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was 14*1 %. This experiment shows that water-insoluble protein, if present,
can be detected in presence of electrolytes and other soluble protein. There-
fore, a large proportion, at least 61 % (8-6 parts out of a total of 14* 1) of the
euglobulin found present at the close of dialysis must be regarded as formed
from the pseudo-globulin by some process of denaturation taking place after
separation from the serum. The remaining 39 % may, on the other hand,
be regarded as euglobulin which was present from the first, although it could
not be separated in presence of salt. But, from the fact that immediately
after preparation only a trace of insoluble protein could be detected in the
salt-containing pseudo-globulin, it seems probable that part of this may also
be derived from the pseudo-globuUn, showing that the denaturation of this
protein is accelerated by dialysis.

Experimental methods.

In the experiments set forth in Tables I to III pseudo-globulin was pre-
pared in various ways by salt-precipitation from horse-serum, the details of
themethod employed being given at the top of each table. Estimations
of the denaturated protein were made before, after, and, in some cases



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408 H. CHICK

(Experiments I and II, Table I), during the dialysis, by determining the
amount of protein which was precipitated by dilution and adjustment to the
iso-electric point.

The method of estimation was as follows. A small portion of the material was taken for a
preliminary trial, and largely diluted, so that the protein-content did not exceed 0-1 or 0-2 %.
If electrolytes are present the dilution should be as large as possible. 10 cc. of this diluted
solution were then placed in each of a series of test tubes and various small quantities of N/100
acetic acid added. If any denaturated protein were present an opalescence or slight turbidity
was usually apparent on mere dilution and, when the solution was adjusted to the iso-electric
point of the protein by addition of the required amount of acid, a precipitate was formed which
aggregated and settled. The series of trial test tubes was always allowed to stand several hours
and the composition of the material in the tube in which the precipitate was largest and agglutina-
tion most perfect was taken as the pattern for the estimation.

5, 10, or 15 cc. (according to the protein-content) were taken for analysis, diluted suitably
and addition made of dilute (N/10 or N/100) acetic acid in the amount necessary for complete
precipitation of the denaturated protein, as calculated from the results of the preliminary trial.
The whole was allowed to stand 24 hours and then centrifuged, the deposit being transferred to
a small, weighed, centrifuge tube, spun down again, washed with distilled water slightly acidified
and centrifuged a third time. The final deposit was dried in the weighed tube at 106^ until
constant in weight.

It is not advisable to permit the estimation to extend over more than one day, as, in some
cases, the denaturation of the pseudo-globulin seems to be continued at an appreciable rate in
the dilute solution and, when several days are spent over the estimation, higher values are fre-
quently obtained.

If the material to be analysed is slightly acid, small quantities of dilute ammonia must be
substituted for the dilute acetic acid. In cases where the material has been dialysed for a long
time and the reaction is approaching that of the iso-electric point, dilution with distilled water
containing about one-thirtieth of its volume of water saturated with carbon dioxide is often
found to be a quick and efficient method of precipitating all the denaturated protein.

If electrolytes are present, denaturated protein may be detected by the above method, but
precipitation, under certain circumstances, is incomplete (see above p. 406). In any case it is
advisable to make the dilution as great as is possible.

The above method has been described in detail, because the estimation
of denaturated protein in presence of other, water-soluble, protein is by no
means an easy matter even in the absence of electrolytes, the difficulty being
to ensure complete precipitation of the former. When this has not been
accomplished, faint turbidity can be detected in the supernatant fluid after
centrifuging ; in a successful estimation the aggregation of the precipitate
should be complete and the top liquor clear. Some of the earlier values
for concentration of " denaturated " proteins given in Tables I to III were
obtained when the method was not perfected and may be a little inaccu-
rate ; the later ones were more satisfactory, as may be seen in some cases
where the results of duplicate analysis are inserted.

It was possible that this water-insoluble protein, like euglobulin [Freund
and Joachim 1902, Gibson 1906, Banzhaf 1911], might be thrown out by
addition of sodium chloride to saturation, arid this would have proved a
convenient method of separation and estimation. It was tried in Exp. II,



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H. CHICK 409

Table I, but without success. After diluting the material 10-fold and satur-
ating with sodium chloride the residual protein (calculated on the original,
undiluted, material) was 10-78 %, the total being 11*12 % ; that is to say
only 0-34 % protein had been thrown down. The concentration of " de-
naturated" protein as separated by the above method was, however, found
to be 0-96 %.

Precipitation by saturated sodium chloride is of little value for the accurate
separation of proteins, the amount precipitated depending, to a greater
degree than is the case with other salts, upon external conditions, such as
the total concentration of protein and the reaction of the material. For
example, on one occasion, with horse-serum, the precipitate obtained on satura-
tion with sodium chloride was four times greater when the serum was diluted
1 in 10 than when the dilution was only 1 in 2 ; in the latter case the higher
concentration of total protein protected more than three-quarters of the
otherwise precipitable protein. Further, if diluted serum is made slightly
acid in reaction, the whole of the pseudo-globulin and albumin is precipitated
as well as the euglobulin.

Discussion of Results.

The results of the experiments in Tables I-III suggest that the pseudo-
globulin undergoes a progressive denaturation after its separation from the
serum. For example, in Exp. I, Table I, the pseudo-globulin immediately
after precipitation from the serum showed only a small amount of water-
insoluble protein capable of separation by dilution and adjustment of the
reaction to the iso-electric point. At the end of dialysis for 10 and 18 days,
however, 9-1 % and 12-9 % respectively, of the total protein was found to be
in the insoluble condition. It is probable that the ammonium sulphate
present interfered with a complete separation of any euglobulin present at
the beginning of the experiment, but it is unlikely that the difference in
amount of the traces of salt present after 10 and 18 days' dialysis respectively
could be responsible for the large difference in amount of insoluble protein
found on these two occasions.

Experiment IV, Table III is a second instance. Here material dialysed
for five days against tap water contained ITl % denaturated protein ; after
a further dialysis against distilled water lasting 29 days this proportion was
increased to 181 %.

Bioch. vm 27



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410



H. CHICK



TABLE I.

Spontaneous formation of euylobftlin from pseiido-globfdin at ordinary
teynperature ; jpsendo-glolndin prepared by half-saturation of undiluted
horse-serum with ammonium sulphate, after separation of euglobulin by
one-third saturation unth ammonium sulphate.



Exp.


Material


Date of

analysis


Len^h

of

dialysis,

days


Total
protein,

o
(1


Water-
insoluble
protein,

o
.o


0. water-
insoluble

protein

per 100 g.

total

protein


Phospborufl-content, g. P
per 100 g. protein

in water- in water-
soluble insoluble
fraction fraction


1.


Prepared 2. 12. 13; re-

final precipitate dis-
solved in water. Di-
alysed in presence
of toluene against
distilled water,
changed daily, from
6. 12. 13 to 23. 12.


5. 12. 13






small amount










13


15. 12. 13


10


14-56


1-33


9-11










14. .3. 14*


18


13-55


1-75


12-9


0K)18


0-171


II.


As in Exp. I but not


















re-precipitated


6. 12. 13
26. 1. 14«


3
3


1117
1112


present
0-964


8-67








(a) dialyscd against
dist. water from 13.


















2. 14 to 25. 3. 14


3. 4, 14«


43


4-77


0-654


13-7










9. 7. 14


43


4-77


0-700


14-7







(6) as above, but di-
alyscd against dist.
water saturated with
COa from 13. 2. 14
to 25. 3. 14 26.



3. 14 43



5-51



(0-652) 4

10-660 •



11-9



1 Kept in cold room from 23. 12. 13 until 14. 3. 14.
' Kept in cold room from 6. 12. 13 until 13. 2. 14 ;

• Kept in cold room from 25. 3. 14 until 9. 7. 14.

* Duplicate analyses.



dialysis then recommenced.



TABLE IL

Spontaneous formation of euglobidin from pseudo-globidin at ordinary
teynperature ; pseudo-globulin prepared from diluted (1 in 20) horse-
serum bif half-saturation with ammonium sulphate, after successive
removal of euglobidin precipitates obtained (1) by acidification of the
serum (1 in 10 dilution), and (2) by one-third saturation of the serum
(1 in 15 dduiion) with ammonium sulphate: once re-precipitated and
dissolved in a small quantity of water.



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H. CHICK



411





Date of


^.xp. Material


analysis


II Control, immedi-




ately after pre-




paration


4. 2. 14


{a) Control, allowed




to remain at labo-




ratory tempera-




ture, in presence




of toluene


3. 4. 14


(ft) Dialysed against




distilled water.




changed daily, in




presence of tolu-




ene from 2. 2. 14




to 25. 3. 14


30. 3. 14



G. water-
insoluble
Length Concen- Water- protein

of trationof Total insoluble per 100 g.
dialysis, (NH4)., protein, protein, total
days S0,,% % % protem



51



6-23 12-86



5-23



2-86



trace



1107



J 0-688 j 1
(0-681/



Duplicate analyses.



TABLE III.



8-61



141



Phosphorus-content, g. P
per 100 g. protein



in water-
soluble
fraction



in water-
insoluble
fraction



0-012



0-262



Spontaneous formation of eughbvlin from pseiido-globulin at ordinary
temperature; pseiido-globulin prepared from horse-serum (diluted 1 in 4)
by precipitation with one-half saturated ammonium sulphate, this first
precipitate extracted with saturated brine, brine-extract jrrecipitated by
addition of acetic acid to a concentration of 0*25 %^ ; this second
precipitate drained, pressed and mixed with washing soda to a con-
centration 0/ 3 % and dialysed for five days against tap ivater.



Exp. Material

IV. Control

(a) Dialysed further
against dist. water,
changed daily, in
presence of toluene
from 6. 3. 14 to
4. 4. 14

(ft) as (a) the distilled
water being satu-
rated with CO from
6. 3. 14 to 4. 4. 14



Date of
analysis

3. 3. 14



Length

of

dialysis,

days

(5)«



Total

protein,

0/

/o

11-72



Water-
insoluble
protein,
0/
/o

1-301



G. water-
insoluble
protein
per 100 g.
total
protein

11-10



Phosphorus-content, g. P
per 100 g. protein



in water-
soluble
fraction



4.4.14 29 (+5)



5-87



1-065



17-98



0-0062*



4.4.14 29 (-h5) 6-40 M61 18-14 0-0066*

* Gibson's [1906] process.

* Against tap water.

* Approximate only (less than 1 cc. N/10 NaOH neutralised).



in water-
insoluble
fraction



0-156



0-098



27—2



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412 H. CHICK

The results of Experiments I to IV also suggest in every case that the
degradation of the pseudo-globulin is approaching a limit. For example,
in Exp. II, Table I, after three days' dialysis 8-7 % of the protein was found
to be water-insoluble. After an interval of more than two months, including
a second dialysis lasting 40 days, the proportion had only risen to 13-7 %
and, after having been kept in the refrigerator for a further period of over
three months, to 14'7 %. A similar result is shown in Exp. I. In many
cases, the experiments were continued beyond the date of the last analysis
given in the tables, and the denaturation process was found to be practically
at an end. The degraded material was separated as completely as possible
by a modification of the method described above, and the clear, top liquor
adjusted to the iso-electric point of the denaturated protein and kept for a
considerable time in the cold room under observation. In the case of Exps. Ill
and IV {a and 6), only a trace of insoluble material was deposited after periods
of two months and one month respectively.

Phosphorus-content of this water-insoluble pseudo-globulin.

The resemblance between the denaturated pseudo-globuUn obtained in
these experiments and euglobulin led me to make estimations of the phos-
phorus contained respectively in the insoluble and unchanged pseudo-globulin.

Hardy [1905] demonstrated the presence of phosphorus (0-07 to 0-08 %)
in euglobulin (ox-serum) and considered it to be one of the characteristics
marking off this protein as a chemical entity. The pseudo-globulin (separated
from the serum by saturation with magnesium sulphate) he found to contain
a trace only (about 0-009 %) of phosphorus. Haslam [1913] also found
phosphorus to be a constant constituent of euglobulin (ox-serum), even after
repeated purification, and to be absent from the purest samples of pseudo-
globulin which he was able to obtain. In consequence, he used the absence
of phosphorus as a criterion of the purity of pseudo-globulin.

In the present case phosphorus was determined by Neumann's method,
using N/10 alkali and acid for the final titration, the quantity to be estimated
being very small. In some cases where the ammonium molybdate pre-
cipitate was only just visible and the total amount of standard sodium
hydrate neutralised less than 1 cc, the values obtained must be regarded
as approximate only ; such values are indicated in the tables by an
asterisk.

The results of the phosphorus estimations showed the resemblance between



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a CHICK 413

the denaturated pseudo-globulin and euglobulin to be maintained in this
respect also. The former was found to remove almost the whole of the small
amount of phosphorus contained in the original pseudo-globulin preparation.
In Exp. I, Table I, the water-insoluble protein at the close of the experiment
contained nearly 10 times, in Exp. Ill, Table II, more than 20 times, as
much phosphorus as the unchanged pseudo-globulin ; in both cases that
present in the latter was reduced to a mere trace. In Exp. IV the contrast
was even greater ; the denaturated pseudo-globulin contained 0'16 % phos-
^phorus, while that remaining with the soluble protein was too small in amount
to be estimated with any degree of accuracy.

Two different explanations might be advanced to resume these facts.
According to the first, the process described in the last paragraph is merely
the separation of the last traces of phosphorus- containing euglobulin derived
from the original serum and present from the beginning. On this theory the
cessation of the gradual precipitation of insoluble protein would naturally
coincide with the disappearance of the phosphorus originally present in the
solution. On the other hand, we may regard this precipitate containing
protein and phosphorus, which gradually makes its appearance, as the product
of a gradual '' denaturation " of the pseudo-globulin which requires the
co-operation of some phosphorus-containing body (probably a serum-lipoid)
which is present in small quantity in the preparations employed. When
this serum-lipoid is exhausted, the process is at an end.

The second explanation would appear to be the correct one for the following
reason. After the removal of phosphorus from the pseudo-globulin solution,
by the separation of the insoluble protein precipitate, addition of a weak
emulsion of lecithin, in presence of salt, causes additional formation of a
protein resembling euglobulin which can be precipitated on subsequent
dialysis.

Handovsky and Wagner [1911] showed that, when emulsions of lecithin,
and other lipoids extracted from serum, were added to dialysed serum, a
precipitation of the protein took place, which was prevented if salts were
present. I have been able to confirm these observations. Addition of a
dilute lecithin emulsion produces only slight turbidity when added to solutions
of pure egg- or serum-albumin, a fact also noticed by Handovsky and Wagner.
In the case of a dialysed pseudo-globulin, an immediate precipitation
of protein takes place, which is prevented if a small concentration of salt
(1 % sodium chloride), acid, or alkali is present.

I have prepared artificial euglobulin from pseudo-globulin in the following



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414 H. CHICK

manner. A small amount of a watery emulsion of lecithin ^ was added to the
salt-free material obtained at the close of Exps. Ill and IV, in which the
denaturation process had ceased and from which the insoluble protein had
been separated. An immediate precipitate occurred, which was prevented
by the presence of alkali in minute proportion or of salt (NaCl), to a con-
centration of about 1 %. On subjecting the solution to dialysis for 14 days
precipitation occurred of a protein, containing phosphorus, and with the
characteristics of euglobulin.


Suggestions as to the origin of euglobulin in serum.

On consideration of the results of the foregoing experiments it seems not
improbable that euglobulin in serum is a complex material, formed from
pseudo-globuHn by association with some serum-lipoid, to the presence of
which it owes its phosphorus-content.

Hardy [1905], on the other hand, has regarded phosphorus as an
integral part of the euglobuHn molecule. He [1905, p. 331] was unable
to remove all the phosphorus from heat-coagulated euglobulin (ox-serum)
by treatment with strong acetic acid and subsequent extraction with alcohol
and ether. He therefore concluded that it could not be " due to entangled
lecithin." Haslam [1913, p. 514] found that extraction of euglobulin (ox-
serum) with boihng alcohol removed a yellow, fatty substance which con-
tained phosphorus, the proportion of the latter remaining in the euglobulin
being reduced to about one-half the original. It is, however, quite
possible that complete extraction with alcohol or ether is rendered very
difficult by the state of aggregation of the protein and that if fresh surfaces
could in succession be exposed to the action of these solvents a larger propor-
tion of phosphorus could be removed.

In this connection it is worthy of note that the available analyses of
euglobulin, some of which are collected in Table IV, show no approach to
constancy in the proportion of phosphorus present. Hardy found 0-07 to
0-08 % phosphorus in euglobuhn prepared from ox-serum by dilution and
acidification; Haslam found 0-108% and 0-105% respectively present in
two different samples. In three specimens prepared by the same method
from three separate samples of horse-serum, I found 0-12 %, 0*032 %, and
0-065 % phosphorus respectively.

* For this preparation I am indebted to the kindness of Dr H. Maclean. The lecithin was
prepared by his own method [1914] from heart muscle.



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H. CHICK



4\i



TABLE IV.

Phos'phorus-content of eughlubin.



Dosoription of material and mode of
Na preparation from serum



1. Euglobulin

2. Euglobulin

3. Euglobulin
(horse-serum A)

4. Euglobulin
(horse-sorum A)

5. Euglobulin
(horse-serum A)



6. Euglobulin
(horse-serum B)

7. Euglobulin
(horse-scrum C)



(ox-serum) by dilution and
acidification

(ox -serum) by dilution and
acidification

by dilution and acidification
once re-precipitated and
washed

after three subsequent re-
precipitations
as (4), but after extraction
of the euglobulin suspen-
sion with acetone followed
by dry ether

by dilution and acidifica-
tion, twice re- precipitated
by dilution and acidifica-
tion, four times reprecipi-
tated



Method
Neumann
Carius
Neumann

Neumann
Neumann



G. phosphorus

per 100 grms.

protein



Oil I

Oil j

0-07 )

008 f

0122)
01171

0-092)
0096)



Oil



0-075



0119



' 0-094



0-020 }<^*^20



Neumann 0033 )
0032 f



0-032



Neumann



n^ol 0-065
00621



Authority

Haslam
Hardy

Chick

CJhick

Chick

Chick
Cliick



♦ Less than 1 cc. of N/10 NaOH neutralised.



TABLE V.



Propartian of phofspkorus contained in a sample of whole horse-serum and
in the various proteins after separation by different methods.



No.



Material



1. Horse-serum ; whole serum dried in vacuo at 37°

2. Total proteins of horse-serum, precipitated by dropping into

a large volume of absolute alcohol ; washed with alcohol
and ether ; and dried at 105°

3. Total proteins coagulated in hot water, coagulum washed

with water, alcohol and ether; dried at 105"

4. Euglobulin prepared from No. 6 by one- third saturation with

ammonium sulphate (re-precipitated six times)

5. Euglobulin prepared by dilution and acidification of the

original serum (re-precipitated four times and washed twice
with distilled water)

6. Total globulin, prepared by one-half saturation with am-

monium sulphate (re-procipitated six times)

7. Serum albumin precipitated in the filtrate from No. 6 by satu-

ration with ammonium sulphate (ro-precipitatcd five times)



G. P. per
100 g.
protein

0098 1
01031

0-027 )
001 8* )

0-004*1
0-008*1

0071

0-069 ^
0062 »



0023
0-032



Mean
100

0027

0-006*

0071
0065

0023
0032



* Approximate only, less than 1 cc. N/10 NaOH neutralised.



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416 H. CHICK

A further set of analyses, see Table V, was made of the total globulin,
and euglobulin of horse-serum, as well as the total protein precipitated
by various methods. The specimens used were all prepared by Dr P.
Hartley with great precautions and carefully purified and I am greatlv in-
debted to him for generously placing this valuable material at my disposal.
The results showed that none was free from phosphorus. In some cases the
proportion contained showed great variation according to the method
employed in preparation {e.g. nos. 1, 2, and 3, Table V). The euglobulin
contained the greatest amount of phosphorus, and the content was about the
same whether precipitated with ammonium sulphate or by dilution and
acidification of the serum. This would indicate that the phosphorus contained
in euglobulin was in closer association than the traces found present in the
purified samples of the other proteins.

Any direct evidence regarding the character of the union of phosphorus
in euglobulin is at present scanty, but the following results are worthy of
consideration. Haslam [1913] was able to remove about half the total
phosphorus from a sample of euglobulin by means of extraction with alcohol
and ether. Absolute alcohol must, however, in case of proteins be regarded
as a powerful reagent, causing serious changes ; all are rendered permanently
insoluble if contact is long enough and the temperature is allowed to rise above
a low maximum. If previous to extraction with ether, acetone^ in place of
alcohol, is employed to remove the water, no damage appears to be suffered.

The material can then be extracted with ether, three or four changes being
employed to remove completely all acetone, and the euglobulin remaining in
contact with ether at room temperature for one or two days. The last ether
extract being removed, the euglobulin is shaken up once more with ether,
which is evaporated by pouring the euglobulin suspension on to a warmed
porcelain basin and allowing a gentle current of warm air to play over it, the
whole operation taking place in a hot room (about 36°). The extracted
euglobulin is then obtained as a fine, white powder, which is exceedingly
hygroscopic. The solvents employed should all be carefully purified and
freed from water.

The extracted euglobulin was found to have lost nearly all its phosphorus ;
on one occasion it was reduced to less than one-quarter of the original (see
Table V, original phosphorus-content 0*094 % ; final 0-02 %). It is possible
that the care taken to free all materials from water may account for the



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