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6-60
5-56


6-626


3


0-9467
0-9653


^.t2|x 10=20250
fo:^{x 10=206-00


0-05670
0-05768


6-00
6-04


6-020


4


0-9026
0-9896


20;^(x 10=20650
22:25j^ 10 = 222-60


005782
0-06230


6-401
6-3o/


6-360


5


0-9373
1-0038


2J:75| ^10 = 218-00
23'30Jx 10 = 233-75


006104
006660


6-61
6-62


6-565


6


1-2733
10307


24-70 X 10=248-00


0-08694
006944


6-741
6-83J


6-785



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163



III. Estimation of Amino-nitrogen. B. Egg albumin,
25 CO. of the standard volume (250 cc.) of liqaid were titrated in each ease with N/50 soda.



Time of

hydrolysis

in boors

0-25



0-50



Weight of

egg albumin

used, g.

0-9942
1-0086

0-8607
0-9895

0-8164
1-0576

0*8806
0-7636

1-0956
0*9860

0-7748
1-0865

1-0181
0-9522

1-0098
1-0429

0-9884
0*8228



Number of cc. of
N/50 soda used



500 (
4-80(

i



515
5-05

9-60

9-50

10-30,

10-501

14-40
14-30
18-20
18-30



xlO= 4900



xlO= 51-00



|xlO= 95*50
X 10 = 10400

X 10 = 143-50
X 10 = 182-50



n3^i<>=i72-^<>



15-40
15

22*90
23

20-90
20-80

16-40
16-60
2410
24

22-^^}xl0-
28-30P^"~

22-00

22



15300
23000



I x 10 = 208-50

1x10 = 165-00
;Jg|x 10 = 240-75



2-00) ,rt

2.ior^^=

23-80



23100
220-60



X 10=238-26
I X 10 = 24400

23-805^^^ = 237-^^
25:^1x10=199*00



23-85
24-30
24-50

23-70/



Equivalent Percentage Mean of
nitrogen, g. nitrogen duplicates



0-01372
0*01428

002674
002912

0-04018
0-05210

0-04816
004284

0*06440
0-06838

004620
0-06741

0*06496
0-06174

0*06671
0-06832

0*06650
0*05672



l-38j
I-42J

3-09|
3-00)

4-93)
4-93/

■"}

•61 1

I

6-00 1
6-20/

S-38|

6-48|

6*66 1
6*55)

6*741
6*77/



5-471
5*<

5-88
5-92

6-00)
6-1

6-381
6-^



1-400



3*045



4-930



5-540



5*900



6-100



6-430



6-605



6*765



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164



W. W. P. PITTOM




0-25 0-5 1

Time of Hydrolysis in hours

Fig. 2.
Curves showing: A. Bate of Liberation of Amino-Nitrogen.

B. Bate of Liberation of Nitrogen precipitated by Phosphotangstio acid.

IV. Estimation of Nitrogen precipitated by Phosphotungstic Acid.







A. Ca


seinogen.






75 00. of the liqnid were


nsed.








Time of

hydrolysis

in hoars


Weight of
caseinogen,


Namber of cc.
0-lNHCl
ased (actual

titration x 3-33)


Nitrogen
eqaivalent,


Percentage
nitrogen


Mean of
duplicates


0-25


0-9994
10618


35-84
36-67


0-04947
005131


4-96 )
4-84


4-900


0-50


11179
0-9648


29-17
25-67


004083
0-03594


3-66 i
3-73 1


3-695


1


1-0627
1-0769


26-16
26-84


0-03662
003687


3-40 \
3-48 J


3-440


2


0-9842
11830


21-50
26-50


0-03010
003710


306 1
3-14


3100


3


0-9467
0-9563


20-83
20-50


002912
0-02870


8-08 1
8-00


3-040


4


0-9026
0-9896


19-00
21-67


0-02660
003034


2-95 \
8-07 f


3 010


5


0-9378
1-0038


1900
20-67


0-02660
0-02894


2-88 )
2-84


2-860


6


1-2783
1-0307


25-67
20-83


0-08694
0-02916


2-84
2-82


2-830










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W. W. p. PIT^M



165



IV. Estimation of Nitrogen precipitated by Phosphotungsiic Acid.

B. Egg albumin.



Time of
lydrolysis
in hours


Weight of
egg albumin,


Number of cc.
0-lNHCl
used (actual

titration X 8-83)


Nitrogen

equivalent,

g-


nitrogen


Mean of
duplicates


0-25


0-9942
1-0086


42-34
44-34


006922
0-06202


5-96
6-15


1


6*065


0-60


0-8607
0-9896


26-67
28-67


004018
003694


4-17
4 06


I


4-110


1


0-8164
10676


21-67
28-34


0-03034
0-03966


3-72
3-76


1


3-735


2


0-8806
0-7636


22-60
19-16


0-08160
0-02684


8 -58
3-52




8-550


8


1-0956
0-9860


25-67
23-67


0-08594
0-08314


3-28
8-36


1


8-320


4


0-7748
1-0866


18-17
26 00


02541
0-03600


8-28
8-24




3-260


5


1-0181
0-9522


22-67
22-00


0-03174
003080


3-14
3-23


1


8-186


6


1-0098
1-0429


21-34
23-00


0-02988
0-08220


2-96
8 09




8-025


7


0-9884
0-8228


21-34
17-34


0-02988
0-02428


802
2-96





2-990



Discussion of results.

I. Ammonia. In the very early stages of the hydrolysis the rate of
liberation of ammonia is very rapid, and consequently the curves (Fig. 1)
rise very steeply. This early period is then followed by one in which the
ammonia comes off at a steady slow rate, so that the curves during this
second period rise slowly. Finally the amount of ammonia liberated reaches
a limit and the curve becomes a straight line parallel to the time axis.

The ammonia liberated on protein hydrolysis is always considered as
being combined in the protein in the form of an amide, and Osborne and
his fellow workers have shown that the amount of ammonia liberated on
complete acid hydrolysis of a protein roughly corresponds to what would be
obtained from the half amides of the two dibasic acids, glutamic and aspartic,
contained in the protein. The results here obtained are in accord with such
an explanation.

The experimental error in the estimation of ammonia is probably rather
high, chiefly owing to the smallness of the quantities measured, but still
there is no doubt that the curves indicate the rate at which the ammonia
is set free.



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166 W. W. P. PITTOM

II. Humin Niti^ogen. From the mean of all the values obtained it
appears that 0*31 per cent, nitrogen in the case of egg albumin and
0*29 per cent, nitrogen in the case of caseinogen is removed by the magnesia.

The values for 0*25 hour's hydrolysis are not included here as evidently
in these cases the magnesium oxide carries down with it bodies other than
humin substances.

III. Amino-nitrogen. The Wo curves (Fig. 2 A) obtained showing the
rate of liberation of amino-nitrogen are very similar, although of course by no
means identical. Moreover, they resemble in a striking manner the curves
obtained for the rate of liberation of amino-nitrogen when the hydrolysis of
the protein has been accomplished by means of an enzyme, such as trypsin.
Hence the process of hydrolysis is the same whether the hydrolysing agent
be a vigorous one, such as hydrochloric acid, or a slow one, such as trypsin.
These curves for the amino-nitrogen illustrate very clearly the difference
in composition and behaviour of the primary products of hydrolysis of the
two proteins. The curves twice intersect, indicating that the rate of libera-
tion of amino-nitrogen from the primary products of hydrolysis of the two
proteins, twice changes.

In the first phase of the hydrolysis the amount of amino-nitrogen set free
is greater in the case of caseinogen, indicating that more amino-acids and
polypeptides are formed during this period in the hydrolysis of caseinogen
than in that of egg albumin. In the second phase of the hydrolysis this
state of aflfairs is reversed, showing that during this period the complex
polypeptides derived from egg albumin are broken down more readily than
those formed in the primary decomposition of caseinogen.

Finally after the second intersection of the curves the rate of liberation
of amino-nitrogen is greater again in the case of caseinogen, proving that the
simpler polypeptides derived from caseinogen are more easily broken down
to amino-acids than those produced from egg albumin. This of course is
what one expects when it is remembered that the complete hydrolysis of egg
albumin takes longer to accomplish than that of caseinogen.

IV. Nitrogen precipitated by phosphotungstic ivdd.

Here again the two curves (Fig. 2 B) are very similar in form, but
throughout the whole period investigated the caseinogen curve lies below
that of the egg albumin.

In the early part of the hydrolysis there is a large amount of nitrogen
in the phosphotungstic acid precipitate in both cases, showing that there



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W. W. p. PITTOM



167



is a good deal of polypeptide nitrogen in the precipitate. Afterwards, how-
ever, the phosphotungstic acid contains very little polypeptide nitrogen, for
from three hours onwards the curve runs nearly parallel to the time axis,
though it does fall just a little.

The big difference between the values of the phosphotungstic acid
nitrogen after 0*25 and after 0*5 hour's hydrolysis, is undoubtedly due to the
decomposition of complex polypeptides into simpler ones, which are not
precipitated by phosphotungstic acid. This point is brought out more
clearly by drawing the curve total nitrogen accounted for, against time.

In the figures which follow no account is taken of the &ct that
the diamino-acids, which are precipitated by phosphotungstic acid, contain
a certain amount of amino-nitrogen and hence some of the nitrogen is
accounted for twice. This of course has no bearing on the point at issue.



Total Nitrogen accounted for in the above Estimations.



Time of


Percentage


Peroentage


Peroentage


Percentage




hydrolysis


ammonia


amino-


phosphotung-


hnmin


Total


in hours


(amide nitrogen)


nitrogen


stic nitrogen


nitrogen


peroentage


A. Caseinogen.










0-25


1-340


3-190


4-900


0-63


10-06


0-50


1-360


3-705


3-695


0-29


9-06


1 •


1-386


4-886


3-440


0-29


1000


2


1-406


5-526


3-100


0-29


10-33


3


1*440


6-020


8-040


0-29


10-77


4


1-460


6-350


3-010


0-29


11-11


5


1-605


6-666


2-860


0-29


11-22


6


1-495


6-786


2-830


0-29


11-40


B. Egg


alfmmin.










0-26


0-940


1-400


6050


1-13


9-52


0-60


0-975


3-045


4110


0-31


8-44


1


1060


4-930


3-735


0-31


10-02


2


1100


5-640


3-560


0-31


10-60


3


1150


5-900


3-320


0-31


10-68


4


1-200


6-100


3-260


0-31


10-87


5


1-245


6-430


3-180


0-31


11-16


6


1-260


6-605


3-025


0-31


11-20


7


1-260


6-766


2-990


0-31


11-32



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168



W. W. P. PITTOM



%
12



11



10-



9-



8-





1 1 1


1 1


^-.,


■ \ /

\ 1

8






-


-JL-I 1


1 1




. .



0-25 0-5 12 3 4 5 6 7

Time of Hydrolysis in hours

Fig. 3. Carves showing total nitrogen aocounted for.

These figures and the curves (Fig. 3) plotted from them show that the total
amount of nitrogen accounted for rises steadily with the time except in the
case of 05 hour, where the total nitrogen accounted for is considerably less
than that at 025 hour.

This proves definitely that the huge decrease in the amount of nitrogen
precipitated by phosphotungstic acid at these two times of hydrolysis, is not
nearly accounted for by the increase in the amount of amino-nitrogen found,
or in other words it is clear proof that between 0*25 and 0*5 hour's hydrolysis
some complex pol)rpeptides which were precipitated by phosphotungstic acid
after 025 hour's hydrolysis, have been broken down into simpler products,
which are not precipitated by this reagent.

The fact that at no time is the whole of the nitrogen of the proteins
accounted for shows that there are many polypeptides which are not pre-
cipitated by phosphotungstic acid, a point which is further emphasised by



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W. W. p. PITTOM 169

the fact that the phosphotungstic nitrogen curves run nearly parallel to the
time axis after a few hours' hydrolysis, i.e. long before the hydrol)rsis of the
protein is nearly complete.

Another point which the phosphotungstic nitrogen curves indicate is
that the diamino-acid content of caseinogen is less than that of egg albumin,
a result which is in direct opposition to the published figures, viz.:







Hugonenq et Galimard




Hart [1901]


[1906]




Caseinogen


Egg albamin


Lysine


6-80 0/,


2-16 %


Arginine


4-84


214


Histidine


2-69


0-00



from which figures it appears that caseinogen should have 3*36 per cent, of
diamino-nitrogen and egg albumin 1*10 per cent.

Although in the above experiments the hydrolysis was not complete, the
curves show that it is impossible to obtain, on complete hydrolysis, results
compatible with the published figures, and the indicated diflferences seem
large enough to warrant a re-determination of the diamino acids in both
cases.

Summary.

These results prove definitely that many of the simpler polypeptides are
not precipitated by phosphotungstic acid, and moreover indicate that a
definite point exists at which the more complex peptides are broken down
into simpler ones, most of which are not precipitated by phosphotungstic
acid, at which therefore it is advantageous to stop the hydrolysis so that the
complex pol)^ptides may be isolated. The isolation of such bodies, and
the study of their properties, it is my intention to essay in the near future.

Moreover the experiments here described emphasise the diflference in the
constitution of caseinogen and egg albumin and the consequent difference in
composition of their primary products of hydrolysis.

Finally, I wish to thank Professor T. B. Wood for suggesting this work,
and to say how much I appreciate the interest he has shown in its progress.

BEFEBENGES.

Hausmann (1899 & 1900), ZeiUch. pkysioL Chem. 27, 95 ; 29, 136.
Sorensen (1908), Biochem. ZeiUch. 7, 45.
Hart (1901), Zeitsch. physiol. Cherts. 38, 347.
Hugonenq et Galimard (1906), Compt. rend. 143, 242.



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XXIV. DIAGRAMMATIC CO-ORDINATION OF
PHENOMENA RELATING TO AGGREGATION
OF SOLS.

By GEORGE STANLEY WALPOLE.

From the Wellcome Physiological Research Laboratories^ Heme Hill, S,E.

{Received March 10th, 191j^)

The published investigations of the following phenomena embrace a
considerable portion of the facts so far accumulated on the causes inhibiting
or actuating the aggregation of sols, especially when they are mixed with
proteins and electrolytes :

1. The action of electrolytes on sols — causing the running together of sol
particles when added to the sol in sufficient concentration.

2. " Protection " — the property of proteins and certain other substances
iti preventing the aggregation of sols by electrolytes.

. 3. Chamge of sign of the electrostatic charge on the protected sol particle.

4. Non-coincidence of point of maximum flocculation and point where
sign of observed particle changes.

5. Change- of sign of charge on the protein at its isoelectric point.

6. Mutual predpitaiion of dissimilarly charged colloids in solutions free
from electrolytes, including the particular case wheipe one of the colloids is
amphoteric and has the properties of "protection," e.g. is of a protein nature ;
also the solubility of the precipitate formed in excess of either constituent.

7. " Irregular series " (Bechold) and " Pre-zone phenomenon " (Buxton).

8. " Reversible and irreversible aggregation."

Up to the present these phenomena have been generally investigated
one at a time. Each worker has naturally chosen experimental conditions
particularly suitable for the study of one phenomenon only. Little or no
co-ordinated work has been done, linking up on a quantitative basis the
known £Bicts, and demonstrating them as parts of a well-ordered scheme of
things, easy to remember, and, on the surface, easy to understand.

The plotting of results, obtained in this class of work, in chart form,
undei-taken primarily for my own use, has proved so helpful to me in



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G. S. WALPOLE 171

following out these relationships that I feel in a position to recommend them
for general use in expressing the results of experiments of this type.

In working with a very finely divided oil emulsion, the particles of which
were so small that, without misapplying the term, it could be called an
"oil sol," I found, in common with previous experience, that the addition
of hydrochloric acid in sufficient strength caused the particles to run together
and the oil to separate. The presence of a sufficient quantity of gelatin,
however, prevented this from happening; the sol was "protected" in this
case. When the gelatin was in certain concentrations, not too small and not
too great, the oil particles aggregated immediately if just the right amount
of acid was added, but not if the acid was too strong or too weak.

This seemed to point to some new and peculiar phenomenon where the
gelatin acted as an " activator " to the acid, for in this case the acid was far
too weak to aggregate the sol alone. It was not till later that it was seen
that this fitted into the general scheme of things and was capable of expression
in terms of things known, and that the invention of a new " phenomenon "
and a corresponding vocabulary was unnecessary. In other cases where
gelatin of a certain strength caused immediate aggregation of the oil in the
presence of a small quantity of acid incapable of doing this alone, it was
found that all strengths of acid greater than this brought about the same
result.

To simplify the matter of making and studying these mixtures it was
decided that each should contain 2 cc. of oil sol, 2 cc. of gelatin solution and
2 cc. of hydrochloric acid solution. Rows of these mixtures were put up
in test tubes. In any one row the 2 cc. of acid placed in each tube was
of the same strength throughout ; the concentration of the 2 cc. of gelatin
solution added varied ftx)m tube to tube. The strength of acid used was
changed from row to row.

As very dilute solutions were sometimes used it was found necessary
from the outset to express their concentrations as powers of ten, either of
actual concentration or normality. For instance the strength of a gelatin
solution was expressed in terms of its concentration. A 1 in 10 solution
was expressed as "gelatin 10"*," for example, and a 1 in 15,000 solution
as 10-^-». Similarly 0001 N hydrochloric acid was written 10-» N HCl.

After the mixtures had been made two hours they were examined
carefully, and notes made of those which showed to the eye no change of
state of the sol, those in which partial aggregation had occurred, and those
in which the separation of the oil was complete.

Observations in the electric field using the microscope method showed

Biooh. vin 12



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172 G. S. WALPOLE

that the oil particles in some of these mixtures were positively and in others
negatively charged. The sign of the charge did not depend upon the state
of aggregation — either charge was observed in tubes containing mixtures
which did not aggregate just as in those that did.

When the results of these two sets of observations were expressed in
tabular form little that was intelligible could be made of them ; in the form
of a chart they are all summarised in Fig. 1.

Aggreoation Diagram No. 1.

It will be remembered that each mixture consisted of 2 cc. of oil sol,
2 cc. of gelatin solution and 2 cc. of hydrochloric acid. As ordinates are
expressed negative exponents of normality of the hydrochloric acid put into
the tube ; as abscissae the negative exponent of the concentration of gelatin.
The tubes were considered one at a time and a mark plotted on the chart
at the point corresponding with the concentration of acid and gelatin in the
tube indicating (1) that no change had taken place in the distribution of

o

I

i
1

o

I

o

I

H

Exponents of gelatin concentration
Fig. 1. Aggregation diagram No. 1. Oil sol-gelatin-hydrochloric acid.

the oil particles, (2) that complete aggregation and separation of the oil had
occurred, or (3) that aggregation was only partial.

The sign of the oil particles, whether aggregated or not, was indicated
by a small + or — . Lines were now drawn on the diagram by the help of these
distinguishing marks. The first, LHJF, enclosed all points where aggregation,
whether partial or complete, was observed. Another, CABE, enclosed all
points where separation of the oil was complete.



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G. S. WALPOLE 173

The line XY was drawn through all points where the oil particles
appeared to have no electric charge; that is, they did not wander in the
electric field. In all mixtures represented by points to the right of this
line, the particles were negatively charged. All points to the left of this
line correspond to mixtures in which particles were positively charged.

The chart — called for convenience an " aggregation diagram " — ^represents
the state of aggregation and sign of the dispersed particle, two hours after
mixings of every possible mixture of equal volumes of oil sol, gelatin solution
and hydrochloric acid. Mixtures stronger in HCl than 0*33 normal were
not plotted in the present instance simply to avoid unnecessary complication
of the diagram.

At this stage it will be well to examine this chart with reference to the
list of phenomena given at the beginning of this paper.

1. The action of electrolytes (m sols. The action of electrolytes in the
absence of protein is seen in the extreme right of the diagram. In the
particular case shown in Fig. 1 the protein in a dilution of one in one
hundred million or more has no effect. The lines bounding the aggregation
area become parallel to the abscissae, and may be produced to infinity to the
point where the protein is infinitely dilute. The ordinate DEFG represents,
therefore, the behaviour of mixtures of 2 cc. sol, 2 cc. water and 2 cc. of every
strength of electrolyte solution, in this case HCl. No aggregation occurs
with extremely dilute acid, but when the strength of acid used is 10~* N
a slight separation of the sol occurs. As the strength of acid increases the
amount of sol separation increases until at 10~~^*" N and all concentrations
above this it is described as " complete."

In the case of an electronegative sol and an electrolyte with a polyvalent
cation the ordinate DEF would be much shorter — a result obtained equally
if the sol were electropositive and the electrolyte contained a pol)rvalent
anion.

At present the actual mechanism of electrolyte precipitation of sols is not
very well understood. It is hoped that in the course of this work some
phenomena may be observed which can be co-ordinated with the facts already
known about the conditions determining the stability or otherwise of sols.

2. " Protection" Any point in the area WPLR corresponds to a mixture
in which the gelatin has prevented the acid fix)m aggregating the oil sol.
Enough acid is present to bring this about completely if the gelatin were
not present. It may therefore be referred to as the "area of complete
protection against aggregation." In the same way the area WPST is " the
area of complete protection against partial aggregation," for in the mixtures

12—2



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174 G. a WALPOLE

the behaviour of which is represented by points in this area the acid alone
would only partially aggregate the sol in two hours.

Areas of "partial protection against partial aggregation" and "partial
protection against complete aggregation " are to be found in the diagram and
may some day become matters of study.

3. The change of sign of the ''protected'' sol particle. It is interesting
to note that the " area of protection " lies entirely inside the area where the
sign of the sol particles, or rather the sol-gelatin complexes, is positive. Also,
and this is shown better in Fig. 2 where mastic sol was used, it is possible
to have a sol protected by gelatin in a mixture containing as much as one-
third of its volume of N HCl, while a mixture exactly similar except that the
acid is 6000 times more dilute, flocculates immediately. A result of this
kind was observed by Mines [1912] using a gold sol.

4. Non-coincidence of point of maximum flocculation and point where
the sign of the observed particle changes. The type of flocculation which may
be observed in extremely dilute acid concentration if the concentration of
the protein be adjusted with great care, is remarkable in that no change
of sign of the electronegative sol particle necessarily takes place.

In Fig. 1 the ordinate GPHJ passes through points representing a series
of mixtures in which are placed say 2 cc. of oil sol, 2 cc. gelatin 10"*, and
2 cc of hydrochloric acid decreasing in concentration from tube to tube.
From normal acid to decinormal acid (10~^) complete flocculation is observed,
from there to 10"^"^ the flocculation is incomplete, and as far as can be made
out in solutions containing so much electrolyte the sign of the visible



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