Estimation of Serum Proteins by Electrophoresis
on Cellulose Acetate
J. W. Keyser and G. L. Watkins
We have evaluated
results for albumin obtained
by a
standard
procedure
for cellulose
acetate
electrophoresis
of proteins
in serum. The Ponceau
S-stained
.albumin
and globulins
were eluted and the albumin
was calculated by the generally
accepted
formula
[(albumin-bound dye absorbance/absorbance
of total
albumin
tionation
content
of which had been determined
by fracwith Na2SO4 (260 g/liter)
and determination
of
the trichioroacetic
acid-precipitated
protein (N x 6.25) in
the filtrate. Identical treatment
of tests and standard was
x
ensured
by running
them together
on single sheets (12
12 cm), which were processed
entire,
and all determina-
protein-bound dye) X total serum protein concn] and
by the formula (absorbance of albumin-bound dye in
test/absorbance
of albumin-bound
dye in a reference serum)
X concn of albumin in reference
serum. The ratio of values by the first and second
methods ranged from 0.93 to 1.30, the first giving
the higher results in cases of discrepancy. These
findings confirm the limitations in accurately calculating any serum-protein fraction by the first method.
The second method appears to be the more accurate.
tions were done in duplicate.
Sheets were stained for 10
mm in a mixture consisting of Ponceau S 0.09 g, trichloroacetic acid 1.34 g and sulfosalicylic acid 1.34 g in water,
the final mixture
being diluted to 100 ml. (In the case of
three specimens,
electrophoresis
was in buffer of ionic
strength
0.05 and the strips were stained
in a solution
of 2
Additional
rection
Keyphrases:
binding by proteins
.
serum
albumin
dyecalculation of results of electrophore.
S’s
In the quantitative
electrophoretic
measurement
of protein fractions after their separation
either on filter paper
or cellulose acetate, the calculation must either assume uniform binding of dye by the various fractions or use a factor to correct for differences between albumin and globulin dye-binding
capacity. With filter paper electrophoresis, this factor can vary widely for different
sera (1). Cellulose acetate does not appear to have been investigated
so thoroughly
in this
respect,
although
albumin
and
-y-
globulin are reported to have equal binding characteristics
for Amido Black (Naphthol Blue Black) (2).
Because the globulin fractions obtained on cellulose acetate electrophoresis
of serum consist of complex mixtures
of proteins,
pendently,
ing results
the concentrations
of which may vary indethe scope of this study was limited to evaluatfor albumin,
as obtained
after a commonly
used procedure
for electrophoresis
on cellulose acetate.
g of Ponceau
S in trichioroacetic
acid solution,
5 g/
100 ml.) Excess dye was removed with dilute (5 volumes
per 100 volumes) acetic acid. After drying, strips were cut
into portions,
albuminand globulin-bound
dye separately
eluted with 0.1 molar sodium hydroxide,
and the absorbance of the resulting
solutions
measured
at 546 nm. Cor-
was made for an appropriate
a portion
of the strip
Method
on cellulose
acetate
from
as the
Serum albumin
Method
=
(Ar/Ti’)
X T.P.
b
Serum albumin
reference standard
(AT/As)
=
concentration
X
of albumin
in
serum
where
AT
TT
As
=
absorbance
of albumin-bound
=
absorbance
of total protein-bound
=
T.P.
dye in test,
absorbance
of albumin-bound
dard, and
= totalprotein
concentration
Method
tion.
a is the generally
b has
been
with
those
dye in test,1
dye in reference
in test serum.
accepted
shown
method
(3) to give
obtained
stan-
of calcula-
results
in close
by immunoprecipitation
(4).
Thirty-eight
sera, some from healthy people and others
from patients on whom the determination
of serum protiens had been requested by clinicians, were electrophoresed
prepared
dimensions
a
Method
and Methods
blank,
the same
eluted band(s). Elution was complete under these conditions, and control experiments
showed absorbance
to be
linearly proportional
to albumin concentrations
well above
those encountered
in this series.
Albumin was calculated by two different formulas:
agreement
Materials
of about
in barbital
buffer
(pH
8.6,
75
mmol/liter).
The serum was accurately
measured and a
standard was run in parallel with each batch of test samples. This standard consisted of pooled normal serum, the
From the Department
of Chemical Pathology, The Welsh National School of Medicine, Royal Infirmary, Cardiff CF2 1SZ, U.K.
Received Mar. 6, 1972; accepted Sept. 15, 1972.
the
Because
value
most
cases
tained
when
by Methods
Total
with
in Method
a the result critically
for total protein,
this was carefully
protein
depends
checked
on
in
significantly
different results were oba and b.
was determined
by the biuret method,
the AutoAnalyzer
and with
bovine
serum
albumin
as
‘In most cases this was the sum of the absorbances for albumin
and globulin, pre-albumin
being ignored. In the case of twelve
sera, however, pre-albumins
were included in the calculation of
total protein-bound
dye. Similar results were observed in both series.
CLINICAL
CHEMISTRY,
Vol. 18, No. 12, 1972
1541
standard,
except
for three specimens
for which the micro60
Kjeldahl
method was used (protein
N
X
6.25).
Results and Discussion
The results are presented in Figure 1. Although
cases there was excellent
agreement
between
methods of calculation,
this was not invariably so
ferences as great as 11.5 g/liter were found. The
gave the higher results.
viously
well-recognized
fact
that
This confirms
albumin
binds
as a whole
in these
cases.
more
30
20
Alternatively,
10
there
ratios obtained for normal healthy persons averaged 1.17.
Nevertheless,
the correlation with the electrophoretic
pattern was by no means clear cut, and since the ratios even
for normal sera ranged from 1.05 to 1.30, it would appear
that more detailed analysis would be necessary to account
of the discrepancies
we have
in biuret chromogenic
power
consequent
upon a change in the relative proportions
of
the individual serum proteins; this would of course affect
the figure obtained for total serum protein and hence also
the albumin
results as calculated
by Method a.
In view of all these considerations
it is hardly surprising
that no single correction
factor is applicable
to albumin
results obtained by Method a. What we do find surprising
is the almost universal lack of appreciation
of this possibility, and the general assumption
that a single correction
factor may be used in all circumstances.
Our findings confirm that, as previously stated (5), it is
not possible to calculate accurately by Method a the confractions
of any serum
protein
bind dye equally
fraction
or unless
unless
to cor-
observation
We do not
suggest that this consideration
necessarily invalidates
the
many diagnostically
useful correlations
observed between
electrophoretic
changes and clinical conditions;
but only
that
the
limitations
of these
in quantitative
be borne in mind. Nor do we suggest
the only or even the most important
terms
must
that dye-binding
is
factor defining the
accuracy of filterpaper and cellulose acetate
electrophoresis, because
other
variables
have to be carefully
controlled.
It is possible
that different
results
would be obtained if other dyes were used, and it is conceivable
that a
dye might be found that has an equal affinity
for all the
main protein fractions.
for this at present.
1542
CLINICAL
There
CHEMISTRY,
appears
10
20
30
40
50
60
(b)
Fig.
1.
Serum albumin values (g/liter) obtained
ods a and
the points
result
b (see
would
by Methtext). The diagonal
line is that on which
fall if the two methods
gave the same
Technically, Method
b, which we believe offers the
more accurate results, has the disadvantage
that samples
have to be measured accurately, whereas in Method a this
is unnecessary.
This disadvantage
of Method b might be
overcome by limiting quantitative
measurements
to the
globulin fractions,
these being assumed for purposes of
calculation
to have equal dye-binding power; a measure of
albumin would be obtained by some independent
method
such as, for example, the bromcresol green method. Since,
in many laboratories,
serum total protein and albumin are
routinely determined
by
automated
procedures,
nothing
would be lost by omitting albumin altogether from quantitative electrophoretic
measurements.
Presumably
there
would still remain errors caused by differences
in dyebinding
among the globulins
themselves.
Such errors,
however, would be confined to those fractions;
and at
least albumin would be determined
by a more confidently
controlled method.
the various
it is possible
rect for differences
in dye-binding
power. This
will of course apply equally
to the globulins.
.
dye
in the cases we have studied;
for example,
three of four patients
with carcinoma
and possibly raised a-globulins
showed ratios (Method a/b) of less than 1.0, whereas the
centration
.
the pre-
might be a change in the dye-binding
capacity of the albumin, although in view of previous results obtained by
us (3) this seems less likely. The former suggestion
is in
general accord with the electrophoretic
patterns obtained
for these variations.
Yet another
explanation
noted might lie in variations
40
(a)
than do the globulins. The excellent agreement obtained
with many sera from patients, however, would suggest a
general increase in the dye-binding
capacity of the globulin fraction
.
in many
the two
and dif-
ratio of
the results
obtained
by the two methods
of calculation
(i.e., a/b) ranged from 0.93 to 1.30. Of the cases showing
significant
discrepancies
it was Method a-i.e.,
the one
assuming
equal dye-binding
by albumin
and total globu-
lins-that
50
to be no evidence
Vol. 18. No. 12, 1972
We wish to acknowledge the technical assistance of Mr. S.
Sasitharan, a student of The Hatfield Polytechnic, during a period of employment in this laboratory.
References
1. Keyser, J. W., and Stephens, B. T., Estimation of serum albumin: A comparison of three methods. Gun. Ghem. 8,526(1962).
2. Bartlett,
R. C., Rapid cellulose acetate electrophoresis:
1.
Serum proteins. Clin. Chem. 9, 317(1963).
3. Keyser, J. W., Determination
of serum albumin. GUn. Ghem.
14, 360 (1968).
4. Schultze, H. E., and Schwick, G., Quantitative
immunologische Bestimmung
von Plasmaproteinen.
Gun. Chim. Acta 4, 15
(1959).
5. Keyser, J. W., In Proceedings of the Third International
Symposium on Quality Control, Geneva (Merz & Dade), 1969, pp 52, 56.