PAPER ELECTROPHORESIS OF SERUM PROTEINS
W I T H MICRO-KJELDAHL NITROGEN ANALYSIS OF THE PROTEIN FRACTIONS.
A COMPARISON WITH F R E E ELECTROPHORESIS AND SALT FRACTIONATION
METHODS
B. L E V I N , M . D . , AND V. G. O B E R H O L Z E R , B.A.
Queen Elizabeth
Hospital for Children,
London,
Enyland
The separation and quantitative estimation of the components of serum protein by classical Tiselius electrophoresis is generally impracticable as a routine
procedure. Similar results are obtained by salt fractionation methods,10 but
these also have their disadvantages. A method of electrophoresis on filter paper
has recently been described independently by Durrum 3 in America, Cremer and
Tiselius2 in Sweden, and Turba and Enenkel13 in Germany, and promises to be
a simple alternative procedure to free electrophoresis. The separation of serum
protein into its components is accomplished by placing a small amount of serum
on a strip of filter paper, which has been saturated with veronal buffer solution,
and passing a small current of electricity for several hours. Cremer and Tiselius2
in 1950 showed that by a process of staining the protein on the filter paper,
after electrophoresis, with bromphenol blue, followed by elution and colorimetric
estimation of the dye from successive small segments of the paper, a curve similar
to that from the classical Tiselius electrophoresis could be obtained by plotting
dye concentrations against distance traveled. The relative proportions of each
component forming a peak were measured from this curve, in the usual way.
Good agreement between free and paper electrophoretic analysis of serum protein was found with normal serums, although in nephrosis the concordance was
less satisfactory. Kunkel and Tiselius8 also determined the protein directly
from the small segments of filter paper, by elution and colorimetric estimation
using the Folin-Ciocalteu reagent, with similar results. Turba and Enenkel13
suggested staining the protein after electrophoresis, cutting out the whole of
each protein fraction and estimating the dye after elution. Grassmann and
Hannig, 5 using a somewhat different method, converted the filter-paper strip
into a curve by a direct photometric estimation of the adsorbed dye on the filter
paper.
Analysis of serum protein by paper electrophoresis has obvious advantages
over free electrophoresis. The apparatus is simple, only a small amount of serum
is required and no preliminary dialysis is necessary. The separation is carried
out at room temperature, and several serums may be analyzed simultaneously
in one apparatus. After separation the components are easily fixed, rendered
visible and readily isolated for estimation. Finally, certain very milky serums,
which are not so suitable for free electrophoresis, can readily be analyzed by
paper electrophoresis.
Received for publication Ssptember 14, 1952
D r . Levin is Director of the-Pathologic Laboratories and Mr. Oberholzer is Senior Biochemist at Queen Elizabeth Hospital for Children.
205
206
LEVIN AND OBERHOLZER
This communication is concerned with the analysis of the filter-paper strip
after electrophoresis, a preliminary report of which has already appeared.10 The
protein fractions on the paper are suitably delineated, then cut out as whole
sections, which are analyzed for protein nitrogen by micro-Kjeldahl analysis.
The results have been compared with those obtained by free electrophoresis and
by salt precipitation.
EXPERIMENTAL
Apparatus
The a p p a r a t u s finally adopted (Fig. 1) was a modification of D u r r u m ' s 3 a p p a r a t u s and
was similar to t h a t described by Flynn and de Mayo 4 b u t with certain differences. T h e solid
carbon electrodes entered the tank through glass sleeves, so t h a t they could easily be reGLASS ROD
LID
HOLES
F I G . 1. Apparatus for paper electrophoresis
moved for cleaning; each electrode compartment was connected with its adjacent inner one
by a line of 6 equally spaced holes, about 4 mm. in diameter, placed in the partition 2 cm.
above the floor of the t a n k ; and the 2 inner compartments were joined outside the t a n k by
rubber tubing carrying a screw clip to facilitate leveling of the liquids; and finally a lip
of perspex (lucite), 2 cm. in width, was attached round the outside upper edge of t h e tank,
forming a base on which a perspex (lucite) lid is firmly clamped during electrophoresis. A
thin layer of silicone grease was used to form an air-tight seal. T h e source of direct current
was the mains supply, and incorporated in the circuit were a milliammeter, a voltmeter and
a variable 100,000-ohms resistance.
Reagents
Barbitone buffer, p H 8.6, ionic strength 0.1. Dissolve sodium diethyl b a r b i t u r a t e (10.3
Gm.) and diethyl barbituric acid (1.84 Gm.) in 1 liter of distilled water.
Stain reagent. One per cent bromphenol blue in 95 per cent ethyl alcohol saturated with
mercuric chloride.
PAPER ELECTROPHORESIS OF SERUM PROTEINS
207
Wash reagents. One per cent mercuric chloride in methyl alcohol, followed b y pure methyl
alcohol.
Procedure
Buffer solution is placed in all b u t the middle compartment of the t a n k to a d e p t h of
about 3 cm., keeping the screw clip open until the fluid levels have equalized. Strips of filter
paper (Whatman N o . 31), 4 cm. wide and 39 cm. long are placed in position over the glassrod, with the ends dipping about 2 cm. below the level of the buffer in the 2 inner compartments. T h e buffer is allowed to ascend the paper by capillarity to within 3 cm. of the t o p ,
when the serum (0.16 ml.) to be analyzed is applied to the apex of the paper, at a point previously marked with a pencil line. T h e serum is spread evenly across the paper over as
narrow a band as possible (usually about 1 cm. in width) by deliver}' from the fine tip of a
laboratory calibrated Ostvvald-type pipet. As the buffer meets the protein, the band narrows
appreciably and the boundaries become sharper. T h e soaked paper strips are adjusted t o
hang straight down into the buffer, and the cover is then clamped down into position. After
an interval of at least 15 minutes to allow equilibrium and saturation of air, a potential difference of 110-125 volts is applied across the carbon electrodes, with a current of approximately 0.3 milliampere per centimeter of width of paper, t h e current being adjusted by the
variable resistance. T h e potential difference usually drops slightly, and t h e current strength
increases slightly during electrophoresis. An adequate separation of the protein fractions is
attained during a run of 15 hours.
At the end of the run, the paper strip is removed, placed horizontally over glass-rod supports and dried in an oven at 100 C. for about 20 minutes. T h e whole process of staining
and washing the paper is thereafter carried out in a lj^"-diameter extraction t u b e . T h e
paper is rolled up loosely, placed in the tube and covered with the one per cent solution of
bromphenol blue. After standing several minutes, the liquid is decanted and replaced by
the 1 per cent solution of mercuric chloride, which is in turn removed after several minutes. T h e paper is then washed by a continuous distillation of methyl alcohol into the
extraction tube, from which automatic siphoning occurs. T h e process is continued until
the wash liquid is colorless, which usually takes about half an hour. T h e paper is finally
washed with small amounts of ether, and allowed to dry in the air.
Estimation
of Protein Fractions on the Paper
T h e boundaries of the fractions, defined by the midline of the troughs between the peaks
of the fractions, having regard to the p a t t e r n on both sides of the paper, are marked by ruled
pencil lines. Measurements of t h e width of each fraction are made to enable the respective
blanks to be obtained. T h e fractions are then cut, and the total nitrogen in each separately
estimated by micro-Kjeldahl digestion, using 3 ml. of concentrated sulphuric acid and 1
Gm. of solid sodium sulfate with copper sulfate and selenium dioxide, according to the
previously published procedure. 1 1 Digestion proceeds smoothly, frothing is avoided and
the amount of mineral m a t t e r raised to the correct proportion b\ r the utilization of acid in
the oxidation of the paper, if the solutions of copper sulfate and selenium dioxide are not
added until most of the carbon has been oxidized. Two filter-paper strips 2 cm. and 4 cm.
in width, respectively, and taken from the paper outside the protein spread, were always
digested a t the same time, as blanks, in addition to a reagent blank. F r o m these results,
an average blank for the paper was calculated. T h e total reagent and paper blanks were
appreciable, relative to the low amount of protein in the smallest fractions. Blanks from
untreated filter paper, were shown to be the same as blanks obtained as above, provided
washing with methyl alcohol was adequate.
RESULTS
Reproducibility
In a consideration of the duplicate analyses on 17 serums, it was found that
for albumin, the mean percentage difference between duplicates was 0.65 per
208
LEVIN AND OBERHOLZER
cent, with a range of 0.0-2.2 per cent; for ai-globulin, it was 0.46 per cent, with
a range of 0.0-1.5 per cent; for a2-globulin, it was 0.8 per cent, with a range of
0.1-2.0 per cent; for ^-globulin, it was 0.65 per cent, with a range of 0.0-1.7 per
cent; and for 7-globulin, it was 0.43 per cent with a range of 0.0-1.5 per cent,
all these results being expressed as percentages of total protein. The reproducibility of the method is therefore good, and, indeed, somewhat better than that
obtained by Flynn and de Mayo, 4 using the dye-elution method. Slightly better
results were obtained by Grassmann, Hannig and Knedel, 6 using the direct
TABLE 1
COMPARISON OP P A P E R AND T I S E L I U S
ELECTROPHORESIS
PERCENTAGE OF TOTAL PROTEIN
PROTEIN
Albumin
ctl-
Globulin
aiGlobulin
/SGlobulin
yGlobulin
Gm.l
100 ml.
1
Normal
Paper
Tiselius
6.90
62.4
62.8
3.4
3.7
7.2
7.1
14.3
14.0
12.7
12.2
2
Infectious hepatitis
Paper
Tiselius
S.13
59.2
66.0
3.2
3.7
10.9
9.15
11.4
12.0
15.3
9.05
3
Multiple myeloma
Paper
.11.70
IS.4
1.5
3.5
3.8
72.S
Tiselius
20.6
62.9
16.5
,4
Normal
Paper
Tiselius
7.91
58.5
60.3
3.2
4.6
8.4
5.9
10.3
15.6
19.6
13.6
5
Pregnancy
Paper
Tiselius
6.35
• 50.6
49.7
5.8
3.2
14.2
10.1
16.3
17.8
13.0
19.2
6
Rheumatoid arthritis Paper
Tiselius
6.96
30.8
30.9
7.9
4.6
15.9
15.7
11.2
13.6
34.2
34.9
7
Nephrosis
Paper
Tiselius
4.16
18.0
14.7
3.2
4.3
54.9
41.9
S.l
28.8
15.8
10.3
photometric estimation of the dye on paper. They did not, however, compare
their results with those obtained by free electrophoresis.
As a check on the analysis, an estimation of total nitrogen was always carried
out independently on each ssrum for direct comparison with the sum of the
individual protein fractions of that serum. The 2 estimations were always within
0.2 Gm. per 100 ml. of each other, and most were in much closer agreement.
Comparison of Paper Electrophoresis with Classical Tiselius Electrophoresis and
Salt Fractionation Methods
Seven serums that had been subjected to free electrophoresis were also analyzed by paper electrophoresis, and the results are shown in Table 1. Tiselius
PAPER ELECTROPHORESIS OF SERUM PROTEINS
209
electrophoresis on 5 of the serums was carried out, using veronal buffer pH 8.6
and ionic strength 0.1; for serum No. 4, a phosphate buffer pH 8.0, ionic strength.
0.2, and serum No. 7 a phosphate buffer pH 8.8, ionic strength 0.1 were used.
Seventeen serums, including the 7 mentioned above, were also analyzed by salt
fractionation procedures10 as well as by paper electrophoresis. The results (Table
2) are discussed below.
DISCUSSION
The dye-elution method of Cremer and Tiselius2 depends essentially on the
fact that the amount of dye bound is directly proportional to the amount of
protein present. These authors showed that albumin has a greater binding
capacity for bromphenol blue than has 7-globulin, and to allow for this, they
applied an experimentally determined factor of 1.6, which later 8 appears from
their graph to be 1.3, to their globulin readings. It does not follow that the
same correction factor can be applied to all the globulin fractions. Thus from a
comparison of 10 normal serums, which they analyzed by both free and paper
electrophoresis, Koiw, Wallenius and Gronwall7 calculate empirical factors of
2.8, 1.7, 1.6 and 1.4 for ai-, a2-, /3-, and 7-globulin, respectively. Although these
were not derived from the pure albumin and globulins, nevertheless they suggest
that there are wide differences in the factors for the different globulins. Further,
as Flynn and de Mayo 4 point out, the rates of dye adsorption are different for
the different fractions; that is, the correction factor is constant only for a definite
period of staining and probably for a definite concentration of dye. From these
facts, it would appear that strict control of the technic of staining and washing
is necessary to obtain consistent results.
To convert the paper strip into a curve, it is necessary to determine tin concentration of dye in as many as 40 solutions. The process of ruling the 5-mm.
sections, and the elution and estimation of dye in so many solutions is tedious
and lengthy. Even when the curve is obtained, it is necessary to estimate the
relative proportions of the various fractions by measuring the areas under each
peak. In addition to the possible errors of the dye-elution method, there are
errors in the analysis of the curve. Finally, to convert relative into absolute
concentrations, it is still necessary to determine the total protein of the serum
independently, either by a Kjeldahl analysis or some other method.
These disadvantages are avoided by a determination of the nitrogen on the
paper directly by Kjeldahl analysis. Instead of cutting the paper strip after
electrophoresis into 5 mm. sections, the whole fraction is delineated and cut out,
and the paper digested. This method is quite analogous to the analysis of the
curve obtained in the classical Tiselius method by dropping perpendiculars from
the lowest point between each peak, and assuming that the relative amounts of
each fraction is directly proportional to the areas enclosed between the perpendiculars. The point of cutting between each fraction should theoretically coincide
with the point of lowest concentration of protein. It has been stated 4 that there
is considerable inaccuracy, especially with pathologic serums, in defining the
various fractions. We have, however, found no difficulty in doing so, provided
TABLE 2
COMPARISON O F P A P E R E L E C T R O P H O R E S I S AND S A L T FRACTIONATION
CONCENTRATION I N GM. PER 100 ML.
TOTAL
PROTEIN
Albumin
0Globulin
Globulin
0.95
0.96
1.01
0.90
1.12
1.31
0.91
1.22
0.36
8.13
0.44
8.51
1.19
1.35
0.82
1.56
1.58
0.73
0.89
1.02
0.81
1.58
2.01
1.11
0.78
2.38
2.18
0.65
0.33
0.64
0.79
0.24
0.72
0.60
0.22
1.34
1.63
0.81
0.91
1.60
1.11
0.46
0.73
0.45
0.95
0.58
0.64
0.54
1.76
0.62
0.44
0.56
1.5S
2.58
0.63
0.80
3.01
2.03
Nil
2.18
0.20
0.26
ttl-
«2-
Globulin
Globulin
Gm./
100 ml.
1
2
3
4
5
6
Normal
Infectious hepatitis
Multiple myeloma
Normal
Pregnancy
Rheumatoid arthritis
7
8
" 9'
10
11
Nephrosis
Infant (normal)
Infectious hepatitis
Pregnancy
Pregnancy
e At
12
13
14
Nephrosis
? Chronic leukemia
Nephrosis
Salt
6.90
Paper
7.10
4.43
Salt
8.13
4.76
Paper
8.00
4.74
Salt
11.70
2.13
Paper
11.69
2.15
Salt
7.91
4.25
Paper
7.95
4,65
Salt
6.35
3.18
Paper
6.25
3.17
Salt
6.96
2.20
Paper
6.97
2.15
Salt
4.16
0.56
Paper
4.04
0.73
Salt
5.42
3.65
Paper
5.46
3.76
Salt
8.13
4.34
Paper
8.13
4.57
Salt
5.12
2.79
Paper
5.19
2.60
Salt
4.19
1.98
Paper
4.27
1.56
Salt
3.69
0.45
Paper
3.52
0.44
Salt
8.49
3.15
Paper
8.48
3.91
Salt
3.23
0.43
Paper
3.13
0.35
210
a
4.99
0.24
0.52
0.94
0.26
0.87
1.08
0.41
0.18
1.12
0.25
0.67
0.86
0.36
1.17
0.55
0.77
2.21
0.13
0.74
0.16
0.82
0.24
0.76
0.80
0.61
0.68
0.87
0.66
0.86
0.22
1.86
1.18
0.13
0.77
0.14
PAPER ELECTROPHORESIS OF SERUM PROTEINS
211
TABLE 2—Continued
CONCENTRATION IN GM. P E R 100 ML.
CONDITION
METHOD
TOTAL
PROTEIN
Albumin
02-
Globulin
Globulin
0-
Globulin
Globulin
Cm./
100 ml.
15
16
17
Normal
Hemochromatosis
Celiac disease
Salt
7.01
3.SO
Paper
7.22
3.71
Salt
7.05
4.00
Paper
6.S9
4.06
Salt
6.94
4.14
Paper
7.07
4.09
0.96
0.50
1.06
0.63
0.14
0.80
0.82
0.26
0.99
1.25
1.00
1.05
0.90
1.23
1.19
0.70
1.19
1.08
0.90
0.94
0.79
that the separation of the protein fractions is adequate. It is also not essential
to have a rigidly standardized technic of staining, since it is only necessary for
dye adsorption to be sufficient to define the peaks of the fractions. If the troughs
between the fractions are too deeply stained, it may indeed be more difficult to
define the boundaries. Finally only 5 nitrogen estimations in addition to the
blanks are required in this method, compared with the 40 or more colorimetric
estimations in the dye-elution method.
In order to determine whether the fractions were being correctly defined at the
lowest point of protein concentration between the fractions, 2 serums were
analyzed in the following manner.
Electrophoresis was carried out in triplicate on paper 4-cm. wide, and stained
in the usual way. All 3 papers showed the components in practically identical
positions. Two of the strips were analyzed by the procedure described above;
the third was ruled into consecutively numbered strips 5-cm. wide, starting
from 1 cm. before the outer boundary of the albumin fraction and continuing
to 1 cm. beyond the outer margin of the 7-globulin fraction. These were then
cut out and separately digested for the estimation of nitrogen. In only 1 of these
2 serums was a note made of the points at which the whole fraction would normally have been cut. Those 5-mm. portions of the paper before the albumin and
after the y-globulin fractions were taken as blank readings. When the nitrogen
content was plotted against the number of strip, a curve was obtained similar
to that from the dye-elution method or the classical Tiselius technic (Figs. 2
and 4). The arrows on this curve mark the points at which the paper would have
been cut in the normal procedure. It will be seen that the arrows indicate the
lowest point of the curve between each component, except for that dividing
air from ^-globulin, where it is less than 5 mm. from the true point.
The relative concentrations of the components in the 2 serums were ascertained by analysis of the 2 curves obtained, and measurement of the areas under
the peaks. The results, calculated as percentage of total protein, are shown in
Table 3. I t will be seen that the 2 methods are in close agreement for both serums
212
L E V I N AND
OBERHOLZER
and that the differences are no more than would be expected between duplicate
analyses by the routine procedure. It may therefore be concluded that the method
of cutting and analyzing sections containing whole fractions gives results as
accurate as the longer method of digestion of 5-mm. sections and subsequent
analysis of the curve.
Comparison of Paper Electrophoresis with the Classical Tiselius Electrophoresis
When the results by the 2 methods are compared, it can be seen (Table 1)
that the albumin fraction agrees well, although in 1 case there is a difference of
6.8 per cent calculated on the total protein. In the 7-globulin fraction, several
show differences of over 6 per cent, although the greatest difference (10 per cent)
was shown with the serum from a patient with multiple myeloma. These are
rZ
ui
y
z
o
\
0
z
ul
O
o
0:
h
Z
v
--^ \ U I.-"
Dl S T * N CE
FIG. 2. Curve obtained by micro-Kjeldahl analysis of 5-mm. strips on serum from a patient with rheumatoid arthritis.
unexpectedly wide, since the 7-globulin, like the albumin, is an end fraction and
readily demarcated from /3-globulin. It would therefore be expected that the
7-globulin would show closer agreement with free electrophoresis than the middle fractions, whereas the results are the reverse. Where the proportion of
7-globulin is higher, as in most cases, this may be clue to the fact that some of
the protein may be adsorbed at the point of application on the paper, and does
not move under the influence of the current. With the remainder of the globulin
fractions, there is much better agreement. As a whole, the results by the 2 methods agree well and are similar to those obtained by Flynn and de Mayo 4 and
Kunkel and Tiselius.8 One other serum from a patient with nephrosis, was analyzed by both free and paper electrophoresis, in the latter case by the ordinary
method of whole fractions and also by cutting into 5-mm. strips. This was done
partly because of the difficulty, which had been noticed in previous nephrotic
PAPER ELECTROPHORESIS OF SERUM PROTEINS
213
serums, of demarcating the boundary between the a2- and /3-globulin, and it
was hoped that the correctness of the demarcation would be demonstrated by
the curve. The result is shown in Figure 3 (serum No. 7 in Tables 1 and 2), and
that obtained by free electrophoresis in Figure 5. I t can be said at once that the
curves while similar do not show such good agreement as in the other serums
compared by the same methods. Further, the curve obtained by paper electrophoresis presents as much difficulty in analysis as cutting the paper strip in
whole fractions, owing to the poor resolution of the /3-globulin peak. The discrepancy in the results on this nephrotic serum by free and paper electrophoresis
F I G . 4. Electrophoretic pattern obtained by classical Tiselius technic on
same scrum as Figure 2. (Ascending curve.)
may be due to difficulty in defining the boundary between the 2 fractions, or
may be due partly to the presence of large amounts of lipids. It would be expected
that the Kjeldahl method would give a lower /3-globulin value than the Tiselius
method, since this fraction is mainly associated with the lipids and the latter
method measures the whole of the protein-lipid-carbohydrate complex, while
the former estimates the protein part only. Cremer and Tiselius2 also found that
the agreement in. nephrotic serums using the 2 methods was not so good as in
normal serums, although they felt that this was possibly due to the different
buffers used. A similar discrepancy was noted by Koiw and associates7 in cases
of hyperglobulineniia, and by Flynn and de Mayo4 in the serum from a patient
214
LEVIN AND OBERHOLZER
with abnormal lipids. It is possible that the dye-elution method also gives a
measure of the protein part of the protein-lipid-carbohydrate complex.
Comparison of Paper Electrophoresis with Salt Fractionation Methods
Previous investigation10 has shown that it is possible by a selection of suitable
salt fractionation methods, to carry out an analysis of serum proteins for albuTABLE 3
COMPARISON OF WHOLE FRACTION WITH 5-MM. STRIP METHOD
PERCENTAGE OF TOTAL PROTEIN'
NO.
METHOD
Albumin
m-Globulin
m-Globulin
0-Globulin
7-Globulin
4
Whole fraction
5-mm. strip
58.5
60.3
3.2
3.2
, S.4
7.2
10.3
10.0
19.6
19.3
6
Whole fraction
5-mm. strip
.30.8*
29.7
7.9
9.6
15.9
17.5
11.2
9.2
34.2
34.0
A.
/
*—'
DISTANCE
FIG. 3. Curve obtained by micro-Kjeldahl analysis of 5-mm. strips on serum from a patient with nephrosis.
min, a-, /}-, and 7-globulin, giving results in good agreement with those of free
electrophoresis. I t was therefore thought of interest to compare the salt-fractionation method with paper electrophoresis. In Table 2 are shown the results
obtained in 17 serums, of which 7 were from normal patients, the others being
from patients with a variety of conditions. Generally, the agreement between
the results obtained by the 2 methods was as good as the agreement between
salt fractionation and free electrophoresis previously obtained. The first 7 of
these serums were analyzed by all 3 methods, and a comparison of the results
PAPER ELECTROPHORESIS OF SERUM PROTEINS
215
shows that paper electrophoresis gave on the whole somewhat better agreement
with free electrophoresis than did the salt-fractionation method.
The most marked difference between paper electrophoresis and the saltfractionation method was found in cases of nephrosis, and especially in the
«2- and /3-globulin fractions. Whereas in salt fractionation most of the globulin
appears in the (3-globulin fraction, in paper electrophoresis, the globulin is mainly
aa-globulin. It would appear then that with chemical fractionation a large part
F I G . 5. Electrophoretic p a t t e r n obtained by classical Tiselius technic on
same serum as Figure 3. (Descending curve.)
of the a2-globulin is precipitated as /3-globulin. Whether this is because the
a2-globulin of nephrotic serums is in some way different from that of normal
serum, or whether it is because the former is present in abnormally large amounts,
is not clear. It must be admitted that, as has already been stated, the resolution
of the a2- and /3-globulin fractions is sometimes poor with nephrotic serums, and
it is possible that our results for a2-globulin are too high and for /3-globulin too
low, in such cases. That, however, cannot account for the low a2-globulin fraction found by the salt-fractionation method, since free electrophoresis (Fig. 5)
216
LEVIN AND OBERHOLZER
also reveals a high a 2 fraction, similar to that from paper electrophoresis. It has
previously been reported 1 ' 12 that salt-fractionation methods do not agree with
Tiselius electrophoresis in nephrosis, and this conclusion is supported by our
findings.
Since the separation of protein on filter paper, like free electrophoresis, depends
on the mobility of the components in an electrical field, and since all the fractions are directly analyzed, it is obviously preferable to adopt paper electrophoresis rather than salt-fractionation methods for a complete analysis of serum
protein. Although the classical Tiselius electrophoresis must still remain the
standard method, the advantages possessed by paper electrophoresis, especially
when the simplified procedure of micro-Kjeldahl analysis of whole fractions is
used, make it much more suitable for routine clinical laboratory work.
SUMMARY
A simplified procedure of analysis after paper electrophoresis of serum protein
is described in which the protein is determined by micro-Kjeldahl analysis after
cutting out the whole fraction. The estimation of protein on 5-mm. segments of
the paper is shown to give a curve similar to that obtained by the classical Tiselius or the dye-elution method. A comparison of the procedure described with
that of free electrophoresis for 7 serums, both normal and pathologic, and with
salt-fractionation methods for 17 serums, has been made. It is shown that the
results given by nitrogen analysis are in good agreement with those obtained
by free electrophoresis and at least as good as those that have been reported for
the dye-elution method.
The advantages of nitrogen analysis following paper electrophoresis over the
dye-elution method as well as over chemical precipitation are discussed. It is
concluded that paper electrophoresis is to be preferred to salt-fractionation
methods, and that on the grounds of simplicity, nitrogen analysis following paper
electrophoresis is preferable to dye elution.
Acknowledgments. We wish to thank D r . R. A. Kekwick and D r . Pen-in for providing us
with specimens of serum t h a t they had analyzed b y the Tiselius technics. We are also greatly
indebted to Mr. R. G. S. Johns and D r . B . E . Conway for kindly undertaking the Tiselius
analysis on 5 of our serums, and for their helpful criticism.
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