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Chromatography of serum proteins with special reference to α-globulins
163
CLINICA CHIMICA ACT.4
CHROMATOGRAPHY WITH
SPECIAL
M. P. TOMBS,
OF SERUM
REFERENCE D. C. 0. JAMES
PROTEINS
TO cr-GLOBULINS* AND
N.
F. MACL.4GhS
Westminster Medical School, Lozdon (Great Britain) (Received
July z&h,
1960)
Since PETERSON AND SOBERI introduced substituted cellulose as ion exchange materials for the column chromatography of proteins there have been numerous reports of the application of chromatography to protein fractionation2. It has found less application to serum proteins than to simpler systems and has been most successful in resolving mixtures containing a relatively small number of components. With present techniques the resolving power on serum proteins is poor. It is not
possible to obtain useful quantitative
results, since at the best only one or two of the
chromatographic components are electrophoretically homogeneous. It would appear therefore, that chromatography is not a suitable method at present for the primary fractionation of serum protein, but might give valuable results if, after a preliminary fractionation, it is employed for finer subdivision. We have described elsewhere some results obtainable after a preliminary ammonium sulphate fractionation 3. The protein soluble in half saturated ammonium sulphate could be split into a /%globulin, albumin and a mixture of a-globulins on diethyl-amino-ethyl cellulose (DEAE). We describe here a further investigation of the group of a-globulins soluble in half saturated ammonium sulphate, and their variation in cancer. While DEAE-cellulose offers the opportunity to obtain a quantitative estimate of the a-globulins, carboxy-methyl cellulose (CM) has some advantage for their qualitative analysis and a few cases have been examined using this material. METHODS
Chromatography The apparatus used in this work was similar to that described previously”. DEAE- and CM-cellulose were prepared as described by PETERSON AND SOBERI and packed into columns (2.5 cm diameter) to a depth of IO cm under air pressure of 5 lbs. sq. inch. Flow rates of 150-200 ml/h were maintained with the same air pressure. Stepwise elution was usually employed, using the buffers indicated in the appropriate figures. Suitable buffers were selected from a consideration of gradient experiments. In some runs on DEAE-cellulose the final buffer was applied through a mixing chamber (IOO ml) initially filled with the preceding buffer. Gradient elution was employed here to minimise albumin trailing into the final peak. Protein concentration in the eluent was determined by measurement of the absorption at 280 m,u either on individual fractions or continuously on an automatic recorder. * Based on a paper read at the 8th Colloquium on Protides of Biological Fluids, Bruges, 1960. Cl&. Chipn. Acta, 6 (1961) x63-169
bl. P. TOMBS, D. C. 0. JAMES,
164
iY. F. MACLAGAN
Salt fractionation An equal volume of saturated
ammonium
sulphate
was added to serum (usually
5 ml) and the precipitate separated by centrifugation at 7000 g for 20 min. The prccipitate was then suspended in half saturated ammonium sulphate (271 g/l solution) and re-centrifuged. All experiments were carried out at room temperature. Thr supernatants from both centrifugations were united, dialysed against water to l-t’move salts and finally into appropriate
equilibrating
buffer for chromatography.
Fig. I. Chromatography of supernatant fraction from 5 ml of serum on DEr\F:-cellulose. The column and the applied protein were equilibrated with 0.005 JW phosphate PH 7. Buffers were applied stepwise, except for V which was by gradient (for detail see text). Buffer compositions were: II, 0.02 M phosphate PH 6; III, 0.018 M phosphateo.oz MNaClp~6; IV, 0.06Mphosphate pH 5 ; V, 0.05 iW phosphate pH 5, o. IO M NaCl. Each fraction was of O-ml xwlume. Fractions were pooled into protein fractions a, b, c and d as shown. Below is the result of electrophoresis of the fractions. In each case, at the top is shown the result of bromphenol blue staining of the agar slab, and immediately below the results of immune-electrophoresis.
Electrophoresis After elution from the column, fractions were concentrated by perevaporation and then dialysed against water to remove salts. In some cases concentration dialysis against polyethylene glycol (mol. wt. 20,000) was used, but this led to contamination of the fraction with carbohydrate material. Although this did not interfere with electrophoresis it was felt that perevaporation was a safer procedure. After concentration immuno-electrophoresis was carried out in agar-gel by a slight modification of the method of SCHEIDEGGER 4. Starch-gel electrophoresis was by the method of SMITHIES~. Horse anti-human anti-serum (Institut Pasteur, Paris) was used throughout. The immuno-electrophoresis results shown in the figures were made by tracing a projection of the original slides. RESULTS
Chromatography
OK DEAE-cellulose
Fig. I shows the results of DEAE chromatography of the supernatant fraction from a typical normal individual. Fraction a was completely homogeneous by electro-
CHROMATOGRAPHY phoresis, another
but
spectrum ent
were
in this
from
immuno-electrophoresis
globulin.
The main
the column,
therefore, mained
showed
of transferrin.
Fraction
possible.
1%
presence
of
small
line, pink colour
1.6%
b invariably
of protein-bound
precipitated
and could not subsequently
in solution
the
immune-electrophoretic
characteristic
fraction.
OP SERUM PROTEINS amounts
hexose
immediately
be redissolved.
of
and absorption was pres-
after
Electrophoresis
However,
in haemolysed
sera it had
a reddish
in a large
scale preparation
in which
it was eluted
elution was not,
colour,
and
together
re-
with
at 410, 540 and 577 rnp. This is complex@. Fraction c was at least 957; albumin, but immune-electrophoresis revealed the presence of traces of cr-globulin. It contained about 0.8$;/, protein-bound hexose. Fraction d always contained a trace of albumin, but was mainly a mixture of CY%and cx,-globulin. Orosomucoid, a postalbumin and a part of the For,-complex were detected by starch-gel electrophoresis. The misture had about 11% protein-bound hexose. Fig. 2 shows the result of chromatography of the same ammonium sulphate fraction from three cases of advanced cancer. The pattern was qualitatively similar to
albumin.
Its
characteristic
absorption
spectra
showed
maxima
of a haptoglobin-haemoglobin
o
t
$j $
8
)Tjarea
I
1
1
1
2.
1
I cancer of long cancer of breast
0
0
CD
units
1
0
00
0
mewstatic 00 oa3
0 Fig. z. lose of serum Buffers
fracti%?
number
but the relative
tion c, the albumin,
was greatly
ing edge of the peak. prominent. globulin
The
most
The trace striking
peak d. A rough
and absolute
reduced
estimate
rheumatoid arthritis etc.
co
size of the peaks was changed.
and partial
cr-globulins change
-
Fig. 3, Variation of the size of peak d (see Figs. I, 2) in cancer and other conditions. The size of the peak was measured in area units, where one area unit is equal to approximately 0.5 mg of protein. Ail results have been calculated to IOO ml of serum.
Chromatography on DEAE-ceIlusupernatant fraction from 5 ml of in three cases of advanced cancer. and other conditions were the same as for Fig. I.
that of the normal
0
CDOCD
I 200
0
0000
caT%?a other cancer
was
resolution
normally a marked
of the amount
found
in the
in fraction
Frac-
on the trail-
in this fraction
increase
of protein
appeared
size
were more of the cc-
d was obtained
the area under the peak. The results in eighteen cases covering a wide range of cancer are shown in Fig. 3. In some cases a five-fold increase over normal levels was found. Similar increases were found in a variety of other conditions, however, and this increase is by no means specific for cancer. The qualitative composition of fraction d is shown in Fig. 4. Compared with the normal several extra components appeared, there was great individual variation and no single pattern characteristic of cancer. from
Clin. Chim. Acta, 6 (1961) 163-169
166
M. P. TOMBS,
Chromatografihy
D. C. 0.
JAMES,
N. F. MACLAGAX
on CM-cellulose
Typical patterns from a normal and a cancer supernatant are shown in Fig. j and 6 respectively. It will be seen that although the fractions obtained were less homogeneous than with DEAE, the cc-globulins were distributed over peaks a, b and c, so that qualitative examination was facilitated. In general a gross difference between normal and cancer serum was noted as with DEAE above; albumin was decreased, peak a (cr-globulin) was increased, while peak c showed little variation. Immuno-electrophoresis Immuno-electrophoresis
revealed
the presence
of an a-globulin
(indicated
by an
arrow in Fig. 4) in five out of the twelve cancer cases, which was not found in normals, origin
I
A_.
2
0
4
7
album in
1
O&
5
0
6
0
a -
-
~__
albunin d
Fig. 4. Immune-electrophoresis of the d peak from DEhE chromatography. Examples I to 5 were from cases of cancer, whilst 6 was from a case of nephritis. 7 shows a whole serum. Cases j and 6 show an essentially normal pattern as in Fig. Id. Some of the a-globulin lines are not seen in normal serum, particularly that indicated with an arrow.
Lz-
0 -
l’ig. 5. CXI-cellulose chromatographyof supernatant fraction from j ml of normal serum. The protein and column were equilibrated with 0.02 iVl acetate PH 4.6. Buffers: 1, 0.05 M acetate, pH j.25; II, 0.08 M acetate, PH 6.0, III, 0.5 M NaCl, o. 1 ilf phosphate pi 9. Below is shown the results of simple and immuno-electrophoresis of the fractions.
while in some cases other ccr- and cc,-globulins were detected. Again there was, however, no constant qualitative change which distinguished cancer cases from normal. It soon became evident that there were individual variations in the a-globulins which were independent of disease and fell into three main groups. They occurred in fractions a, b and c. Fig. 7 illustrates the characteristic immuno-electrophoresis patterns. Fraction a always contained orosomucoid, but in nine out of seventeen individuals another component was also present (al) ; this was a post-albumin in starch-gel electrophoresis. Fraction b contained two more post-albumin components which show individual variations since in seven out of seventeen instances one of them was absent. Finally, all those cases, except one, which had a, also had a component
CHROMATOGRAPHY
(c)~ which was part of the F cr,-complex
OF SERUM PROTEINS
on starch-gel
‘67
electrophoresis
in fraction
c.
c4 was only seen in cases also showing the a, component. Chromatography of the material insoluble in half saturated ammonium sulphate was attempted on carboxymethyl cellulose 3, but resolution was never sufficient for quantitative estimation. There was a clear increase in the cr-globulin levels in cancer; one group of four closely related cc,-globulins, which were unique in possessing elution properties identical with those of the y-globulins, showed a relatively great increase.
E;w f
a
0
\ 0
%ion
aj, /-LT=oid
a
nurn b%?
b
b
o
O ?Tztx5l o p05~tyxmn
b
0
I 0
C
-
/
tmnsferrin d
o 0 ---
m
l
c
Fig. 6. CiWchromatography of supernatant fraction from 5 ml of serum from a case of advanced cancer. Conditions and buffers were as for Fig. 5.
V&
Fig. 7. Typical individual variation in fractions isolated by CM-cellulose chromatography of serum supernatant fraction. a, b, and c refer to the appropriate peaks in Figs. 5 and 6. For further explanation see text.
DISCUSSION
There have been very few reports describing the use of ion-exchange chromatography of protein in which quantitative estimations have been made from the chromatogram. This is doubtless a reflection of the insufficient resolution obtainable using these materials. Fortunately in this case a considerable simplification was possible since the cr-globulins of interest were soluble in half-saturated ammonium sulphate, and could be fairly clearly resolved from the other soluble components. The cr-globulins soluble in half saturated ammonium sulphate form a fairly homogeneous group. They all migrate rapidly in starch-gel electrophoresis and probably have low molecular weights. The results shown in Fig. 3 give an indication of the increase in the components which occurred in cancer. The increases found here are greater than have previously been reported for the cr-globulins as a wholelo. Inspection of starch-gel electrophoresis patterns of whole serum similarly indicate that in cancer these soluble cr-globulins show a more marked increase than those which migrate more slowly79 8 and it was concluded that a large part of the general rise in tcglobulins was due to the rapidly migrating group, although it only comprises about one-fifth of the total m-globulins. Elevated values for this group of globulins were constantly found in advanced Clin. Chim. Acta, 6 (1961)
163-169
3333
M. I'.TOMBS,
Il.C. 0, JAMES,
N, F. M.$CLAG,SN
cancer. 18 such cases are illustrated in Fig. 3 (DEAE) and 12 further cases showed similar increases when examined on CM-cellulose. This finding was not specific for cancer, since a proportion of cases of rheu~toid arthritis, nephritis and infections showed a similar increase. Qualitative examination of the components by immuno-electrophoresis, of which the examples shown in Fig. 4 are representative, revealed up to four components which were not detectable in normals, although in many instances no extra components were found. Some of the cr-globulins appeared in the supernatant from half saturation with ammonium sulphate when their level in whole serum was elesated. They thus appeared in the soluble fraction although in normal cases they wei-e confined to the insoluble fraction. Others, particularly the globulins marked with ;m arrow in Fig. 4, may be present in normal supernatants but at too low a lcscl to permit detection. The individual variations shown in Fig. 7 occurred in both normal and cancer cases. SMITNIES% s has reported two post~albunlins present in some iildi~r~d~~ls ant1 three in others. We find four post-~lbunl~ns in all, one of which (ce) was always present while the other three were of variable occurrence. There was a strong association between the incidence of post-albumin a1 and c, (Fig. 7). Although cJ was part of the FE,-zone, it was not a haptoglobin; it may be part of the system recently described by HIRSCHFELD ll. It was concluded that at least 6r.e a-globulins (three post-albumins, two haptoglobins and component c,) may show individual variation. While some combinatio~~s probably do not occur or are very rare, there are many varieties of normal ct-globulins. In our cases we were unable to show any correlation between these variations and the ABC) blood groups. While the potential capacity of the a-globulins in any indi\+dual to vary in disease is presumably determined partly by the genetic coIlstitution, me have found evidence to suggest that cancer can cause at least a q~antitati~~e increase in the production of ~-globulins. The variety of normal sc-globulins is such, however, that it would be difficult to obtain any direct evidence of qualitative change due to disease. This group of cr-globulins described here show? a marked increase in several disea5;c.s apart from cancer. Their origin is unknown, and the rise in disease might be duta either to tissue destruction or possibly to an anabolic process which may be enhanced in patients with tumours. The latter suggestion arises from the work of DARCYxl' and C.4MPRELI.13 who both demonstrated increased concentrations of an a-globulin in the serum of tumour bearing rats.
We are indebted to the British Empire Cancer Gmpaign for their support work. We also thank Mr. I<. B. COOKE for valuable discussion.
of this
(I) A method for the chromatographic fractionation of serum proteins to obtain a group of low molecular weight a-globulins is described (peak d). A rough estimate of this group by quantitative chromatography was possible, and this approach was used to investigate their variation in cancer.
CHROMATOGRAPHY
(2)
In 30 cases of cancer an elevation
169
OF SERUM PROTEINS
in serum a-globulin
up to ten-fold normal values. Other conditions and nephritis caused a similar rise.
was found, sometimes
such as rheumatoid
arthritis,
infection
(3) Qualitative investigation of peak d by immuno-electrophoresis and starchgel electrophoresis showed that orosomucoid, post-albumin and fast cc,-globulin were always present. In cancer cases other components frequently appeared, and the significance of this observation is discussed. (J) Individual variation of the post-albumin and fast cr,-components was encountered which made interpretation of qualitative changes difficult. REFERENCES E. X. PETERSON AND H. A. SOBER, J. Am. Chem. Sec., 78 (1956) 751. H. A. SOBER AND E. A. PETERSON, Federation Proc., 17 (1959) 1116. Ii. B. COOKE, M.P. TOMBS, R. D. WESTON, F. SOUTER AND N. F.MAcLAGAN,CZ~)Z. Chim.dcta, -I (1959) 779. J. J. SCHEIDEGGER, Z>ztenl. Arch. A1lerg.y .4$pl. Znzmuxol., 7 (1959) 0. SIVIITHIES, Biochem. J., 61 (1955) 629. CHI-CHIN LUNG, Biockent. J., 66 (1957) 552. L. BECKMANN, LanCet, 277(1959) 952. L. BECKMANN, Lalzcet, 278 (1960) 229. 0. SMITHIES, Biochem. J.. 71 (1959) 585. R. J. WIXZLER, .4duances iiz Cancer Research, I (1953) 503.
103.
J. HIRSCHFELD, B. TONSSON AND M. RASZVIUSON,9dzcve. 185 (1960)932. D. A. DARCY, Brit. J. Cancer, 11 (1957) 137. P. N. CAMPBELL, B. A. KERNOT AND 1. M. ROITT, Biochem.J., 71 (1959) Ijj Clin.Chiwz.Acta, 6 (rg61)163-1bg
CLINICA CHIMICA ACT.4
CHROMATOGRAPHY WITH
SPECIAL
M. P. TOMBS,
OF SERUM
REFERENCE D. C. 0. JAMES
PROTEINS
TO cr-GLOBULINS* AND
N.
F. MACL.4GhS
Westminster Medical School, Lozdon (Great Britain) (Received
July z&h,
1960)
Since PETERSON AND SOBERI introduced substituted cellulose as ion exchange materials for the column chromatography of proteins there have been numerous reports of the application of chromatography to protein fractionation2. It has found less application to serum proteins than to simpler systems and has been most successful in resolving mixtures containing a relatively small number of components. With present techniques the resolving power on serum proteins is poor. It is not
possible to obtain useful quantitative
results, since at the best only one or two of the
chromatographic components are electrophoretically homogeneous. It would appear therefore, that chromatography is not a suitable method at present for the primary fractionation of serum protein, but might give valuable results if, after a preliminary fractionation, it is employed for finer subdivision. We have described elsewhere some results obtainable after a preliminary ammonium sulphate fractionation 3. The protein soluble in half saturated ammonium sulphate could be split into a /%globulin, albumin and a mixture of a-globulins on diethyl-amino-ethyl cellulose (DEAE). We describe here a further investigation of the group of a-globulins soluble in half saturated ammonium sulphate, and their variation in cancer. While DEAE-cellulose offers the opportunity to obtain a quantitative estimate of the a-globulins, carboxy-methyl cellulose (CM) has some advantage for their qualitative analysis and a few cases have been examined using this material. METHODS
Chromatography The apparatus used in this work was similar to that described previously”. DEAE- and CM-cellulose were prepared as described by PETERSON AND SOBERI and packed into columns (2.5 cm diameter) to a depth of IO cm under air pressure of 5 lbs. sq. inch. Flow rates of 150-200 ml/h were maintained with the same air pressure. Stepwise elution was usually employed, using the buffers indicated in the appropriate figures. Suitable buffers were selected from a consideration of gradient experiments. In some runs on DEAE-cellulose the final buffer was applied through a mixing chamber (IOO ml) initially filled with the preceding buffer. Gradient elution was employed here to minimise albumin trailing into the final peak. Protein concentration in the eluent was determined by measurement of the absorption at 280 m,u either on individual fractions or continuously on an automatic recorder. * Based on a paper read at the 8th Colloquium on Protides of Biological Fluids, Bruges, 1960. Cl&. Chipn. Acta, 6 (1961) x63-169
bl. P. TOMBS, D. C. 0. JAMES,
164
iY. F. MACLAGAN
Salt fractionation An equal volume of saturated
ammonium
sulphate
was added to serum (usually
5 ml) and the precipitate separated by centrifugation at 7000 g for 20 min. The prccipitate was then suspended in half saturated ammonium sulphate (271 g/l solution) and re-centrifuged. All experiments were carried out at room temperature. Thr supernatants from both centrifugations were united, dialysed against water to l-t’move salts and finally into appropriate
equilibrating
buffer for chromatography.
Fig. I. Chromatography of supernatant fraction from 5 ml of serum on DEr\F:-cellulose. The column and the applied protein were equilibrated with 0.005 JW phosphate PH 7. Buffers were applied stepwise, except for V which was by gradient (for detail see text). Buffer compositions were: II, 0.02 M phosphate PH 6; III, 0.018 M phosphateo.oz MNaClp~6; IV, 0.06Mphosphate pH 5 ; V, 0.05 iW phosphate pH 5, o. IO M NaCl. Each fraction was of O-ml xwlume. Fractions were pooled into protein fractions a, b, c and d as shown. Below is the result of electrophoresis of the fractions. In each case, at the top is shown the result of bromphenol blue staining of the agar slab, and immediately below the results of immune-electrophoresis.
Electrophoresis After elution from the column, fractions were concentrated by perevaporation and then dialysed against water to remove salts. In some cases concentration dialysis against polyethylene glycol (mol. wt. 20,000) was used, but this led to contamination of the fraction with carbohydrate material. Although this did not interfere with electrophoresis it was felt that perevaporation was a safer procedure. After concentration immuno-electrophoresis was carried out in agar-gel by a slight modification of the method of SCHEIDEGGER 4. Starch-gel electrophoresis was by the method of SMITHIES~. Horse anti-human anti-serum (Institut Pasteur, Paris) was used throughout. The immuno-electrophoresis results shown in the figures were made by tracing a projection of the original slides. RESULTS
Chromatography
OK DEAE-cellulose
Fig. I shows the results of DEAE chromatography of the supernatant fraction from a typical normal individual. Fraction a was completely homogeneous by electro-
CHROMATOGRAPHY phoresis, another
but
spectrum ent
were
in this
from
immuno-electrophoresis
globulin.
The main
the column,
therefore, mained
showed
of transferrin.
Fraction
possible.
1%
presence
of
small
line, pink colour
1.6%
b invariably
of protein-bound
precipitated
and could not subsequently
in solution
the
immune-electrophoretic
characteristic
fraction.
OP SERUM PROTEINS amounts
hexose
immediately
be redissolved.
of
and absorption was pres-
after
Electrophoresis
However,
in haemolysed
sera it had
a reddish
in a large
scale preparation
in which
it was eluted
elution was not,
colour,
and
together
re-
with
at 410, 540 and 577 rnp. This is complex@. Fraction c was at least 957; albumin, but immune-electrophoresis revealed the presence of traces of cr-globulin. It contained about 0.8$;/, protein-bound hexose. Fraction d always contained a trace of albumin, but was mainly a mixture of CY%and cx,-globulin. Orosomucoid, a postalbumin and a part of the For,-complex were detected by starch-gel electrophoresis. The misture had about 11% protein-bound hexose. Fig. 2 shows the result of chromatography of the same ammonium sulphate fraction from three cases of advanced cancer. The pattern was qualitatively similar to
albumin.
Its
characteristic
absorption
spectra
showed
maxima
of a haptoglobin-haemoglobin
o
t
$j $
8
)Tjarea
I
1
1
1
2.
1
I cancer of long cancer of breast
0
0
CD
units
1
0
00
0
mewstatic 00 oa3
0 Fig. z. lose of serum Buffers
fracti%?
number
but the relative
tion c, the albumin,
was greatly
ing edge of the peak. prominent. globulin
The
most
The trace striking
peak d. A rough
and absolute
reduced
estimate
rheumatoid arthritis etc.
co
size of the peaks was changed.
and partial
cr-globulins change
-
Fig. 3, Variation of the size of peak d (see Figs. I, 2) in cancer and other conditions. The size of the peak was measured in area units, where one area unit is equal to approximately 0.5 mg of protein. Ail results have been calculated to IOO ml of serum.
Chromatography on DEAE-ceIlusupernatant fraction from 5 ml of in three cases of advanced cancer. and other conditions were the same as for Fig. I.
that of the normal
0
CDOCD
I 200
0
0000
caT%?a other cancer
was
resolution
normally a marked
of the amount
found
in the
in fraction
Frac-
on the trail-
in this fraction
increase
of protein
appeared
size
were more of the cc-
d was obtained
the area under the peak. The results in eighteen cases covering a wide range of cancer are shown in Fig. 3. In some cases a five-fold increase over normal levels was found. Similar increases were found in a variety of other conditions, however, and this increase is by no means specific for cancer. The qualitative composition of fraction d is shown in Fig. 4. Compared with the normal several extra components appeared, there was great individual variation and no single pattern characteristic of cancer. from
Clin. Chim. Acta, 6 (1961) 163-169
166
M. P. TOMBS,
Chromatografihy
D. C. 0.
JAMES,
N. F. MACLAGAX
on CM-cellulose
Typical patterns from a normal and a cancer supernatant are shown in Fig. j and 6 respectively. It will be seen that although the fractions obtained were less homogeneous than with DEAE, the cc-globulins were distributed over peaks a, b and c, so that qualitative examination was facilitated. In general a gross difference between normal and cancer serum was noted as with DEAE above; albumin was decreased, peak a (cr-globulin) was increased, while peak c showed little variation. Immuno-electrophoresis Immuno-electrophoresis
revealed
the presence
of an a-globulin
(indicated
by an
arrow in Fig. 4) in five out of the twelve cancer cases, which was not found in normals, origin
I
A_.
2
0
4
7
album in
1
O&
5
0
6
0
a -
-
~__
albunin d
Fig. 4. Immune-electrophoresis of the d peak from DEhE chromatography. Examples I to 5 were from cases of cancer, whilst 6 was from a case of nephritis. 7 shows a whole serum. Cases j and 6 show an essentially normal pattern as in Fig. Id. Some of the a-globulin lines are not seen in normal serum, particularly that indicated with an arrow.
Lz-
0 -
l’ig. 5. CXI-cellulose chromatographyof supernatant fraction from j ml of normal serum. The protein and column were equilibrated with 0.02 iVl acetate PH 4.6. Buffers: 1, 0.05 M acetate, pH j.25; II, 0.08 M acetate, PH 6.0, III, 0.5 M NaCl, o. 1 ilf phosphate pi 9. Below is shown the results of simple and immuno-electrophoresis of the fractions.
while in some cases other ccr- and cc,-globulins were detected. Again there was, however, no constant qualitative change which distinguished cancer cases from normal. It soon became evident that there were individual variations in the a-globulins which were independent of disease and fell into three main groups. They occurred in fractions a, b and c. Fig. 7 illustrates the characteristic immuno-electrophoresis patterns. Fraction a always contained orosomucoid, but in nine out of seventeen individuals another component was also present (al) ; this was a post-albumin in starch-gel electrophoresis. Fraction b contained two more post-albumin components which show individual variations since in seven out of seventeen instances one of them was absent. Finally, all those cases, except one, which had a, also had a component
CHROMATOGRAPHY
(c)~ which was part of the F cr,-complex
OF SERUM PROTEINS
on starch-gel
‘67
electrophoresis
in fraction
c.
c4 was only seen in cases also showing the a, component. Chromatography of the material insoluble in half saturated ammonium sulphate was attempted on carboxymethyl cellulose 3, but resolution was never sufficient for quantitative estimation. There was a clear increase in the cr-globulin levels in cancer; one group of four closely related cc,-globulins, which were unique in possessing elution properties identical with those of the y-globulins, showed a relatively great increase.
E;w f
a
0
\ 0
%ion
aj, /-LT=oid
a
nurn b%?
b
b
o
O ?Tztx5l o p05~tyxmn
b
0
I 0
C
-
/
tmnsferrin d
o 0 ---
m
l
c
Fig. 6. CiWchromatography of supernatant fraction from 5 ml of serum from a case of advanced cancer. Conditions and buffers were as for Fig. 5.
V&
Fig. 7. Typical individual variation in fractions isolated by CM-cellulose chromatography of serum supernatant fraction. a, b, and c refer to the appropriate peaks in Figs. 5 and 6. For further explanation see text.
DISCUSSION
There have been very few reports describing the use of ion-exchange chromatography of protein in which quantitative estimations have been made from the chromatogram. This is doubtless a reflection of the insufficient resolution obtainable using these materials. Fortunately in this case a considerable simplification was possible since the cr-globulins of interest were soluble in half-saturated ammonium sulphate, and could be fairly clearly resolved from the other soluble components. The cr-globulins soluble in half saturated ammonium sulphate form a fairly homogeneous group. They all migrate rapidly in starch-gel electrophoresis and probably have low molecular weights. The results shown in Fig. 3 give an indication of the increase in the components which occurred in cancer. The increases found here are greater than have previously been reported for the cr-globulins as a wholelo. Inspection of starch-gel electrophoresis patterns of whole serum similarly indicate that in cancer these soluble cr-globulins show a more marked increase than those which migrate more slowly79 8 and it was concluded that a large part of the general rise in tcglobulins was due to the rapidly migrating group, although it only comprises about one-fifth of the total m-globulins. Elevated values for this group of globulins were constantly found in advanced Clin. Chim. Acta, 6 (1961)
163-169
3333
M. I'.TOMBS,
Il.C. 0, JAMES,
N, F. M.$CLAG,SN
cancer. 18 such cases are illustrated in Fig. 3 (DEAE) and 12 further cases showed similar increases when examined on CM-cellulose. This finding was not specific for cancer, since a proportion of cases of rheu~toid arthritis, nephritis and infections showed a similar increase. Qualitative examination of the components by immuno-electrophoresis, of which the examples shown in Fig. 4 are representative, revealed up to four components which were not detectable in normals, although in many instances no extra components were found. Some of the cr-globulins appeared in the supernatant from half saturation with ammonium sulphate when their level in whole serum was elesated. They thus appeared in the soluble fraction although in normal cases they wei-e confined to the insoluble fraction. Others, particularly the globulins marked with ;m arrow in Fig. 4, may be present in normal supernatants but at too low a lcscl to permit detection. The individual variations shown in Fig. 7 occurred in both normal and cancer cases. SMITNIES% s has reported two post~albunlins present in some iildi~r~d~~ls ant1 three in others. We find four post-~lbunl~ns in all, one of which (ce) was always present while the other three were of variable occurrence. There was a strong association between the incidence of post-albumin a1 and c, (Fig. 7). Although cJ was part of the FE,-zone, it was not a haptoglobin; it may be part of the system recently described by HIRSCHFELD ll. It was concluded that at least 6r.e a-globulins (three post-albumins, two haptoglobins and component c,) may show individual variation. While some combinatio~~s probably do not occur or are very rare, there are many varieties of normal ct-globulins. In our cases we were unable to show any correlation between these variations and the ABC) blood groups. While the potential capacity of the a-globulins in any indi\+dual to vary in disease is presumably determined partly by the genetic coIlstitution, me have found evidence to suggest that cancer can cause at least a q~antitati~~e increase in the production of ~-globulins. The variety of normal sc-globulins is such, however, that it would be difficult to obtain any direct evidence of qualitative change due to disease. This group of cr-globulins described here show? a marked increase in several disea5;c.s apart from cancer. Their origin is unknown, and the rise in disease might be duta either to tissue destruction or possibly to an anabolic process which may be enhanced in patients with tumours. The latter suggestion arises from the work of DARCYxl' and C.4MPRELI.13 who both demonstrated increased concentrations of an a-globulin in the serum of tumour bearing rats.
We are indebted to the British Empire Cancer Gmpaign for their support work. We also thank Mr. I<. B. COOKE for valuable discussion.
of this
(I) A method for the chromatographic fractionation of serum proteins to obtain a group of low molecular weight a-globulins is described (peak d). A rough estimate of this group by quantitative chromatography was possible, and this approach was used to investigate their variation in cancer.
CHROMATOGRAPHY
(2)
In 30 cases of cancer an elevation
169
OF SERUM PROTEINS
in serum a-globulin
up to ten-fold normal values. Other conditions and nephritis caused a similar rise.
was found, sometimes
such as rheumatoid
arthritis,
infection
(3) Qualitative investigation of peak d by immuno-electrophoresis and starchgel electrophoresis showed that orosomucoid, post-albumin and fast cc,-globulin were always present. In cancer cases other components frequently appeared, and the significance of this observation is discussed. (J) Individual variation of the post-albumin and fast cr,-components was encountered which made interpretation of qualitative changes difficult. REFERENCES E. X. PETERSON AND H. A. SOBER, J. Am. Chem. Sec., 78 (1956) 751. H. A. SOBER AND E. A. PETERSON, Federation Proc., 17 (1959) 1116. Ii. B. COOKE, M.P. TOMBS, R. D. WESTON, F. SOUTER AND N. F.MAcLAGAN,CZ~)Z. Chim.dcta, -I (1959) 779. J. J. SCHEIDEGGER, Z>ztenl. Arch. A1lerg.y .4$pl. Znzmuxol., 7 (1959) 0. SIVIITHIES, Biochem. J., 61 (1955) 629. CHI-CHIN LUNG, Biockent. J., 66 (1957) 552. L. BECKMANN, LanCet, 277(1959) 952. L. BECKMANN, Lalzcet, 278 (1960) 229. 0. SMITHIES, Biochem. J.. 71 (1959) 585. R. J. WIXZLER, .4duances iiz Cancer Research, I (1953) 503.
103.
J. HIRSCHFELD, B. TONSSON AND M. RASZVIUSON,9dzcve. 185 (1960)932. D. A. DARCY, Brit. J. Cancer, 11 (1957) 137. P. N. CAMPBELL, B. A. KERNOT AND 1. M. ROITT, Biochem.J., 71 (1959) Ijj Clin.Chiwz.Acta, 6 (rg61)163-1bg