PRECIPITATION OF SERUM-ALBUMIN AND GLUTIN

BY ALKALOIDAL REAGENTS.
BY PAUL J. HANZLIK.
(From the Laboratory of Physicochemical Biology, University of Vienna.
Director: Prof. Wolfgang Pauli.)

(Received for publication, November 1, 1914.)

Downloaded from http://www.jbc.org/ by guest on May 9, 2019

The study of the mechanism of the precipitation of proteins by
so called alkaloidal reagents (tannin, iodine potassium iodide, po-
tassium ferrocyanide, potassium mercuric iodide, tungstate, and
phosphomolybdate) has received practically no attention from the
standpoint of physical chemistry, especially the isoelectric point.
According to Michaelis,1 the isoelectric point of serum is the point
where the number of positive and negative charges of electricity
held by the protein ions are about equal; that is, at this point
there is a maximum of neutral particles of protein. This lies ap-
proximately at the concentration of 2. 10d of acid, such as ob-
tains in a mixture of equal parts (+) of & acetic acid and G
sodium acetate. Certain other phenomena which are exhibited by
proteins at this point are, according to Pauli and his pupils, a
minimum of viscosity as shown by serum and a maximum of pre-
cipitation by ethyl alcohol. It would be interesting to know the
behavior of other precipitants, such as the alkaloidal reagents,
with respect to this point. It might also indicate whether the
mechanism of precipitation by all alkaloidal reagents is the same
or different. This has hitherto been regarded as identical.
In the following experiments attempts have been made to as-
certain where precipitation of serum and glutin takes place with
respect to their isoelectric points, and how it is influenced by differ-
ent degrees of acidity and the addition of salt. The conditions
under which precipitation was observed have been expressed in a
quantitative manner; that is, definite quantities of serum and
glutin of known protein content were mixed with definite quanti-
1 L. Michaelis and B. Mostunski: Biochem. Ztschr., xxiv, p. 79, 1910.
2 W. Pauli: Ztschr. j. Chem. u. Ind. d. Koll., xii, p. 222, 1913.
13
 14 Precipitation of Serum-Albumin and Glutin
ties of known strengths of acids, and to this, the same quantity
of the reagent of known strength was added. The total volume
of the mixture was always the same. The effect of salt was studied
in a similar manner.
All experiments were performed at ordinary room temperature
in test-tubes. The precipitates were observed when. freshly
formed and at the end of twenty-four hours. However, it is to
be noted that, as a rule, the precipitates remained unchanged on
standing, and the data have been compiled without reference to
t,his factor. Precipitation was regarded to have taken place when
6he mixture upon the addition of the reagent became non-trans-
parent. The different degrees of precipitation have been expressed

Downloaded from http://www.jbc.org/ by guest on May 9, 2019

TABLE I.*

Degree of acidity represented by acetic-acetate (“bufer”) mixtures.

A I ‘ib
-I : o.o;.i-.
IH+l
PIi
1o.45;;w
1o.22;;Fs
1,zT+
0.27 X lo-4t
4.57
1.8 X 10-S
’ 4.74

*In the tables, the various signs and abbreviations used have meanings as follows: the plus
sign (+) = precipitate present; the minus sign (-) = no precipitate; sl = slight; st = strong;
m&y = maximum; (?) = precipitate doubtful; tr = trace. The order in which the solutions
were 6xed and the quantities used are appended aa footnotes to each table.
t Calculated.

in ordinary terms as indicated in the various tables. The reagents

were previously rendered neutral to litmus by the addition of
sodium hydroxide, and the strengths of these were usually 5 per
cent. The horse serum and glutin used in these experiments had
been previously dialyzed for a period of five to six weeks, and were
practically salt-free. The end concentration represented in the
different experiments will be found appended to each table. The
standard “buffer” mixtures of acetic acid and sodium acetate were
prepared according to Sorensen,3 and the degrees of acidity that.
t,hey represent are shown in Table I. In my work the concen-
3 Sorenson: Ergebn. d. Physiol., xii, p. 393, 1912.
 Paul J. Hanzlik 15
tration of sodium acetate in all experiments is always the same,
only the variation in the acetic acid being present. The different
concentrations of acetic and hydrochloric acids were prepared by
dilution of standard solutions in the usual manner, and the con-
centrations of these are indicated in the various tables.

1. Precipitation of serum and glutin in mixtures of acetic acid and

sodium acetate.

In mixtures of acetic acid and sodium acetate with proteins,

the dissociated hydrogen ions are not bound by the protein, but
TABLE II.

Downloaded from http://www.jbc.org/ by guest on May 9, 2019

Precipitation
-
of serum and glutin by alkaloidal
acetic-acetate mixtures.
Pcm.4ss1UY 7
reagents in the

Is
MEBCURIC POTASSIUM

IODIDE,
iPER
CENT
BEIlRO-
CY.&.nDE,
5 PER CENl
TANNn-4,
1 PER CENT

--
-
S i
_‘-
s lG - _- I
-I-
G S
_- __
S G ST G

I- ++ ++Sl+ ++ stleasst + + st
++81
+
+
f
+ :+81
:: /+
+ least
+
+
f
+ st
+
+ -tsl :
+

-
++q+
/+ +-I-?
I -
-
msx
+ 81
+St +

+81 -
+ sl
-
Z?
+ st
+

- - - - --
- L - I L
“S” refers to horse serum; “G” to glutin. Order of mixing solutions: aceticgcetate
mixture 1 cc.. water 1 CO., reagent 1 drop and serum (0.5 per cent) orglutin (0.5 per cent) 1 drop.

are made available for the precipitation reaction with the alka-
loidal reagents. It is thus possible to ascertain if precipitation is
concerned (1) with neutral particles of protein, (2) with positive
protein ions, or (3) whether it depends upon the liberation of the
free acid of the reagents. With this in view, experiments with
horse serum and glutin were made according to the method de-
scribed above, the details of which are appended to the results pre-
sented in Table II.
From this it is to be seen that the precipitation of serum by the
following reagents, iodine potassium iodide, potassium mercuric
iodide, potassium ferrocyanide, sodium tungstate, and phos-
16 Precipitation of Serum-Albumin and Glutin
phomolybdate, takes place above the isoelectric point, and in the
direction of higher concentrations of acetic acid, and that it in-
creases as the acidity increases. Precipitation by the different
reagents begins practically with the same mixture of acid and
acetate, and the small variations which occur are probably within
experimental error. Inasmuch as the dissociated acid is not
bound to the protein ion under these conditions, it would seem to
indicate that a certain amount of free and excess of acid is neces-
sary for the formation of the insoluble protein compounds.
On the other hand, with tannin, the maximum of precipitation
occurs at about the isoelectric point, and then diminishes on either
side of it; that is, with either increased or decreased concen-

Downloaded from http://www.jbc.org/ by guest on May 9, 2019

trations of free acid. This would indicate that the mechanism
of precipitation by tannin is different from that of the other alka-
loidal reagents used.
With glutin, precipitation takes practically the same course as
with serum. No precipitation was observed with iodine and po-
tassium iodide and the ferrocyanide. Tannin exhibits practically
the same differences from the other reagents as with serum.

2. Precipitation of serum and glutin by alcohols in mixtures of acetic

acid and acetate.

From the preceding section it appears that the mechanism of

the precipitation of proteins by tannin is different from that with
the other reagents. It is possible that the mechanism is similar
to that of certain alcohols; for ethyl alcohol also produces a max-
imum of precipitation at the isoelectric point. This analogy was
tested out by observing the precipitation of serum and glutin by
different alcohols with respect to the isoelectric point. The dat.a
are presented in Table III.
It is seen that the maximum of precipitation of serum by t,he
different alcohols used occurs at about the isoelectric point; i.e.,
with mixtures of one to two (3) and one to four (2) parts of
acetic acid and acetate, respectively. With the exception of
propyl alcohol, the precipitates diminish in intensity on either side
of this point. There appeared to be no ‘difference in the pre-
cipitates produced by propyl alcohol. It is also to be noted that
 Paul J. Hanzlik 17
TABLE III.

Precipitation of serum bu alcohols in the acetic-acetate mixtures.

+4 ...................... - - - I -
f ...................... - - - -
9 ...................... - - - -
l-51 ....................
+? +?
+ ...................... + + + tr
+ ...................... + st + St + St
+ ....................... + st + St + less st + st
+ ...................... + less st + less st + still less + St
st
iq1 . . . . . . . . . . . . . + still less + least st

Downloaded from http://www.jbc.org/ by guest on May 9, 2019

.., ... -I- tr + St
st
Serum and water (equal
parts). . . . . . . + least st + least st + tr
-/
Order of mixing solutions: horse emurn (0.5 per cen?J 1 cc., acetic-acetate mixture 1.cc., and
alcoho! reagent 1 00.

serum alone without the acid-acetate mixture gave slight precip-

itates with all alcohols except propyl alcohol. The data strongly
indicate that the mechanism of precipitation by tannin more
closely resembles the alcohols than the other alkaloidal reagents.
The results obtained with glutin (Table IV) lead to practically,
the same conclusions.
TABLE IV.

Precipitation of glutin by alcohols in mixtures of acetic acid and acetate.

f ...................... - + less st - +
i ...................... + + St + -I-
7i . . . . + + St + +
*. . . .. - + v. sl -
iI. . ..... .
m . ... . . . . . . . . . . . . . . .. .1 1 j - I
Order of mixing mlutiom: glutin (0.5 per cent) 0.5 cc., acetic-acetate mixture 0.5 CC., water
1 cc.. and alcohol 2 cc.
18 Precipitation of Serum-Albumin and Glutin

3. Precipitation of serum and glutin in acids.

Thus far it is seen that the precipitation of serum and glutin

by the various reagents occurs when acid protein is present; that
is, it takes place in the presence of a concentration of hydrogen
ions higher than the isoelectric point, and, apparently, is aug-
mented when these are in excess. However, it still remains to be
shown whether precipitation depends upon the liberation of the
acid of the reagent, or the formation of protein salts with added
acid and the reaction of these with the reagent.
According to Pauli, the dissociated hydrogen ions of such acids
as acetic or hydrochloric are bound by protein with the formation
of dissociated protein ions with positive electric charges. The

Downloaded from http://www.jbc.org/ by guest on May 9, 2019

number of these charges increases with the addition of more acid
until all of the protein is, saturated. This occurs even in the lowest
concentrations of acid. Such bound hydrogen ions do not become
available for other reactions unless the quantity of protein remains
constant and an excess of acid is added. If, then, precipitation
in a mixture of protein and acid by a reagent occurs only during
the phase of excess acid and not when protein ions only are pres-
ent, the conclusion must be drawn that precipitation depends upon
the presence of free acid and not upon dissociated protein ions
alone; that is, the precipitate is a combination of the free acid of
the salt with the protein or acid protein. This, indeed, is the case
with iodine potassium iodide, mercuric iodide, ferrocyanide, tung-
state, and phosphomolybdate, and to a certain extent with tannin.
The experiments were performed by adding small and constant
quantities of serum and glutin to different concentrations of acetic
and hydrochloric acids and noting where precipitation occurred
by the further addition of constant quantities of different reagents.
The data obtained, as well as the details in which the experiments
were performed, are presented in Table V.
It is seen that in the lowest concentrations of both acids, where
dissociated protein ions occur, no precipitation of serum with any
of the reagents took place. Precipitation occurred only with the
higher concentrations; that is, in the presence of an excess of acid.
The beginning of precipitation with the different reagents varies,
and small but negligible variations are to be noted in the same
reagent with different acids. With tannin, a maximum of pre-
 Paul J. Hanzlik I9
TABLE V.

Precipitation of serum and glutin by alkaloidal reaaents in dilute acids.

-- -- -I-- I- I-.
- --
5.10-6
- - -
7.5.1w
-- - -
- I-

Downloaded from http://www.jbc.org/ by guest on May 9, 2019

fsl -
lO.lO-5
1.5.10-r +? -
:+ +++- - -
-
-
-
+
+st
+?
+
2.5.1W
4.10-r
+ 81
;:
+ 81
+ 81
+ ++
+
-
-
-
-
+ St
+1ess st
-k?
+ ?
5.10-4
10.10-C
-
+ 1st ++ ++ + 81
- fsl +1essst -
1.25.10-1
2.5.103
5.10-r
-
-
:f, ++++ +
+
-+s1+
+s1 +1essst
+ + St
+
+kast
-
-
-

7.5.10-s - -- -
lO.lO-6 - - -- - -
1.5.10-r - -- -
2.5.10-r - - f- - - +
5.10-4 + - - _- +s1 +
lO.lW -. + : - - _- - +more +
1.25.10-s + f- fsl - - + still +
more
2.5.10-Z + + +s1 +I31 - - +.&II +
more
5.1w + + +s1 fmore - +s1 + sl + sl +

Order of mixing ~011Iti ions: acid I cc., water 1 cc., glutin or home serum (0.5 per Cent)
1 CC., and reagent 1 drop.

cipitation in both acids occurred at the concentration of 2.5.10 -4.

In the higher concentrations the precipitates dissolved. This was
also true of iodine and potassium iodide, which at no time gave
absolutely distinct precipitates. This phenomenon perhaps de-
pends upon the formation of soluble acid salts and requires further
investigation.
With glutin, practically the same results were obtained as with
serum.
20 Precipitation of Serum:Albumin and Glutin
It is to be concluded that the precipitation of serum and glutin
in hydrochloric and acetic acids by iodine potassium iodide, mer-
curic iodide, ferrocyanide, tungstate, and phosphomolybdate, takes
place only in the presence of an excess of free acid and depends
upon the formation of insoluble compounds with the free acid
of the salt.

4. The e$ect of salts on the prec&tation of serum and glutin by

alkaloidal reagents.

The addition of salt to an acid might conceivably alter the pre-

cipitation reaction by alkaloidal reagents and alcohols. This was
studied in the following manner: To constant and definite con-

Downloaded from http://www.jbc.org/ by guest on May 9, 2019

centrations of glutin and serum in the same volumes of acetic and
hydrochloric acids of different concentrations were added con-
stant volumes of potassium chloride and potassium sulphocyanide,
and finally to this mixture constant and definite quantities of the
reagents. Each series of experiments with the different salts was
compared with a series without salt; that is, with distilled water
as a blank. The results from all of the experiments are to be
found in Tables VI, VII, and VIII.
From these data it is seen that the two salts, chloride and sul-
phocyanide, exerted practically no influence upon the formation
of the precipitate by the various reagents used. Practically no
precipitation occurred in any ca.seuntil the concentration of 4. 10m4
was reached. The small variations which occurred fall within
the experimental error. This was true of both serum and glutin.
With tannin, precipitation in the presence of the chloride and
sulphocyanide occurred in the lowest concentrations of the hydro-
chloric acid and before 4.10 -4. Thus a small difference from the
other reagents is indicated.
With alcohols, the addition of salts also had practically no in-
fluence upon the precipitation. The slight variations exhibited
by the sulphocyanide are too small to permit the drawing of any
conclusions. The same can be said of glutin in acetic acid-ace-
tate mixtures (Table IX).
 TABLE VI.
Effect of salts on the precipitation of serum by alkaloidal reagents.
BODIUY PHOS-
- TANNIN,
I PxKn!“NGsT*TE,
1 PER CENT
5 PER CENT I
- --- --- -
KC1 :scn RIO :sm I HZ0 KC1 ;SCN KC1 CSCN Hz0 KC1 ;SCN Hz0 1KC1 3 CSCN
-.- - HZ0 .- _
/I-KC1 - -
-i.- - -
- - - + 81 + +
- + - - - - - - + + +
2.5.10-h - - - + - - + - - + St + +
4.10-r +* + + sl + +? - + St + st
5.10-4 + 8’ + + *I + : + : + sl - + 81 + *l + less :*I + St
st
10.10-4 + +? + + *t + + I + + sl + sl + + + still - +
less 8 t
1.25.1~3 + + + + + + + 4-T + + + + still +
~.
less s t !
2.5.10-3 - + + + + + + + + + -t’ + + still - I
z lee.78 t
5.10-a - - + sl + + + + i- -+ + st + st + *t + leas t - -
st
.- -- _-
D
WCETIC *a I-
-_ --
7.5.10-s - - -
- - - -
10.10-S + 51
1.5.10-4 - - - - - -
-t? + + sl
2.5.10-d + *I : + - + - -
+ st i- -
- - -
4.1cw + + + St + -t-
5.lcP’ :; - + + 1 - - + leas + +
st
- - -
lO.llv + *t + + i + + + + less + + st
st
1.25.10-3 - + + + + sl +s + st
+ I + + 8’
2.5.10-s - -L? + : + i + + s’ +* 3? : 181 +
5.lck3 - - + + + 1 + + +S I- : + 81 -I-? + sl
-L i I - -
Order of mixing solutions: acid 1 cc.. $, salt solution (KC1 or KSCN) 1 cc., horse serum (0.5 per cent) 1 drop. and reagent 1 drop.

Downloaded from http://www.jbc.org/ by guest on May 9, 2019

 TABLE VII.
Effect of salts on the precipitation of utin bv alkaloidal reagents.

KC1 --I KC1 1SCN KCI KC1 KC1 Ha0 KC1 KSCN

2.10-a
1 .lOW
++
2.10-s
7.5.10-G :
10.10-S - -
1.5.10-4 -
2.5.104
5.10-a -
+1+
10.10-r -
1.25.10-8 + 81 - -
++
k 2.5.10-s + 81 + 81
5.10-a - - + + sl -I? + + 81 + sl 4-1
++?
-- _-
-- .-
2.10-s - - -
1.10-S - -
2.104 - - -
7.5.10-6 - - - -
10.10-6 - -
1.5.1w - - - - -
2.5.10-a - - -
5.10-4 - - - -
lO.lO- 4 - - - -
1.25.10-s - + 81 - -
2.5.10-a + 81 + 81 + - - -
5.10-1 - + 81 + 81 + + 81 + sl + sl
- -I -!- - __
Order of mixing eolutions: acid 1 cc., water or +? salt (KC1 or KSCN) solution 1 cc., glutin (0.5 per cent) 1 drop, and reagent 1 drop.

Downloaded from http://www.jbc.org/ by guest on May 9, 2019

 Paul J. Hanzlik

TABLE VIII.

Effect of salts on the precipitation of serum and glutin by alcohols.

-i-
END
CONcENTRdTION
i ‘I I:
cm HCl
8
3 8
P
1.25.W’ - i - -
0.2.10-4 - - - +a1
0.3.10-r - - - +a1
0.4.1~’ +s1 - + sl f? + 81 fsl -
1.25.10-4 + +st - + St + + St i-at -
0.62.10-a +a1 + - + -
-
- f? -
1.25.10-a
-
-
-
+
- -
+- -
-
-
-
-
0.62.W*
1.25.10-2 - - - - - - -

Downloaded from http://www.jbc.org/ by guest on May 9, 2019

0.62.10-’ - - - - - - -
1.25.W-1 - - - - - - -
_~
Acmxc ACID

0.5.10-4 -- -
1.25.10-r + st -
0.62.10-8
1.25.1W
+ sl +? ++ 81 ::i
+

0.62.10-*
1.25.10-o
+
- :1
+-
- -
-
-

0.62.lW -
1.25.10-’ - -
order of mixing mlutions: acid 0.5 cc.. glutin (0.5 per cent) 0.5 cc., water or N/5 ssk
(KC1 or KSCN) solution 1 cc., and alcohol reagent 2 cc.

TABLE IX.

E$ect of sulphoqanide on the precipitation of glutin by alcohols in acetic-

acetate mixtures.

N/10 *.CETIC *CID BESORCIN,~ PHENOL, HYDROOUWONE, PBOPYL

5 PEB CENT 5 PER CENT 5 PER CENT d‘coHOL
N/10 SODImd *cJvlwm Hz0 KSCN Ha0 KSCN Hz0 KSCN I&O KSCN
_________~~---
t . . . . . . . . . . . . . . . . . . . . . . +s1 +st - + - + + -
i .... . . . . . . . . . ... ... + st + St’ + st + + st + sl + st + ?
+. .. . . + st + sl + st - - - +st +?
* . . . . . . . . .._.__........ +s1 - - - - - +
+,.. . - . - - - - - - 1;
$+i . . . . . . . . . . .._...._.. - - - - - - - +?

Order of mixing solutions: 2 per cent glutin (1:4) 0.5 DC., acetic-acetate mixture 0.5 cc., :
KSCN 1 OC,, and alcohol 2 cc. The end concentration of KSCN is &; of glutin 0.5 per cent.
24 Precipitation of Serum-Albumin and Glutin

5. E$ect of concentration of protein on the precipitation.

This was tested out by using two different concentrations of
serum, 0.017 per cent and 1 per cent (as end concentrations of
protein) in constant volumes of hydrochloric acid of different
strengths and precipitated by the further addition of one drop
of t,he reagent. The data have been placed in Table X.
As the results with the different concentrations of serum are
practically identical, it is to be concluded that wide differences
in protein content have no marked influence on the precipitation.
TARLEl X.
Ejtfect of concentration of serum-albumin on the precipitation by
_-

Downloaded from http://www.jbc.org/ by guest on May 9, 2019

I

T i
.017 1 0.017 1
%, % % %
I
I -- i- _.
7.5.10;s - - -- - /- -
-- _ - I- -
1O.W-’
1.5.10-4 - - ++ - -
2.5j.10-4 - ++ - - -
-
5.104 + + sl + +s1 - I-
ld.iW + + + +st - i- + 81
1.3.10-3
2.5.10-a
5.104
+
+
- ”
+
+
+st
+ st
j+st
+s1!-
+ sl + sl
+st~+sy ,I + 81
+st
+ st max
+ 81
+
+

Order of mixing solutions: horse serum 1 cc., acid 1 cc., and reagent 1 drop. The per-
centages under “serum-albumin” represent end concentrations of albumin.

SUMMARY.

The mechanism of the precipitation of dialyzed horse serum and

glutin,by tannin is different from that of certain precipitants com-
monly known ?s “alkaloidal reagents.” With these a certain
amount of free acid (hydrogen ion concentration) is necessary
for bhe formation of the complex insoluble compounds.
Tannin behaves like certain alcohols, e.g., resorcin, phenol,
hydroquinone, and propyl alcohol, since the maximum of precip-
itation in both cases corresponds to the isoelectric point in serum-
albumin and glutin.
Precipitation of serum is uninfluenced by wide differences of
concentration and the addition of such neutral salts as chloride
and sulphocyanide.
PRECIPITATION OF SERUM-ALBUMIN
AND GLUTIN BY ALKALOIDAL
REAGENTS
Paul J. Hanzlik
J. Biol. Chem. 1915, 20:13-24.

Access the most updated version of this article at

http://www.jbc.org/content/20/1/13.citation

Downloaded from http://www.jbc.org/ by guest on May 9, 2019

Alerts:
• When this article is cited
• When a correction for this article is posted

Click here to choose from all of JBC's e-mail

alerts

This article cites 0 references, 0 of which can be

accessed free at
http://www.jbc.org/content/20/1/13.citation.full.html
#ref-list-1