Loeb claims. Using the
same solution of protein the experiment clealy indicates, that the
nobler the metal is the higher must be the hydrogen ion con-
centration to prevent the precipitation of metal proteinate from
the solution.
3. The dissolution of precipitate of metal proteinate by
excess of the salt might be accounted for by the increase of
acidity, but not by f.)rmation of a complex compound.

REFERENCES.

Bancroft, w. D. (1921): Applied Colloid Chemistry, 256.

524 K. Kodama.

Galeotti, G. (1904): Zeit. f. physiol. Chem. 40, 492.

Galeotti, G. (1901): .. ," 42, 330.
Kakiuchi and Iioganei (1922): Jonrnrel of Biochemistry 1, 405.
Loeb, J. (1922): Protein and Theory of Colloidal Behavior.
Michaelis and Runs 1922): Die Wasserstoffionenkonzen trat ion 202.
Pauli, W. (1905): Beitr. z. chem. Physiol. and Path. 6, 256
Pauli. W. (1912): Fortschr. nat. Forschung, 4, 258.
Powis (1915): Trans. Chem. Soc. 107, 818.
Robertson (1918): The Physical Chemistry of the Proteins, p. 1.38.
Zsygmondy, (1920): Kolluid Chemie, p 350.