Isolation
of Albumin from Human Serum by Means
of Trichioroacetic
Acid and Ethanol
A Comparison of Methods
Takuzo lwata,*
Hiromi Iwata,*
and James F. Holland
A rapid method for nearly quantitative isolation of human serum albumin is described.
The present technic (1) is a modification of the Schwert method (2). In the present
method 4 ml. of serum is precipitated with trichloroacetic acid in a final concentration
of 5% (w/v). The precipitate is washed with TCA, and after aqueous suspension,
ethanol is added to a final concentration of 80%. The undissolved globulin is washed
with ethanol and the solubilized albumin used for quantification, characterization,
and isotope counting.
Recovery of added human serum albumin was approximately 98%; recovery of
added 1311 albumin was 90%.
Cellulose acetate electrophoresis showed a single band, and agar diffusion produced
one precipitate line. Immunoelectrophoresis with anti-human serum revealed one or
two very faint arcs in the a-, or a- and ?-globulin regions, in addition to the major
arc of albumin.
The method has shown excellent reproducibility in replicate analyses of normal and
pathologic serums.
S
led us to search for a rapid technic
of isolation.
An additional
requirement
was that the separated
albumin
be available
in sufficient quantity
to detect isotope incorporation
despite
low doses of isotopic
amino acids.
The latter requirement
excluded
elect rophoretic
separation
technics
which
might
otherwise
have
been con sidered,
i.e., immunoelectrophoresis
(3) and acrylamide
disc electrophoresis
(4).
Starch
gel and
zone electrophoresis
did not produce
adequate
separation
of albumin
TUDIES
OF ALBUMIN
BIOSYNThESIS
From the Department
of Medicine
A, Roswell Park Memorial
Tiistitute,
Buffalo,
N. Y. 14203.
Supported
by U. S. Public
Health
Service
Researchm Grant C-5834 from the National
Cancer
Institute
and Grant T-231 from time American
Cancer Society.
Presented
at the 17th Annual
Meeting
of The American
Association
of Clinical
Chemists,
Chicago,
III., August
1965.
The authors
are indebted
to Dr. George
L. Tritsch
who performed
the microzone
electrophoresis
using a cellulose
acetate
membrane.
Received
for publication
Dec. 2, 1965; accepted
for publication
July 17, 1967.
*present
address:
Aiehi Cancer Center, Nagoya,
Japan.
22
Vol. 14. No. I, 1968
23
ISOLATION OF ALBUMIN
from
other serum protein
components.
Fractionation
of human
serum
by cold ethanol
was found unsatisfactory
because
of the time
involved
for multiple
analyses
within
the first hour after
isotope
administration
and because
of the volume
of serum required.
protein
We have tested
various
isolation
technics
which make use of the
solubilizatioll
of albumin
in ethanol
when the protein
is 1)OUIidl to au
anion. Trichloroacetic
acid (TCA)
has long been used as a proteinprecipitating
agent. In the preeiie
of certain
organic
solvents,
however, the TCA-alhumnin
complex
is soluble. Race (5) used SO7e acetone
containing
TCA to separate
albumin
from globulin
in serum.
Levine
(6)
observed
that TCA-precipitated
bovine albumin
is readily
soluble
in ethanol
and other organic
solvents,
and he called attention
to possible albumin
losses during procedures
for defatting
tissues
after TCA
precipitation.
Korner
and Debro (7) extended
these observatiouis
and
demonstrated
that precipitated
albumin
is soluble in etilallol if a small
amount of acid is preselit.
Schwert
(2)
found that tile I)rotein recovered
from
TCA-ethanol
fractionation
is identical
with native
protein
in
regard
to sedimentation
and electrophoretic
behavior,
soluhility,
and
crystallization
characteristics.
He reported
that the TCA precipitate
dissolves
niore readily when the organic
solvent
is added if the precipitate is suspended
ill a small
volume
ot water.
I)elaville
et al. (8) and
Foster
(9)
added
TCA-ethanol
simultaneously,
separating
globulin
from ahl)umin
in OIIC step.
Recently,
Rodkev
(10, ii)
described
a simple
method
for a quantitative
separation
of globulin
from
albumin.
using
this approach,
he was able to precipitate
albumin
from
the TCA-ethanol
with sodium acetate.
Hradec
(12)
had described
quantitative
precipitation
of albumihl
from
TCA-ethanol
by diethyl
ether.
Tile present
report
describes
the optimal
coliditions
we have
found
for a quantitative
extraction
of albumin
from serum by ethanolic
TCA.
Efficiency of the separation
has been checked by recovery
of added 131J
labeled
albumin
and added
human
serum
albumin.
In addition,
the
purity
and
phoretically
identity
of the
isolated
albumin
has
been
studied
electro-
and immunologically.
Materials and Methods
Normal
human
serum
was separated
by centrifugation
from the
clotted blood of healthy
adults. Human
serum albumin
was obtained
as
the 25% solution from Cutter Laboratories.
It contained
0.02 M sodium
caprylate
and 0.02 M sodium acetyltryptophanate.
Rabbit,
horse, and
goat antiserum
against
human serum and human
serum albumin
were
obtained
from Hyland
Laboratories.
24
IWATA ET AL.
Clinical
Chemistry
1311-labeled human
serum
albumin
(RIHSA)
was purchased
from
Abbott Laboratories
or from Sorin, Saluggia,
Italy. It had a specific
activity
of either
55.2 or 80 c./mg.
protein,
respectively.
131J
was
counted in a well scintillation
counter
with a 2-in, crystal
to a standard
deviation
of less than 2%.
Microzone
electrophoresis
using a cellulose
acetate
membrane
in a
Beckman
cell (Model RiOl)
was done at room temperature
in barbital
buffer
(pH 8.6, 0.075 ionic strength)
at 250 v for 20 miii. Agar
diffusion
analysis
was done on a microscopic
slide at room temperature
in 1.5%
agar
(13). Immunoelectrophoresis
using an LKB instrument
was performed at room temperature
under a potential
7-8 v/cm. for 75 mm.
Albumin
separated
from serum by TCA-ethanol
in sonic instances
was dialyzed
in a Visking
membrane
against
two changes
of 1000
volumes
of distilled
water at 4#{176}
for 2 days. A small amount
of protein
precipitated
as a flocculent
cloud during
dialysis
hut iii nearly
every
case redissolved
as dialysis
continued
(7).
The dialyzed
albumin
was
lyophilized
and dissolved
in barbital
buffer (pH 8.6, 0.1 ionic strength).
The TCA used was the 20% (w/v)
solution
from
Fisher
Scientific
Company.
Protein
content
was determined
according
to Lowry
et a!.
(14).
Albumin
was estimated
in some specimens
by methyl orange
(15).
Procedures for Albumin Separation
Modified Schwerf Method
The present method is modified from Korner
and IDebro (7) and from
Schwert
(2). The reaction.
was carried
out ill 23-ml. lusteroid
tubes
in
ice water:
4 ml. of serum was diluted to 15 ml. with distilled
water, and
5 ml. of 20% TCA was added dropwise
from a i)uret, with constant
stirring,
over a period of 2 mm. The glass stirring
rod and the side wall
of the tube were washed
with 5% TCA. The tube was centrifuged
at
23,000 g at 0#{176}
for 5 miii. The precipitate
was washed with 20 ml. of 3%
TCA. The supernatant
fluids were collected
for determination
of acidsoluble isotope.
The packed
precipitate
was first suspended
with 3 ml. of distilled
water using a new glass rod for stirring,
and then was dissolved
in
16 ml. of 95% ethanol.
The glass rod and side wall of the tube were
washed with 80% ethanol-to
a total volume of 20 ml. These steps were
carried
out at 00. After a minimum
of 30 mm., tile undissolved
protein
was centrifuged
at 25,000 g for 20 miii. The clear supernatant
fluid
containing
albumin
was decanted
into a 30-nil, volumetric
flask. The
precipitate
was resuspended
in water
and the same ethanol
extraction
procedure
performed.
This superriatant
fluid was also added
to the
V01. 14,
Jo. I, l96
ISOLATION
50-mi. flask. Volume
was adjusted
with
counting
and foi’ protein determinations
Modified
Delaville
25
OF ALBUMUI
80% ethanol,
were taken.
and
aliqiiots
for
Method
At room temperature,
ethanol
containing
1%
collected
by centrifugation
albumin
was piecipitated
2 nil, of serum were mixed with 20 ml. of 80%
TCA. The supermiatant
and wash
fluids
were
at 1370 g for 20 mm. at 3#{176}.
Ethanol-soluble
with excess TCA.
Rodkey Method
A total of S ml. of 2% TCA iii 85.3% ethanol
was placed
in a small
centrifuge
tul)e. Then 0.2 ml. of seim
was added, the tube was shaken
vigorously,
and was then allowed
to stand at room temperature
with
intermittent
shaking
for 2 hr. C’entrifugation
was done at 800 g for
10 mm. to sediment
the globulin.
Globulin precipitate
was washed again
with 5 ml. of 2% TCA in ethanol.
Albumin
was precipitated
from the
supernatant
fluid and wash solutions
by addition
of 2 moles of sodium
acetate
for each mole of TCA (ii).
Albumin
was thus obtained
as a
white precipitate,
and free isotope was present
in the supernataiit
fluid.
Results and Discussion
RIHSA
migrate
albumin
was
subjected
to
indistinguishably
iii human
serum.
and
immunoelectrophoresis
and
symmetrically
proved
to
when
compare(1
with
was added to human
serum
of tile technic
of separation.
RIHSA
(0.1 sc.)
and its distribution
measured
as a function
In our modification
of the Schwert
method,
the best recovery
of ‘31T
was obtained
when the 80% ethanol
used to solubilize
TCA-precipitated
albumin
was 5-10 times the volume of the original
serum
(Table
1).
Furthermore,
the protein
recovery
was optimal
when the ethanol
Table 1.
RECOVERY
RIHSA
ANI) PROTEIN CONTENT
UsmNc. Mol)IFIEI)
SCHWERT METImOI)
OF
R!HS.4
Serum
(ml.)
*
the
Ethanol,
80 %
(ml.)
FRACTIONATION
Protein
(“
contents
(%)f
------------
TCA
soluble
Ethunol
soluble
Ethanol
mao!.
4.2
2
3
4
5
5
20
20
20
20
10
3.1
4.1
4.3
4.3
4.2
91.4
90.7
89.5
85.9
76.5
5
5
4.3
59.5
Using
AFTER
4.7
6.0
8.7
17.4
.35.l
- --
Total
TCA
soluble
Ethanol
soluble
Ethanol
moo!.
98.7
99.5
99.8
98.9
98.1
0.7
0.6
0.6
0.5
0.6
72.5
67.8
64.0
58.2
49.8
26.8
3L0
35.4
38.5
98.9
0.6
38.7
-
-
-
Total
100.0
99.4
48.6
60.6
100.0
97.2
99.0
999
0.06 ize. of “I.
f Protein
content
of serum
total protein
(15).
was
7.83 gm./lOO
ml. (14) and alhumiim
concentration
was 63.8
of
IWATA El AL.
Clinical
Chemistry
1
4.
2
b
B:
-
-
I
3A
-
Fig. 1. Cellulose
acetate electrophoresis.
Normal
human
serumn
(Specimen
1).
Specimens .3, 3, anti 4 are supernatant
fluids
(A)
and
precipitates
(B)
after
ethamiolic
TCA
fraetionatjoll.
Albumin
concentrations
ill Sped.
Incas
3, 3, and 4 were
50, 78, and 30 mg./ml.,
respectively.
Specimen
5
is
eeau’s
B
4A
4
r
B
.i-
f
commercial
human
serum albumin:
50 mg./
ml. 80%
ethanol
containing
5% TCA. Beckmmmanfixative
dye; Ponstain.
Vol. 14, No. I, 1968
ISOLATION
27
OF ALBUMIN
volume was 5 times that of the original
serum.
These volumes
were
thus adopted
and used in further
studies and comparisons
of the method.
The time allowed
for solubihization
of albumin
in ethanol
was not
critical.
Identical
recoveries
were obtained
at 30 and 120 mm.; lesser
intervals
led to erratic
results.
Within broad limits, the alcohol concentration
was not critical
(Table 2). We conducted
all experiments
in an
ice bath for uniformity.
However,
whether
the alcohol-solubilization
proceeded
at Dry Ice-acetone
bath temperatures
or at room temperature, no difference
was found
in RIHSA
recovery,
immunoelectrophoretic
characteristics,
or albumin
content.
In addition
to RIHSA,
we measured
the recovery
of added human
serum
albumin.
Over
a concentration
ramre
of 43-GO m./ml.
of
albumin,
recovery
was excellent
(Table 3), whereas
it was 93% by the
Delaville
method,
and 90% by Rodkey’s
rapid
method
for isolation
of globulin.
A comparison
of tile present
method with two other teehmcs
of TCAethanol
separation
is shown in Table 4. Albumin
measurement
by the
adopted
technic was satisfactory
when judged by ethanol
soluhilization
Table
2.
EFFECT
OF
ALCOHOL
CONCENTRATION
of
Recovery
Ethanol
cone.
(#{176}%)
TCA
10
25
40
60
80
95
*
Using
0.6pc.
of
3.
13h1;
RECOVERY
A d,ied HSA
(mg)
protein
t
of serum
OF HUMAN
Protein
soluble
SERUM
in ethanol.
fraction #{149}
(my.)
172
172
196
195
48
219
217
266
268
96
content.
Ethanol
insol.
Total
t
93.8
94.1
93.3
94.6
93.6
content
24
Protein
soluble
t
1.0
1.0
1.2
0.9
US
was 7.8 gm./l00
99.0
99.3
98.7
99.8
99.3
ml. (14).
-
fluid after centrifugation.
0
*
Ethanol
4.2
4.2
4.2
4.2
4 .3
4.2
t Cloudy supernatant
Table
soluble
(%)
RINSA
was determined
by Lowry
ALBUMIN
(HSA) ADDED TO 4
ML.
OF SERUM
Recovered
(my.)
Theoretical
recovery
1%)
-
-
-
-
24
23
100
96
47
45
94
96
98
94
98
100
ci a!. method
(14).
Fig. 2. Immumiodiffusion
assays of the TCA-ethanol-extracted
albumin
(B and F, 0.5 mg./
mmil.), precipitated
globulin
(I amid M, 6 mmig./mnl.), and normal human
seruma
(C, E, .1, and L,
.1.0 mg./mmml. ) against
goat amiti-liuman
serumim ( G and N) amid goat ammti-hiiimmm
aim serum albumin
(D and K). Commteimts of holes A amid 11 are 80% ethanol
comitaimmimmg s%
‘CA flS control.
Naphithiol blue black stain.
Fig.
3. Immunoelectrophoresis
agaimist goat anti-human
serum. Normaal human
serum (SpedSpecimen
2 is extracted
albumin
(50 mg./mmml.). Note faint
arc intersecting
arm. Speciniemm 4 is commmmercial humaami serum
albumin:
50 mug/mimI. 8(1% etlnmiiol con5% TCA. Napluthol
blue black stain,
memms 1 amid 3).
trailing
taining
Vol. 14, No. I, 1968
ISOLATION
29
OF ALBUMIN
or recovery
of RIHSA
(Table 4). It is apparent
that sonic globulin
was
solubilized
in the 1)elaville
method.
The extracted
albumin
ni each of time above procedures
(Table 4) was
further
studied
by physical
means.
The extracted
albumin
using the
present
method showed a single band on cellulose
acetate
(Fig. 1) and
Table
4.
RECOVERY
OF
RIHSA
FRACTIONATION
‘UHSA
*
Method
TCA
soluble
Present
I)elaville
Rodkey
4.3
4.2
5.2
Analysis
of the same
human
Ethanol
soluble
89.5
88.Ot
S6.l
serum
ANt)
PROTEIN
BY 1)IFFERENT
CONTENT
AFTER
METH(mDS
Protein
3)
Ethanol
in.sol.
6.0
7.8
7.8
TC.4
soluble
Total
99.8
100.0
99.1
specimuemu a.s in Table
I
Ethanol
soluble
Ethanol
insol.
Total
0.6
1.0
64.0
87.Ot
3.U
575
35.4
11.1
37.1
100.0
99.1
98.0
I, stored
at S#{176}.
t
Precipitated
by TCA.
Precipitated
by sodium
acetate.
§ Percent of counts or protein content
fraction
soluble in TCA-ethanoh.
remaining
soluble
after
addition
of so(Iium
acetate
to
precipitate
line on agar diffusion
(14)
as seen in Fig. 2. immunoelectrophoresis
of tile lyophilized
albumin
redissolved
in
veronal
buffer
revealed
one or two very faint arcs in the
or
and $-globulin
regions,
in addition
to tile major
arc of albumin
(Fig. 3). Immunoelectrophoretically,
the extracted
albumin
in the other methods
appeared more contaminated
with traces of other substances.
Three additional arcs were found in the Delaville
method,
and four were found in
Rodkey’s
technic. All were seen in the
or $-globulin
regions.
A number
of normal
and abnormal
serums
have been repeatedly
fractionated
by the present
technic. The method
is relatively
free from
interference
caused by lipemic serums, a characteristic
already
reported
for oilier etiianolic-TCA
separations
(10)
Others
have reported
that
hemoglobin
is soluble
in ethanolic-TCA
(10),
but with faintly
pink,
slightly
hemnolyzed serums
we
were
unable
to recogmiize differences
in
albumin
concentrations
from serum from the same patient
but without
hemolysis.
Iii 34 replicate
determinations
of serums
analyzed
by the
present
technic or by bromcresol
green, a significantly
greater
albumin
content
was found by the latter method.
Rodkey
had reported
no significant
difference
of his bromcresol
green method
and TCA-ethanol
fractionization
methods.
The present
data suggest caution in accepting
the present
technic as
precisely
quantitative
despite
excellent
recovery
curves
for added
albumin.
Nonetheless,
it offers a rapid technic for nearly
quantitative
isolation
of all)umin
of high purity
with electrophoretic
and immunologic characteristics
unimpaired.
one
-,
-
.
-
30
IWATA
fT AL.
Clinical
Chemistry
References
1.
2.
Iwata,
T., Iwata,
H., and Holland,
J. F.,
means of trichloroacetie
acid and ethanol.
Schwert,
G. W., Recovery
of native
bovine
chloroacetie
3.
Grabar,
P.,
acid
and
williams,
electrophor#{233}tiques
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Biochim.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
15.
solution
in
C. A.,
organic
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Clin.
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albumin
d’un
after
from humuan serum
11, 798 (1965).
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by
tn-
J. Am. Chein. Soc. 79, 139 (1957).
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m#{233}lamigede
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B. ,J., Disc electrophoresis:
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J., The determination
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S., Solubilization
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Bloc/tern.
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A., and Debro, J. B., Solubility
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1067 (1956).
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.J., Caraet#{232}re de solubilite
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Rodkey,
F. L., Direet
spectrophotomaetric
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Chin. C/tern. 11, 478 (1965).
Rodkey,
F. L., Separation
amid determnimuation
of time total
globulins
of human
serum.
Chin. C/tern. 11, 488 (1965).
Hradec,
J., Metabohismmmof serum albumin
in tumaor-bearing
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12, 290
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Nace, G. w., Suyama,
T., and Smith,
N., “Early
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In
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and
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Pallanza,
Ranzi,
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Lowry,
0. H., Rosebrough,
N. J., Farr, A. L., and Randall,
R. ,J., Protein
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Failing,
J. F., Jr., Buckley,
M. W., and Zak, B., Automatic
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Tech. Bnhl. Ileg. Med. Tech. 29, 205 (1959).