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The chromatography of serum proteins on cellulose ion exchange columns
CLI?\‘ICACHIMICA ACTA
THE
CHROMATOGRAPHY
ON CELLULOSE Ii.
B. COOKE,
Department
M. I’. TOMBS,
10X
OF SERUM EXCHANGE
R. II. \VESTOS,
of Chemical Pathology,
779
PROTEINS
COLUMNS*
F. SOUTER
AND N. F. MACLAGAS
Westminster Medical School, I..omlo~a(Gut-at Hvi:ain)
The synthesis, by PETERSON AND SOBER’, of modified cellulose ion exchangers in a form suitable for column chromatography opened up a new approach to the problem of fractionating the serum proteins. Although these materials have mainly been applied to the final purification of proteins already separated by other techniques SOBER et aL2 demonstrated that the anion exchanger diethylaminoethyl cellulose (DEAE) could also be used for the preliminary separation of serum proteins. Electrophoretic analysis, however, demonstrated the complex nature of the chromatographic peaks obtained. We, therefore, decided to investigate the behaviour of serum proteins on columns of the cation exchangers carboxymethyl cellulose (CMC) and cellulose phosphate. APPARATUS For this work we used the gradient elution system described by SOBER et al. with minor modifications. With our normal columns of z.5-cm diameter and approx. IO-cm column length we used a mixing chamber of IOO ml capacity stirred magnetically. The fixed volume mixing chamber, whilst not producing an ideal gradient curve has been used to obtain the same pattern on 2 different sets of apparatus. Our automatic scanner was built around a flow cell of o.g-cm light path fitted with tangential side arms to prevent streaming at high flow rates. A Uvispek spectrophotometer was used as a source of collimated light, and the output from the photocell fed into a Vibron 33B electrometer. Appropriate Vibron input ranges and Uvispek slit-widths were then chosen to give full-scale deflection of the recorder at IOO~~ transmission. The tracing obtained with this system could then be plotted on inverted semi-log graph paper to give a plot in good agreement with direct spectrophotometer readings up to O.D. 3.5. As a further refinement an auxilliary L.T. supply was provided to balance the photocell dark current. This supply could be shorted out by the fractioqcutter operating relay thus producing a mark on the recorder tracing whenever the fraction cutter plate moved. For scanning at 280 rnp we have now replaced the spectrophotometer by a simple photometer incorporating an interference filter with subsidiary filters of nickel salt solution and Chance OX7 glass. RESULTS We have found that the elution pattern for serum globulins on CM cellulose remains essentially the same for a given weight of exchanger irrespective of column * Paper presented at the 7th Colloquium on “Protides May 1959. References p.
783
of the Biological
Fluids”,
Bruges,
Ii. R. COOKE d d
780
TABLE SOk,?E CHARACTERISTICS
Exchanger
TYPe .%nion Cation Cation
I
MODIFIED
CELLULOSE
ION __
EXCHANGERS __.~~
8.0
8.0-5.0
4.0
4.5-8.0
-COOH SH -XH,
6.7
+.0-7.0
-- -SW,
b See ref. 2.
_
Buger
ph-a
Diethylamino ethyl cellulose Carbosymetbyl ceiluiose Cellulose phosphate
a See ref. 1.
OF
x-OL*4 @959)
_....
systems
Phosphateb Acetatephosphatec Phosphate (0.02-1.0 iM)
C See ref. 3.
shape and have recently confirmed a similar behaviour for cellulose phosphate. This would appear to exclude conventional adsorption chromatography as a major means of separation with cationic columns. With serum albumin a partitioning of the protein between z peaks which is dependent on column length is observed below PH 5.5 and we have suggested that this may be due to the isomeric forms of albumin known to exist at acid pH having differing equilibria with the column material”. E 280
Fig. I. Elution proteins.
patterns
of
human
serum
DEAF equilibrated with 0.005 ilI phosphate pH 7.0; II. 0.02 M phosphate, PH 6.0; III. 0.05 M NaH,PO,; IV. 0.05 M NaH,PO, + 0.02 M XaCl; V. 0.05 M NaW,PO, + 0.05 M NaCl; VI. 0.05 N NaH,PO, + 0.10 M NaCl. Gradient elution throughout.
OIETHYtAMINOfTHYL CELlULOSE
CM equilibrated with 0.02 M acetate pi 4.6; II. 0.05 M acetate, pH 5.2, applied stepwise; III. 0.08 M acetate, pw 6.0; IV. 0.10 M phosphate, pi 7.0; V. 0.10 M phosphate+o.go M NaCI, pw 9.2. Gradient elution except for buffer II.
-
Cellulose phosphate equiiibrated with 0.02 M XaH,PO,; II. 0.04 M phosphate, PH 5.8; III. 0.05 M phosphate, PH 6.2; IV. 0.06 M phosphate, PH 6.6; V. 0.10 M phosphate, PH 7.2; VI. o. 10 M phosphate + 0.50 M NaCl, PH = 9.5. Buffers applied stepwise throughout. Shaded areas indicate the presence of serum albumin as a major component of the peak. Other major components of peaks are shown below the relevant pattern.
The general characteristics of the 3 materials used are set out in Table I. It will be seen from this that, whilst the pK’s of CM cellulose and cellulose phosphate differ by nearly 3 pH units, the range of pH over which serum proteins can be adsorbed and &ted is similar for the z exchangers. This would indicate that separations are achieved primarily by the effect of PH and ionic strength on the adsorbed protein rather than on the adsorbent column. h’eferertces p. 783
CHROMATOGRAPHY OF SERUM PROTEISS
VOL. 4 (1959) Elution
from CM cellulose
from pH 4.6-6.0
columns
followed by phosphate
was achieved
with acetate
781 buffer
ranging
buffers up to pH 9.2. In the case of cellulose
phosphate elution was attempted entirely with phosphate buffers, ranging from pH 4.5-9.5, in order to provide a buffer system which did not absorb at 210 m/Aand which could be used for small scale runs (0.1 ml serum and less) (TO~IBS, SOUTER AND MACLACAX~). Typical elution patterns are shown in Fig. I for DEAE cellulose, CM cellulose and cellulose phosphate together with the elution schedules employed, Each system resolves, basically, into 6 main peaks, the first of which is usually a single component elcctrophoretically. The similarity in pattern between CM cellulose and E
n
1.5
Fig. 2. Elution patterns of supernatant fraction (LO ml bank plasma half-saturated with ammonium sulphate). Paper electrophoretic results are shown diagramatically below the relevant chromatographic peaks. Black areas indicate major components, cross-hatching minor constituents. .-\ll fractions are of 6 ml volume.
cellulose
phosphate
is most striking
with the albumin
partitioning
previously
men-
tioned occurring over the same pH range on the z materials. In view of the difficulty of obtaining single component peaks from whole serum it was decided to carry out a preliminary separation by half saturating the serum with ammonium sulphate at room temp. followed by centrifugation at 7000 x g. This procedure gave, in our hands, a supernatant which contained 4 components (by paper electrophoresis) : (a) Nearly all the albumin present in the original serum. (b) A variable amount of qglobulin. (C) An a,-globulin. (d) A ,&globulin. The precipitate, after one washing, also contained 4 components: (a) c(, globulin. (b) An cc,-globulin. (c) A @-globulin. (d) y-Globulin. Chromatography of the supernatant fraction on DEAE cellulose, as will be seen from Fig. 2, yielded: (a) A trace of y-globulin well resolved from other components. (b) /3-Globulin. This occurred mainly as a discrete peak with the characteristic pink
h’efcrem2s p. 78.1
I<. B. COOKE t?td.
782
VOL.
4 (Igjg)
colour and spectrum of siderophillin. Furthermore this peak was homogeneous by starch gel electrophoresis6and also when electrophoresed on cellulose acetatesin trisborate-sequestric with a,-globulin.
acid buffer 7 and stained with nigrosine. (c) Albumin contaminated (d) A double peak containing mainly fast cr,-globulin but with pre-
albumin and traces of ctr present. There was little advantage to be obtained
from running the supernatant
fraction
on CM cellulose. The dotted peak (Fig. 2) when present is electrophoretically pure a,-globulin (running under the albumin on starch gel) but its reproducibility is poor.
D.E.A.E. IL1 -
1.5
CM.
1.5 t
n-
Fig. 3. Elution patterns of precipitate fraction. (20 ml bankplasma half-saturated with ammo nium sulphate). Paper electrophoretic results are shown diagramatically below the relevant chromatographic peaks. Black areas indicate major components, cross-hatching minor constituents. All fractions are of 6 ml volume.
When the precipitate is similarly treated it will be seen from Fig. 3 that DEAE cellulose gives no greatly improved separation. These DEAE runs were carried out on unwashed precipitate-hence the presence of albumin. A better pattern is obtained with CM cellulose, although here again no new single component peaks appear. In recent experiments the separation of the slow a2- and /&globulins appearing between fractions 50 and 150 in Fig. 3 has been improved by a modified elution schedule but we have not succeeded in eliminating the contaminating fast y-globulin. Preliminary experiments using an initial rja saturation with ammonium sulphate pH 7.0 at 4” overnights have confirmed that this precipitates mainly y-globulin and some or,-globulin with a consequent improvement in the chromatographic resolution of the globulin fractions. This procedure also appears to stabilise the occurrence of
Iirfevems p.
783
“0~
4 (1959)
CHROMATOGRAPHY
cr,-globulin in the 507; saturated have some evidence that cellulose globulins.
OF SERUM
PROTEINS
783
ammonium sulphate supernatant. In addition we phosphate is better than CM cellulose for resolving ACKNOWLEDGEMEST
The work was supported by generous Westminster Hospital and from the British
grants from the Endowment Empire Cancer Campaign.
Funds
of
From the results it is concluded that single chromatographic runs of whole serum on modified cellulose columns are of limited value. However, when applied after initial salt fractionation this type of chromatography appears to be a promising technique for the preparation of individual proteins and possibly for diagnostic use. The best combination which we have investigated consists of a preliminary salt fracammonium sulphate followed by chromatography of tionation with 5oqb saturated the supernatant on DEAF, cellulose and the precipitate on CM cellulose or cellulose phosphate. Experimental details for these procedures are given. REFERENCES 1 E. il. PETERSON AND H. A. SOBER, J. Am. Chem. Sac., 78 (1956) 751. 2 H. A. SOBER, F. J, GUTTER, M. J. WYCKOFF AND A. E. I’ETERSON, J.
.Im.
Chem.
(1956) 756. 3 M. P. TOMBS, N. F. MACLAGAN AND I<. B. COOKE, J. Clin. Pathol., II (1958) 5jZ. 4 M. P. TOMBS, F. SOUTER AND N. F. MACLAGAN, Biochem. J., (1959) in the press. 5 M. D. POULIK AND SMITHIES, Biochem. J., 68 (1958) 636. 6 J. KOHN, Clin. Chim. Acta, 3 (1958) 450. 7 T. ARONSSON AND A. GRBNWALL, Stand. J. Clin. & Lab. Invest., 9 (1957) 338. 8 B. V. JACER AND M. NICKERSON, J. Biol. Chem., 173 (1948) 683.
sot
THE
CHROMATOGRAPHY
ON CELLULOSE Ii.
B. COOKE,
Department
M. I’. TOMBS,
10X
OF SERUM EXCHANGE
R. II. \VESTOS,
of Chemical Pathology,
779
PROTEINS
COLUMNS*
F. SOUTER
AND N. F. MACLAGAS
Westminster Medical School, I..omlo~a(Gut-at Hvi:ain)
The synthesis, by PETERSON AND SOBER’, of modified cellulose ion exchangers in a form suitable for column chromatography opened up a new approach to the problem of fractionating the serum proteins. Although these materials have mainly been applied to the final purification of proteins already separated by other techniques SOBER et aL2 demonstrated that the anion exchanger diethylaminoethyl cellulose (DEAE) could also be used for the preliminary separation of serum proteins. Electrophoretic analysis, however, demonstrated the complex nature of the chromatographic peaks obtained. We, therefore, decided to investigate the behaviour of serum proteins on columns of the cation exchangers carboxymethyl cellulose (CMC) and cellulose phosphate. APPARATUS For this work we used the gradient elution system described by SOBER et al. with minor modifications. With our normal columns of z.5-cm diameter and approx. IO-cm column length we used a mixing chamber of IOO ml capacity stirred magnetically. The fixed volume mixing chamber, whilst not producing an ideal gradient curve has been used to obtain the same pattern on 2 different sets of apparatus. Our automatic scanner was built around a flow cell of o.g-cm light path fitted with tangential side arms to prevent streaming at high flow rates. A Uvispek spectrophotometer was used as a source of collimated light, and the output from the photocell fed into a Vibron 33B electrometer. Appropriate Vibron input ranges and Uvispek slit-widths were then chosen to give full-scale deflection of the recorder at IOO~~ transmission. The tracing obtained with this system could then be plotted on inverted semi-log graph paper to give a plot in good agreement with direct spectrophotometer readings up to O.D. 3.5. As a further refinement an auxilliary L.T. supply was provided to balance the photocell dark current. This supply could be shorted out by the fractioqcutter operating relay thus producing a mark on the recorder tracing whenever the fraction cutter plate moved. For scanning at 280 rnp we have now replaced the spectrophotometer by a simple photometer incorporating an interference filter with subsidiary filters of nickel salt solution and Chance OX7 glass. RESULTS We have found that the elution pattern for serum globulins on CM cellulose remains essentially the same for a given weight of exchanger irrespective of column * Paper presented at the 7th Colloquium on “Protides May 1959. References p.
783
of the Biological
Fluids”,
Bruges,
Ii. R. COOKE d d
780
TABLE SOk,?E CHARACTERISTICS
Exchanger
TYPe .%nion Cation Cation
I
MODIFIED
CELLULOSE
ION __
EXCHANGERS __.~~
8.0
8.0-5.0
4.0
4.5-8.0
-COOH SH -XH,
6.7
+.0-7.0
-- -SW,
b See ref. 2.
_
Buger
ph-a
Diethylamino ethyl cellulose Carbosymetbyl ceiluiose Cellulose phosphate
a See ref. 1.
OF
x-OL*4 @959)
_....
systems
Phosphateb Acetatephosphatec Phosphate (0.02-1.0 iM)
C See ref. 3.
shape and have recently confirmed a similar behaviour for cellulose phosphate. This would appear to exclude conventional adsorption chromatography as a major means of separation with cationic columns. With serum albumin a partitioning of the protein between z peaks which is dependent on column length is observed below PH 5.5 and we have suggested that this may be due to the isomeric forms of albumin known to exist at acid pH having differing equilibria with the column material”. E 280
Fig. I. Elution proteins.
patterns
of
human
serum
DEAF equilibrated with 0.005 ilI phosphate pH 7.0; II. 0.02 M phosphate, PH 6.0; III. 0.05 M NaH,PO,; IV. 0.05 M NaH,PO, + 0.02 M XaCl; V. 0.05 M NaW,PO, + 0.05 M NaCl; VI. 0.05 N NaH,PO, + 0.10 M NaCl. Gradient elution throughout.
OIETHYtAMINOfTHYL CELlULOSE
CM equilibrated with 0.02 M acetate pi 4.6; II. 0.05 M acetate, pH 5.2, applied stepwise; III. 0.08 M acetate, pw 6.0; IV. 0.10 M phosphate, pi 7.0; V. 0.10 M phosphate+o.go M NaCI, pw 9.2. Gradient elution except for buffer II.
-
Cellulose phosphate equiiibrated with 0.02 M XaH,PO,; II. 0.04 M phosphate, PH 5.8; III. 0.05 M phosphate, PH 6.2; IV. 0.06 M phosphate, PH 6.6; V. 0.10 M phosphate, PH 7.2; VI. o. 10 M phosphate + 0.50 M NaCl, PH = 9.5. Buffers applied stepwise throughout. Shaded areas indicate the presence of serum albumin as a major component of the peak. Other major components of peaks are shown below the relevant pattern.
The general characteristics of the 3 materials used are set out in Table I. It will be seen from this that, whilst the pK’s of CM cellulose and cellulose phosphate differ by nearly 3 pH units, the range of pH over which serum proteins can be adsorbed and &ted is similar for the z exchangers. This would indicate that separations are achieved primarily by the effect of PH and ionic strength on the adsorbed protein rather than on the adsorbent column. h’eferertces p. 783
CHROMATOGRAPHY OF SERUM PROTEISS
VOL. 4 (1959) Elution
from CM cellulose
from pH 4.6-6.0
columns
followed by phosphate
was achieved
with acetate
781 buffer
ranging
buffers up to pH 9.2. In the case of cellulose
phosphate elution was attempted entirely with phosphate buffers, ranging from pH 4.5-9.5, in order to provide a buffer system which did not absorb at 210 m/Aand which could be used for small scale runs (0.1 ml serum and less) (TO~IBS, SOUTER AND MACLACAX~). Typical elution patterns are shown in Fig. I for DEAE cellulose, CM cellulose and cellulose phosphate together with the elution schedules employed, Each system resolves, basically, into 6 main peaks, the first of which is usually a single component elcctrophoretically. The similarity in pattern between CM cellulose and E
n
1.5
Fig. 2. Elution patterns of supernatant fraction (LO ml bank plasma half-saturated with ammonium sulphate). Paper electrophoretic results are shown diagramatically below the relevant chromatographic peaks. Black areas indicate major components, cross-hatching minor constituents. .-\ll fractions are of 6 ml volume.
cellulose
phosphate
is most striking
with the albumin
partitioning
previously
men-
tioned occurring over the same pH range on the z materials. In view of the difficulty of obtaining single component peaks from whole serum it was decided to carry out a preliminary separation by half saturating the serum with ammonium sulphate at room temp. followed by centrifugation at 7000 x g. This procedure gave, in our hands, a supernatant which contained 4 components (by paper electrophoresis) : (a) Nearly all the albumin present in the original serum. (b) A variable amount of qglobulin. (C) An a,-globulin. (d) A ,&globulin. The precipitate, after one washing, also contained 4 components: (a) c(, globulin. (b) An cc,-globulin. (c) A @-globulin. (d) y-Globulin. Chromatography of the supernatant fraction on DEAE cellulose, as will be seen from Fig. 2, yielded: (a) A trace of y-globulin well resolved from other components. (b) /3-Globulin. This occurred mainly as a discrete peak with the characteristic pink
h’efcrem2s p. 78.1
I<. B. COOKE t?td.
782
VOL.
4 (Igjg)
colour and spectrum of siderophillin. Furthermore this peak was homogeneous by starch gel electrophoresis6and also when electrophoresed on cellulose acetatesin trisborate-sequestric with a,-globulin.
acid buffer 7 and stained with nigrosine. (c) Albumin contaminated (d) A double peak containing mainly fast cr,-globulin but with pre-
albumin and traces of ctr present. There was little advantage to be obtained
from running the supernatant
fraction
on CM cellulose. The dotted peak (Fig. 2) when present is electrophoretically pure a,-globulin (running under the albumin on starch gel) but its reproducibility is poor.
D.E.A.E. IL1 -
1.5
CM.
1.5 t
n-
Fig. 3. Elution patterns of precipitate fraction. (20 ml bankplasma half-saturated with ammo nium sulphate). Paper electrophoretic results are shown diagramatically below the relevant chromatographic peaks. Black areas indicate major components, cross-hatching minor constituents. All fractions are of 6 ml volume.
When the precipitate is similarly treated it will be seen from Fig. 3 that DEAE cellulose gives no greatly improved separation. These DEAE runs were carried out on unwashed precipitate-hence the presence of albumin. A better pattern is obtained with CM cellulose, although here again no new single component peaks appear. In recent experiments the separation of the slow a2- and /&globulins appearing between fractions 50 and 150 in Fig. 3 has been improved by a modified elution schedule but we have not succeeded in eliminating the contaminating fast y-globulin. Preliminary experiments using an initial rja saturation with ammonium sulphate pH 7.0 at 4” overnights have confirmed that this precipitates mainly y-globulin and some or,-globulin with a consequent improvement in the chromatographic resolution of the globulin fractions. This procedure also appears to stabilise the occurrence of
Iirfevems p.
783
“0~
4 (1959)
CHROMATOGRAPHY
cr,-globulin in the 507; saturated have some evidence that cellulose globulins.
OF SERUM
PROTEINS
783
ammonium sulphate supernatant. In addition we phosphate is better than CM cellulose for resolving ACKNOWLEDGEMEST
The work was supported by generous Westminster Hospital and from the British
grants from the Endowment Empire Cancer Campaign.
Funds
of
From the results it is concluded that single chromatographic runs of whole serum on modified cellulose columns are of limited value. However, when applied after initial salt fractionation this type of chromatography appears to be a promising technique for the preparation of individual proteins and possibly for diagnostic use. The best combination which we have investigated consists of a preliminary salt fracammonium sulphate followed by chromatography of tionation with 5oqb saturated the supernatant on DEAF, cellulose and the precipitate on CM cellulose or cellulose phosphate. Experimental details for these procedures are given. REFERENCES 1 E. il. PETERSON AND H. A. SOBER, J. Am. Chem. Sac., 78 (1956) 751. 2 H. A. SOBER, F. J, GUTTER, M. J. WYCKOFF AND A. E. I’ETERSON, J.
.Im.
Chem.
(1956) 756. 3 M. P. TOMBS, N. F. MACLAGAN AND I<. B. COOKE, J. Clin. Pathol., II (1958) 5jZ. 4 M. P. TOMBS, F. SOUTER AND N. F. MACLAGAN, Biochem. J., (1959) in the press. 5 M. D. POULIK AND SMITHIES, Biochem. J., 68 (1958) 636. 6 J. KOHN, Clin. Chim. Acta, 3 (1958) 450. 7 T. ARONSSON AND A. GRBNWALL, Stand. J. Clin. & Lab. Invest., 9 (1957) 338. 8 B. V. JACER AND M. NICKERSON, J. Biol. Chem., 173 (1948) 683.
sot