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Calcium phosphate chromatography of normal human serum and of electrophoretically isolated serum proteins
216
s. CHAYKIN, K. BLOCH
VOL.
31 (1959)
REFERENCES x T. T. TCHEN AND K. BLOCH, J. Biol. Chem., 226 (1957) 921. 2 E. C. TAYLOR AND A. J. CROV'ETTI, J. Org. Chem., 19 (1954) 1633. 3 G. R. CLEMO AND H. KOENIG, J. Chem. Soc., (1949) $ 2 3 i . 4 M. L. W u CHANG AND B. C. JOHNSON, J. Biol. Chem., 226 (1957) 799. 6 K. POLLER AND W. LINNEWEH, Ber., 59 (1926) 1362. 6 G. DUNN, J. j . GALLAGHER, G. T. NRWBOLD AND F. S. SPRING, J. Chem. See., (1949) S 126. v G. R. CLEMO AND H. MclLwAIN, J. Chem. Soc., (1938) 4798 G. R. CL]EMO AND A. F. DAGLISH, ] . Chem. Soc., (195o) 1481. 9 M. S. FISH, C. C. SWXELEY, N. M. JOHNSON, E. P. LAWRENCE AND E. C. HORNING, Biochim. Biophys. Acta, 21 (1956) 196. 1o A. MAY, Enzymologia, 18 (1957) 142.
CALCIUM P H O S P H A T E C H R O M A T O G R A P H Y O F N O R M A L H U M A N S E R U M AND O F ELECTROPHORETICALLY
ISOLATED SERUM PROTEINS
STELLAN HJERTt~N
Institute o] Biochemistry, University o] Uppsala (Sweden) (Received M a y 9th, 1958)
SUMMARY
The usefulness of calcium phosphate as an adsorbent for proteins has been demonstrated by experiments with whole serum and electrophoretically isolated serum proteins. The separation of, for example, ~-lipoprotein and 7-globulin from whole serum and caeruloplasmin from as-globulin may easily be attained. A heavy a Sglobulin has been isolated b y cutting a column, utilizing the property of proteins to displace each other.
INTRODUCTION
The development of chromatographic techniques during the recent years has made it possible to achieve extensive purification of proteins without recourse to complicated and expensive equipment, usually employed for this purpose. In a previous paper 1 we described some chromatographic experiments with serum proteins o n columns of calcium phosphate. As the results obtained were interesting and promising, the chromatographic behaviour of the serum proteins has been studied in further detail. This paper deals with these investigations and also with the practical usefulness of the strong mutual displacement often shown by proteins. As model substances, electrophoretically isolated proteins of normal human serum have been used. For comparison, one experiment with whole serum has also been described. For information concerning protein chromatography on calcium phosphate as well as on other adsorbents, the reader is referred to the paper mentioned above and to a survey Re#~ences p. a35.
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of recent progress by Moom~ AI~D STEIN2. Different adsorbents are also discussed by CALMON AND KR:ESSMANm. MATERIALS
The normal human sera used in this investigation were obtained from different blood donors at the Blood Donor Centre, Academic Hospital, Uppsala. No pooled sera were used in any of the experiments. The serum applied to columns for electrophoresis or chromatography, was one day old at the most. The proteins separated b y zone electrophoresis were chromatographed without unnecessary delay. Thus, lyophilization and freezing were omitted to avoid the risk of denaturation of the proteins. METHODS
Chromatography All chromatographic experiments were carried out on the same adsorbent, viz., hydroxylapatite. The preparation of this modified calcium phosphate was described b y TISELIUS, HJERT~N AND LEVINx in a paper in which also practical details concerning the method were treated. The columns were packed under or in the absence of pressure, depending on the flow rate desired. This corresponded to about 3oytfl]h for columns with a length of z o c m and an inner diameter of x.3 era. For the elution of the proteins, sodium phosphate buffers were mostly used. Owing to its higher solubility, potassium phosphate buffer was used when phosphate concentrations of o.3 M or higher were required for the desorption. As abbreviations for these buffers, the notations NaPB 6.8 and K P B 6.8 are introduced; the figures indicate the pH of the solution. Unless otherwise specified, the dialysis of the samples and the pretreatment of the columns was performed with the same buffer concentration as was used in the first step for the ehtion. This concentration is indicated in the corresponding chromatogram. All chromatographic experiments were carried out in the cold room ( + 4 °) with the exception of the gradient elution of a crude preparation of caeruloplasmin which was performed at room temperature ( + 2I°).
A bsorption measurements o/the e~uent The effluent was collected in an automatic time-operated fraction collector, The concentration of the proteins in the different fractions was determined b y measuring the absorption at z8o mt~ with a Beckman DU spectrophotometer, using a x-cm cell. For continuous recording of the effluent, a simple filter photometer was constructed, which will be described in a subsequent publication.
Concentration o/protein solutions The protein solutions, which were to be analysed by free electrophoresis or ultracentrifugation were concentrated b y ultraftltration through a collodion membrane according to MIES3. The collodion sack was supplied b y MembranengeseUschaft, G6ttingen. Protein concentration was determined by measurements of the refractive indices and b y using the conversion factor 1.8. xo-~. The fl-lipoprotein concentration was estimated with the same value of the factor, though this is certainly not correct. RefR~c~ p. ~35.
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Ultracentri[ugation Sedimentation analyses were carried out with a Spinco analytical ultracentrifuge, model E. Prior to the analyses, the protein solutions were dialysed against o.2 M sodium phosphate buffer, pH 6.8. The sedimentation constants were calculated to the density and viscosity of water at 2o °, but not extrapolated to the protein concentration zero. They are represented b y the notation $20, w.
Zone electrophoresis To obtain the main electrophoretic components of serum, zone electrophoresis on cellulose columns according to PORATH4 and GEDIN AND PORATH 5 was used. The column, which had a length of 15o cm and a diameter of 6.5 cm, was loaded with IOO ml of normal human serum, dialysed against veronal buffer, p H 8.6, ionic strength o.o3. The run was conducted in the same buffer for 7 ° h at 145 mA.
Free electrophoresis Some chromatographic fractions were analysed in a Tiselius-Svensson apparatus (type LKB), with cells requiring 12 ml of a protein solution.
Paper electrophoresis All runs were conducted in veronal buffer, p H 8.6 and ionic strength 0.05, with an apparatus of the horizontal, moist-chamber type. The electropherograms were stained with bromophenol blue. For lipoproteins Sudan-Black B was used. In the earlier experiments the concentration of the dilute chromatographic fractions, prior to the electrophoretic analyses, was achieved b y pervaporation, i.e. b y blowing air over a dialysis bag filled with the solution to be concentrated. But as pervaporation involves some disadvantages, another method was desired, preferably one which would permit comparatively large volumes to be applied directly to the paper without previous concentration. A zone-sharpening method fulfilling these requirements was worked out. It will be described in a future publication. EXPERIMENTS AND RESULTS
Whole serum I ml of undialysed serum was filtered into a column, 1. 4 × 21 cm. The first fraction in the diagram in Fig. I is characterized b y the fact that it leaves the column without any adsorption, and that it is stained b y ninhydrin but not by bromophenol blue, which indicates that this fraction contains low-molecular-weight substances. Determination of the molecular weights of these substances has not been undertaken, but it m a y be mentioned here that all of them pass through the pores of a common dialysis membrane. Paper chromatography in butanol-acetic acid and paper electrophoresis in pyridinium formate reveal several components. No further analyses of these substances have been carried out. The other chromatographic fractions have been analysed by paper electrophoresis--after concentration b y pervaporation. The result is represented in Fig. 2. It is evident from these electropherograms that a rather far-going purification is obtained already in one chromatographic experiment. According to SCHULTZE 6, m o r e than one hundred components of whole serum have been identified up to now. It is Re]erences p. 235.
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CtO2M
CHROMATOGRAPHY OF HUMAN SERUM
CtO3M
~04M
~O~M
CtO6M
~08M
; CIIOM
~,15M
2I 9
n 20M
Q30M
~65M
Alb
1.0. CLS.
2bo I
•
tO0 $odiutn p h o s p h a f e buffer. OH 6.8
,
,
300 ~. l u a f e. (toO= ~( ~ofass#urn-~ phosphate buffe~ ~ £ 8
Fig. I. Chromatographic behaviour of human serum in stepwise elution with phosphate buffer, pH 6.8, on a calcium phosphate column, i. 4 × 2z cm. i ml undialysed serum was applied. expected that many of these proteins possess affinities of similar magnitude for the adsorbent, and since the number of fractions eluted from the column must necessarily be very small in comparison with the number of different protein components present in serum, it is quite plausible that many of these chromatographic fractions are of a heterogeneous nature. Some proteins, however, show a very specific adsorption, an advantage which may be utilized for chromatographic isolation directly from whole serum. This is true in the ease oi fl-lipoprotein and 7-globulin, as is evident from the corresponding chromatograms (Figs. IO and z4).
Fig. 2. Paper electropherograms of the different chromatographic serum fractions in Fig. I. The notation O refers to unfractionated serum. The presence of the great number of proteins in whole serum makes the interpretation of the chromatogram somewhat difficult. The work should be facilitated if the proteins of the whole serum were first separated into groups and these groups then chromatographed separately. Since very well defined groups are easily obtainable by zone eleetrophoresis, a separation of the serum proteins by this method was made prior to chromatography. As it is not possible to isolate al-globulin well enough b y zone electrophoresis, this component has not been studied chromatographically. Relerences p. z35.
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S. HJERT~-I~I
Albumin
In a previous paper z the chromatography of bovine serum albumin has been described. The three fractions obtained were homogeneous on rechromatography. The preparation used was a freeze-dried one and contained both monomers and dimers. Some separation with regard to these was obtained. Thus the first fraction contained only monomers. Attempts to find differences in electrophoretic mobility in the three chromatographic fractions obtained were not successful. E2e
1 0,04
0.07/"/
H
ll
111
O.I114
0.65H
Potosslum phox~ohafe I.*~
buffer, p H 6.8
1.0
i
0.5.
{)
0.0
2'5 •
Sodium
phosphate
510 buffer, p H 6.0
75
Frachon number )
Fig. 3. Chromatogram of stepwise elution of h u m a n serum albumin.
With h u ma n serum albumin, however, some correlation between chromatographic and electrophoretic separation is obtained; this will now be described. 19o ml of a 0.2 % solution of human albumin, dia]ysed against 0.02 M NaPB 6.8, was applied to a column, 3.8 × 13.5 cm. The column was equilibrated for 15 h with 0.02 M NaPB 6.8. Elntion was performed with 250 ml 0.04 M N a P B 6.8, 300 ml 0.07 M NaPB 6.8, 250 ml o . I I M N a P B 6.8 and 250 ml 0.65 M K P B 6.8. The chromatogram is given in Fig. 3- Paper electrophoresis in veronal buffer, p H 8.6, ionic strength o.o5, showed that fraction I I I had a smaller mobility than I and I I (Fig. 4). With free electrophoresis in veronal buffer of the same p H but of a higher ionic strength (o.io), this difference in mobility was determined and found to be 6 %. Free electrophoresis of a mixture of the fractions I, I I and I I I resulted in one peak only. A]I three fractions contained lipid material associated with the albumin, as revealed b y paper electrophoresis and subsequent staining with Sudan-Black B. Electrophoretic analysis in acetate buffer, p H 4.1, ionic strength 0.05, was also carried out. At this lower p H two peaks were obtained ~-n. The mobilities of these were different for the three fractions. Thus fraction I I I h a d a rate of migration 50 % higher than that of I. The ratio between the amounts of the two electrophoretic components of these two chromatographic fractions was not of the same order of magnitude as shown in Fig. 5. The eiectrophoretic data are given in Table I. To test the reproducibility of these results, another experiment was performed in the way just described but albumin from another blood donor was used. The three Relerences p. 235.
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22I
chromatographic fractions now showed no differences in mobility at pH 8.6. At pH 4.I, however, the three electropherograms corresponding to the three chromatographic fractions differed in appearance. However, the mobilities and the ratio between the amounts of proteins in the two peaks of the electropherogram were different from those obtained in the first experiment. A plausible reason for this low reproducibility regarding the correlation between electrophoresis and chromatography will be presented under DISCUSSION.
a
6
0
i
//
~
!
6
//I
Fig. 4. Paper electropherogram of the three albumin fractions in Fig. 3.
Fig. 5. Free electrophoresis in acetate buffer, p H 4. i, and ionic strength o.05, of the unchromatographed albumin (O) and the three chromatographic fractions (I, II, III). The peak to the left (6) represents the protein-buffer salt boundary. The photographs are of the ascending boundaries. The potential gradients were io.o, Io.5, 9.9 and 9.7 V/cm respectively. The current was in all 4 runs adjusted to 15 mA. TABLE I ELECTROPHORETIC DATA OF CHROMATOGRAPHIC FRACTIONS OF HUMAN SERUM ALBUMIN
Mobilities are given in cm]sec/V/cm • Io I and based on values from both limbs. Un~k~me~ograpl~d
Frcu$ionI
Fr~ios II
--6. 7
--6.6
Fr~ios III
Mobility in veronal buffer,, pH 8.6,/'[2
ffi o . I o
--6.6
m6.2
Mobility in acetate buffer, p H 4.I, F/2 ~ o.o 5
5.0 5-7
5.~ 6.0
4.I 5.4
7.5 9.0
The ratio between the amounts of the two electrophoretic components at p H 4.I
z.o
Difficult to determine (see Fig. 5)
z,o
o.8
~l-Glob~din Ioo ml of a o.o5 % al-globulin solution, diaiysed against o.o5 M N a P B 6.8 was added in drops to a column x.6 × 25 cm, equilibrated with the same buffer. When Re/erences ~. z 3 5 .
222
s. ~J~RT~N
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the whole protein solution had been filtered into the column a narrow blue band was observed at the top of the column, followed by a broad yellow zone. The lower part of the column was not colored, but from other experiments made it is clear that proteins are adsorbed there also. The chromatogram is shown in Fig. 6. Since the blue band was suspected to contain caeruloplasmin, activity tests for this enzyme were made. These measurements were performed according to BROMAN1= and the values obtained are indicated on the chromatogram. It should be mentioned that during elution with o.20 M NaPB 6.8 the blue band migrated down the column but suddenly stopped, with the result that only part of the band left the column. The rest could be eluted with a higher buffer concentration and thus two peaks with oxidase activity were obtained.
~eo
I1
2. 0
1.5. C~IO M
CXlS M
CX20 M
1.0.
0.65M Pofassiur~ phosphafe buffer, p H 6.6
0,3 0.5.
0. I 0.0
I0 20 30 <----Sodium p h o s p l ~ l e buffer, p H 6 . 8
Krocfion n u m b e r
O.O
Fig. 6. Chromatogram of stepwise elution of ai-globulin. The dashed curve refers to activity test of caeruloplasmin.
Analyses of the three main fractions were carried out with ultracentrifugation. The sedimentation constants are given in the legend to the diagrams in Fig. I5 A. For comparison it may be mentioned that WALLENIUS, TRAUTMAN, KUNKEL AND FRAI~KLINls recently reported that electrophoretically-isolated a2-globulin contains components with the sedimentation constants 4.1, 6.6, 12.1 (traces) and 18.5 S, while BRATTSTEN14 found the values 3.2, 7.0, 12.6 and 15.9 S. The 2.9 S component present in the first chromatographic fraction may be a=-seromucoid, which according to SCI~ULTZE6 is characterized b y a sedimentation constant of 2.6 S. No efforts have been made to obtain more homogeneous fractions in the ultracentrifuge by trying buffer concentrations other than those shown in the chromatogram. It is, however, quite evident that for the ehition of caeruloplasmin a phosphate concentration of 0.65 M Relerences p z35.
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is unnecessarily high. It should be changed to 0.25 M. It may be of value to have caeruloplasmin available in large amounts so that one could dispense with the comparatively time-consuming zone electrophoresis as an intermediate step. Thus a crude preparation of caeruloplasmin, received from AB KABI, Stockholm, Sweden, was chromatographed. Since the main impurity in this preparation (made from the Cohn fraction IV) is albumin, and this protein is eluted with much lower phosphate concentration than caeruloplasmin, it is, of course, very easy to obtain a high degree of purification. Chromatography was carried out as gradient elution at room temperature in the following way. 4 ° mg of the crude preparation was dialysed against 0.05 M NaPB 6.8 and applied to a column 1.3 × 20 cm, treated with the same buffer. The buffer gradient was made in a simple apparatus similar to that used by DRAKEls. The mixing chamber was filled with 25 ° ml 0.05 M NaPB 6.8 and the other chamber with 13o ml 0.4 M NaPB 6.8. Continuous recording produced the chromatogram depicted in Fig. 7. The fractions corresponding to the second peak were characterized b y a blue color. Analyses of these b y paper electrophoresis (Fig. 8) and ultracentrifugation (Fig. I5 B) indicated homogeneity. The sedimentation constant was calculated to be 7.2 S, whereas SCHULTZE15 reported a value of 7.1 S. It may be observed that a concave gradient was chosen for the elution. According to the experience gathered in this laboratory, convex gradients should be avoided in protein chromatography, since they increase the risk of tailing.
Fig. 7- Continuous recording of a gradient elution of a crude preparation of caeruloplasmin. The horizontal arrow (< 7) indicates the fractions which have been combined and analysed with the aid of ultracentrifugation (Fig. I5) and paper electrophoresis (Fig. 8).
Relerences p 235.
Fig. 8. Paper electrophoresis of caeruloplasmin, obtained from the gradient elution in Fig. 7. The method of zone sharpening was used, which made it possible to apply the dilute caeruloplasmin solution to the paper without first concentrating it. The notation 0 refers to t h e unchromatographed, crude caeruloplasmin preparation.
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Further information on the chromatographic behaviour of the a=-globulins will be found under Cutting the columns. How to obtain a crude preparation of caeruloplasmin directly from undialysed whole serum is described in connection with t h e discussion of the results obtained by chromatography of the a=-globulin. [3-Glob~din
The column, x.3 × z o c m was equilibrated with a comparatively high phosphate concentration, viz. o.zo M NaPB 6.8, before the application of 30 ml of a o.z % /3-globulin solution. The chromatogram (Fig. 9) shows two fractions. Pretreatment with the high buffer concentration caused the first fraction to emerge from the column without any adsorption. The second fraction, however, had a very strong affinity to the calcium phosphate. Some of the properties, characterizing the two fractions, are given in Table II, from which it is evident that the first fraction contains ill-metalcombining protein and the second fi-lipoprotein. Electrophoresis and ultracentrifugation indicated that the lipoprotein was somewhat denatured, hence its mobility and sedimentation constant were not calculated. Other indications of denaturation were the facts that the solution had lost its yellow color and that the peak at 280 m/z was very fiat, probably due to considerable light scattering, owing to condensation of lipid material. This denaturation is not caused, however, by adsorption (see DISCUSSION).
No attempts were made to separate chromatographically the two components E2ao O.lOh'l
CUSM
0.20M
t3H Po fassiurn phosp/'lafe
t.O
buffer. pPI 6 . 0
Eluafe (rnlf <'~Sodium DhosDh¢;l~ b u f f e r . ~o~'/~ 8 -'~
Fig. 9. Chromatogram of stepwise elution of H-globulin. The first fraction contains ~z-metal-
combining protein, and the last ~-lipoprotein. TABLE If
ANALYSES OF TWO CHROMATOGRAPHIC FRACTIONS OF H'GLOBULIN
Mob~Cy Colin, o! tl~ $ol'~l~
St~s~ ~ bnm~plumol b l ~
S t 4 i n ~ w~k S~dar.-B~ B
Fraction I
Reddish brown
Strong
No
Fraction II
Opalescent
Weak
Strong
R s l e r e n c e s p. z 3 5 .
im ~ o ~ b ~ t e , pH 8.6 1"1~= o.zo
- - 3.x" x o -~ cm/sec/V/cm --
Sao, w S
5.5 ( m a i n component) 7.0
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(1959)
CHROMATOGRAPHY OF HUMAN SERUM
@-25
which, according to the ultracentrifugation data, are present in the first fraction. The sedimentation constants are similar to those reported by a number of authors ~S-ls, 17,- . A comparison between the chromatograms in Fig. z and Fig. 9 shows that it would be possible to isolate the fl-lipoprotein chromatographically without previous electrophoresis. Therefore a column 1.3 × 2o cm was treated with o.I M NaPB 5.8 and loaded with 8.5 ml undialysed normal human serum. The chromatogram is given in Fig. io. The most strongly adsorbed fraction contained/~-lipoprotein. The solution was yellow and had an absorption maximum at 46o rag. As first pointed out by MEHL19 and later proved by O~cLEY", this color is due to the presence of a carotenoid pigment. The carotenoids, fl-carotene and lycopene, are transported in the body exclusively by the serum lipoproteins". That the lipoprotein is homogeneous when analysed with the aid of paper electrophoresis, is quite evident from Fig. 2. The uncorrected sedimentation constant was found to be 2.I S. The corresponding diagram is given in Fig. IsC. Since it is known 1~ that the value of the partial specific volume of the/3-1ipoprotein differs considerably from that of "ordinary" proteins, and since the author did not determine it, no correction of this sedimentation constant was made. ONCLEY, SCATCHARD AND BROWN 1~ reported a corrected value of 2.8 S for a 0.8 % solution.
E2801 O, I O M
0. 2'~ M
O;(;~M
Pofoss~Jrn p~ospkQte buffer, pl-t ~ 8
2,5.
2.0.
1.5-
1.0-
0.I 0 . 0 ~.-~ ~ " •
. . . . 50 tOO 150 Sodium ohos~'~ b u f f e r , o H (~6
~
0.0 E / u a f e (m/)
Fig. IO. Chromatography of whole serum for preparation of ~-lipoprotein. 8. 5 ml undialFsed serum was applied on a column, x.3 × 2o cm. Only the last fraction can be stained with Sudan-Black B. The dashed line refers to activity test for caeruloplasmin.
Re/eTe.ces p. 235.
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y-Globulin io mg of a fast ?-globulin component, isolated by zone electrophoresis, was chromatographed on a column 1.2 × I 3 cm. Simultaneously a similar experiment with IO mg of a slow 7-globulin component was performed on another column of the same size. A comparison of the chromatograms (Fig. II) shows that the hydroxylapatite is sensitive to the differences between the two y-globulin components. However, since the elution ranges of the two y-globulins differ only rather slightly, several similar experiments were made with y-globulins from other blood donors. Buffers of lower pH ( = 6.8) were tried also. All these experiments gave chromatograms similar to that in Fig. II. In a previous paper z we described a gradient elution of 7-globulin. Only one very extended peak was obtained. Three fractions corresponding to different parts of the peak were analysed by means of paper electrophoresis and rechromatography. We reported that "there is a slight but probably significant difference in electrophoretic mobility on paper between the three v-globulin fractions--the more easily eluted fractions show somewhat faster migration". Thus these previous results agree with those presented in this paper.
E~e°I
o.o ,lo.os,o.o,io,,lo.o,io.o io.,o-
O.o~O" I
50
~
I00
,,%~ 150
E l u a t e [rn/)
Fig. H . Chromatograms showing the stepwise elution of a " f a s t " ( - O - O - O - ) and a "slow" ( - O - O - O - ) ?-globulin iraction. Elution performed with sodium phosphate buffer, pH 7.9.
Cutting the columns When filtering the solutions of colored proteins into a column, the different proteins often appear as sharply defined zones on the column already before ehition is started (see for instance the chromatographic experiment with as-globulin ) . Thus the proteins show a very strong mutual displacement. To find out whether this effect could be used as a basis for separation, the following experiments were carded out. Whole serum was applied on a column, treated with o.oz M NaPB 6.8. All serum proteins were adsorbed at this low buffer concentration. Displacement was achieved with 0.65 M K P B 6.8. This phosphate concentration was high enough to elute all protein material from the column. In the chromatogram obtained only one peak appeared. This peak was, however, extended, and analyses by paper electrophoresis of different parts of the peak indicated that a certain separation had been attained. But this separation was not efficient enough to be useful for practical purposes. It seems probable that proteins with different affinities for the calcium phosphate arranged themselves as distinct zones on the column during the application of the whole serum, but the separation thus obtained was destroyed b y the displacement brought about with the phosphate buffer. An attempt was therefore made to utilize the mutual displacement already taking place when the sample is applied to the column, and to improve the separation only by washing with a weak buffer solution. Under these conditions the zones remain on the column, and therefore a method for cutting the column was devised. References p. z35.
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The chromatographic column was of the common type, with the exception that the glass tube was replaced by a piece of polyvinyl chloride tubing (Fig. 12). The column was pretreated with 0.02 M NaPB 6.8. I ml serum was dialysed against the same buffer. After application of the serum the column was washed with o.o2 M NaPB 6.8. Cutting was carried out with a safety-razor blade in the manner shown in Fig. 12. Each section, the length of which was about I cm was suspended in 1.5 ml 1.3 M K P B 6.8. Analysis of the supernatant, performed with paper electrophoresis, showed that the upper part of the column contained v-globulin, followed by a section containing ~-globulin and albumin. The rest of the column was covered with albumin.
G
-Y
G
8
t ]
o°%
p
Fig. I2. C u t t i n g of columns. G: Glass bulb. P : PVC-tube, o.7 × 2o cm. B: Block of perspex, I c m thick. R :
II II
T
11 []
Razor blade.
[1
Fig. 13. Method of eluting columns in sections. G: Glass bulb. T: Tube, o. 7 × 2o cm, m a d e from perspex. S: Simrit ring. I n a c h r o m a t o g r a p h i c exp e r i m e n t all t h e holes are covered with Simrit rings. Details are given in t h e text. This m e t h o d of elution is useful also for zone electrophoresis columns.
I] H
II
Fig. 12.
~]
I~
Fig. 13.
The last section, however, contained besides albumin, ax-globulin. The ~l-metalcombining protein could not be detected. Probably the column was too short for the amount of protein used, with the result that the fit-metal-combining protein was displaced from the column. Staining with Sudan-Black B showed that ~-lipoprotein was adsorbed at the top of the column. In another experiment, also aimed at investigating the possibility of using the displacement effect for the separation of proteins, the as-globulins were chosen as a2e]erences p. ~35.
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model substances. They were dialysed against 0.03 M NaPB 6.8 and such a large quantity was apphed to the column, equilibrated with the same buffer, that the lower part of the yellow zone (see the corresponding elution experiments with as-globulin ) migrated down to a few centimeters above the outlet of the column. The colorless protein solution that left the column was collected. It should be mentioned here that these colorless proteins are adsorbed at this phosphate concentration and thus emerge out of the column by true displacement. The column was then washed with 0.03 M NaPB 6.8. The calcium phosphate was cautiously pressed out of the glass column with the aid of water suction, and, by cutting, the yellow zone was separated from the blue one. The two zones were dispersed in 1.3 M K P B 6.8, and then the calcium phosphate was separated from the protein solution b y centrifugation. The blue, the yellow and the colorless solutions were analysed in the ultracentrifuge. The sedimentation diagrams are seen in Fig. 15D. From these it may be inferred that the blue zone consisted mainly of caeruloplasmin ($2o, w ~ 7.5 S), and the colorless zone of a component with a higher sedimentation velocity (S2o,tv = I8.2 S). The yellow zone, however, contained several components, perhaps because the yellow zone had not been cut up in spite of its covering the major part of the column. The values of the sedimentation constants of the various globuhns, as reported by other authors, are given under "a~-globuhn". From these two experiments it is evident that the displacement effect is an important factor in the chromatographic separation of proteins. Purification of proteins by displacement chromatography on columns of calcium phosphate has been reported by POLLSAND SHMUKLER40. The cutting of a column has been performed by various authors ~-24 in different ways. Instead of this procedure, which has certain drawbacks, the following can be employed. The glass tube of the column is substituted by a tube made from perspex. A number of holes are drilled in planes perpendicular to the axis of the tube. For details see Fig. 13. The column is packed, with Simrit rings covering the holes. (The Simrit rings are manufactured by C. Freudenberg, Simrit-Werke, Weinheim/Bergstr., Germany.) Each section is eluted in the following way. 0.6 M K P B 6.8 is added to the column. The volume of this buffer should be about 1. 5 times the liquid content of a section. The first upper section can now be ehited by moving the Simrit ring from the top of the column downwards, so that the proteins adsorbed in this section can be displaced b y the buffer solution and collected in the Simrit ring. When the top of the column has become dry, the protein solution is sucked up with a pipette and the Simrit ring is then placed in its original position. The same volume of 0.6 M K P B 6.8 is again added, and the second Simrit ring is moved down, and thus the proteins in the second section are ehited. In this way each section can be eluted without destroying the column. In order to utilize the advantages of the displacement effect it is essential that the packing of the column should be perfect. Ordinary elution of narrow electrophoresis columns causes considerable zone broadening. In order to avoid this broadening, the author successfully eluted these columns also in the manner shown in Fig. 13.
The e~ea o/NaCI upon adsorption The addition of sodium chloride to the buffer causes a decrease in protein adsorption to the hydroxylapatite. Of the serum proteins it is, however, only the Rsl~w~s p. z35.
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CHROMATOGRAPHY OF HUMAN SERUM
229
r-globulins that show a striking effect. The result of ehromatographing 7-giobulins with and without 0.2 M NaC1 in the buffer is given in Fig. 14. The same decrease in adsorption when adding sodium chloride to the buffer is shown by hemoglobin. As v-globulin and hemoglobin are characterized by comparatively high isoelectric points (about pH 7), it may be assumed that the lowering of the elution threshold, which sodium chloride brings about, is connected with the position of the isoelectrie point.
O.Ol h'l O.O,~hl
07/'I 0 . I 0 hl ~0.15 I"1 0.20 I~1
l. I
0.6"
:
t o
o
Q
t
(~:2. 1
~
k 50 (
Sodium
too
p~ospl~afe buffer,
pH 6.8
\ Eluate (ml) >
Fig. 14. Chromatograms of 7-globulin, with ( - O - O - O -) and without ( - O - O - O - ) 0.2 M NaG1 in the phosphate buffer, pH 6.8. The other serum proteins show only a very small decrease in adsorption, when sodium chloride is added to the buffer.
The effect o/temperature upon adsorption To gain an idea as to how the adsorption changes with temperature, two chromatographic experiments with bovine serum albumin were performed at two different temperatures, viz. + 4 ° and + 2 2 °, respectively, other experimental conditions being identical. The chromatograms thus obtained showed that at + 4 ° 5o % of the total amount of protein could be eluted with o.ix M NaPB 6.8, while the corresponding figure for the experiment at -t-22 ° was 7 o %. From both the columns the rest of the protein material could be eluted with o.5 M KPB 6.8. Thus there is an increase in adsorption with decreasing temperature, as expected. The effect is, however, so slight that the temperature variations which may occur during an experiment exert no influence. Recovery Calculations of the recovery were based on determinations of the nitrogen, according to the method of Kjeldahl. Recovery was xo4 % for human serum and 99 % for bovine serum albumin. Thus no irreversible adsorption to the hydroxylapatite was detected. DISCUSSION
Whole serum The chromatography of whole serum shows that there is no clear correlation between the isoelectric points of the proteins and the order of their emergence from Re/erences p. ~ 3 5 .
230
s. I~J~-I~T~I~
voL. 31 (I959)
the column. In this respect calcium phosphate markedly differs from cellulose ionexchangers ~5. Thus these two groups of adsorbents separate proteins on the basis of different principles. This means that chromatography on calcium phosphate combined with ion-exchange chromatography or with zone electrophoresis has possibilities of bringing about extensive purification of proteins. Recently STEEL~ANz6 has purified hog pituitary FSH by chromatographing first on DEAE-cellulose and subsequently on calcium phosphate. When chromatographing undialysed whole serum (Fig. I), it was observed that the first peak contained low-molecular-weight substances without adsorption to the column. This is in agreement with the results obtained by LEVlN1 with amino acids and a number of dye-stuffs. Thus calcium phosphate seems to be a useful tool for the separation of small molecules from proteins. Albumin
Chromatographic fractionation of human serum albumin into components of different electrophoretic mobilities at PH 8.6 was reported b y SOBER et al. ~5. They also showed that a slow albumin component did not appear in the chromatogram after prolonged dialysis, which according to them may be a result of the removal of small molecules from an "albumin complex" during dialysis. This seems plausible, since one of the functions of the albumin is to transport low-molecular-weight substances in the blood. Such a transport is possible only if the protein has affinity to such substances. The binding of, for instance, fatty acids b y albumin is very strong, as was shown by KENDALL27. According to SURGENOR28, the amount of fatty acids bound seems to depend on the time elapsed between the bleeding and the fractionation. Thus it is understandable that experiments with albumin are not quite reproducible. Recently, CANN AND PHELPS29 demonstrated that at pH 4 a solution of bovine serum albumin is an equilibrium mixture of the electrophoretic components, the composition of which is influenced by the composition of the medium, particularly by the concentration of acetate ions. Amino acids appear to displace the equilibrium in one direction and acetate ions in the opposite one 8°. SCHMID31 reported on the successful isolation of a human albumin component which is homogeneous in acetate buffer, pH 4.0 and 1"/2 = o.I. Homogeneity is lost, however, after treatment with cysteine and deionization with ion-exchange resins. All these findings may account for the difficulty experienced in obtaining reproducible electrophoretic analyses at p H 4.1 of albumin fractions obtained by chromatography at pH 6.8. as-Globulin
In connection with the chromatography of as-globulin, it was mentioned that the blue caeruloplasmin zone suddenly stopped migrating down the column. When this phenomenon occurs, the result of the chromatographic experiment will depend on the length of the column. Fortunately, the phenomenon is a rare one, but one should keep this in mind when using chromatography for homogeneity tests. It may also be appropriate to point out that a chromatographic experiment should not be stopped until a migrating zone has emerged from the column. Otherwise it may happen, when the interrupted experiment is resumed, that the zone can be eluted Re[erences p. 235.
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CHROMATOGRAPHY OF HUMAN SERUM
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only by increasing the buffer concentration. This is due to the fact that the adsorption increases with time. From the chromatogram in Fig. IO it will be seen that most of the activity of the caeruloplasmin is found in the second peak. Thus it is possible to separate a crude preparation of caeruloplasmin without previous zone electrophoresis. Analysis with paper electrophoresis revealed that the second peak in Fig. 1o among other substances contained ~,-globulin; however, the latter may be displaced from the column by using o.15 M NaPB 6.8 (Fig. 14). Thus to obtain caeruloplasmin in a purer state directly from whole serum the following elution scheme is recommended. (The buffers indicated in Fig. IO were chosen for the isolation of fi-lipoprotein, rather than of caeruloplasmin.) (i) Equilibrate with o.Io M NaPB 6.8. (ii) Apply undialysed serum. (iii) Elute with o.Io M NaPB 6.8. (iv) Elute with o.15 M NaPB 6.8. (v) Elute with 0.25 M NaPB 6.8. It was shown by MARTIN32 that bilirubin is bound to a=-globulin, as well as to albumin, but it was not reported to which aB-component(s) it is attached. Though the chromatographic experiments mentioned in this paper provide no satisfactory information on this point, yet it may be concluded that the pigment is not bound in great amounts to the heavy a=-component--isolated b y cutting the c o l u m n - since this component was found to be colorless. Isolation of a heavy a2-globulin was described earlier only b y BROWN et al. 38 who, starting from Cohn Fraction III-O, applied salting-out with ammonium sulphate and repeated ultracentrifugation, the product being a glycoprotein with a sedimentation constant of 14.6 S for a 2 % solution. r-Globulin
The llpoprotein of the r-globulin complex is known to be very unstable. The data given by RAY et d . ~ support the view that the denaturation is oxidative in nature. According to ONCLEYet al. 36, also the discoloration which a lipoprotein shows on storage is due to an oxidative process, in this case directed against the pigment. Thus it is very important that the isolation of this protein should be undertaken immediately, as was pointed out recently by ONCLEY~ in a paper describing a rapid method for the bulk isolation of ~-lipoproteins. The separation of the lipoprotein b y chromatography via zone electrophoresis causes denaturation, owing to the long time required for zone electrophoresis. Fortunately the purification can be achieved by chromatography of undialysed whole serum without previous zone electrophoresis. The isolation of the protein in this way is attained in a few hours and does not show any of the indications of denaturation mentioned in the description of the experiment corresponding to Fig. 9. When chromatographing labile substances it would be of some practical value to know the physical meaning of increased adsorption. It may mean an increase in adsorption force between each active site on the adsorbent and the protein molecule. On the other hand, it may involve no considerable change in this force, but instead an increase in the number of active sites per unit volume of the adsorbent. If this !',#fences p. ~35.
232
S. HJERT]~N
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were the case, the risk of denaturation would be independent of the buffer concentration used for the pretreatment of the column. It is also conceivable, however, that a decrease in buffer concentration causes a great increase in the number of active centra and in the number of bonds between the adsorbent and the protein molecule. This may force the molecule to assume a certain modified shape, with denaturation as a result. When the nature of the adsorption to an adsorbent is unknown, it is safest to use a high buffer concentration in the treatment of the columns prior to the chromatography of very unstable proteins. Accordingly, the column was equilibrated with a buffer concentration as high as o.I M in the experiment aimed at isolating the labile fl-lipoprotein.
7-Globulin Gradient elution of 7-globulin gives one flat peak. Rechromatography of fractions corresponding to different parts of the peak indicates that these fractions are characterized by different but fixed elution ranges 1. Thus it is quite evident that these fractions are different when emerging from the column. It is not quite certain, however, whether these differences also exist in the solution that is filtered into the column, even if. this appears most probable, since the adsorption of proteins to the column may cause some denaturation which might give rise to components that did not exist in the original solution. Thus it would be more satisfactory to demonstrate that the fractionation corresponded to heterogeneity present in the original solution. Therefore, an electrophoretically "fast" and an electrophoretically "slow" 7-globulin component were chromatographed and the two chromatograms compared, instead of determining the mobilities of the fractions obtained by chromatography of the whole 7-globulin. To separate v-globulin from whole serum, one can utilize the effect of sodium chloride, which is very specific inasmuch as it affects only the adsorption of y-globulin (Fig. 14). A comparison between Figs. I and 14 shows that the following elution procedure will result in the separation of 7-globulin. (i) (ii) (iii) (iv)
Equilibrate the column with 0.03 M NaPB 6.8. Apply undialysed serum. Elute with 0.o9 M NaPB 6.8. Elute with 0.07 M NaPB 6.8 + 0.2 M NaCl. The last step will desorb the y-globulin.
"False" components Though it was mentioned earlier in this paper that chromatography is a simple separation method for proteins and that it often shows a high power of resolution, the method has some disadvantages, the difficulty of interpreting the chromatograms being perhaps the most serious one. This is true for all adsorbents, and thus one is often confronted with the question, whether or not the various fractions represent different kinds of proteins. During the chromatography of a2-globulin one circumstance was met, which might lead to separation of such false components. It was mentioned (p. 222) that caeruloplasmin suddenly stopped migrating down the column. Only part of the blue band emerged from the column. An increase in the buffer concentration was required to displace the rest of the caeruloplasmin. Re#fences p. 235.
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31 (1959)
CHROMATOGRAPHY OF HUMAN SERUM
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Another procedure that may give rise to false components is the following. Let us assume that a homogeneous and stable protein is to be chromatographed on a column, small in size in comparison with the amount of protein applied. If small increments in the buffer concentration are used for the desorption, the capacity of the column is gradually decreased. This means that the protein zone will broaden but not migrate. At a certain buffer concentration the capacity of the column is exceeded and the first fraction is obtained. At the next displacement step the capacity is further decreased and the second fraction will follow and so on. This way of obtaining false components was first reported by BOMAN3~ when chromatographing proteins on Dowex-2, but it is certainly valid for all protein adsorbents. LEVlN~, using DEAE-cellulose, has made the same observations. It should be pointed out that in such cases the ehition ranges depend on the ratio between the amount of adsorbent and the amount of protein applied. However, if the column is not loaded to excess, proteins are characterized by fixed elution ranges, which are independent of the amount of substance applied. This has been verified for a large number of proteins, and is particularly evident from the rechromatographic experiments reported in a previous paper 1. If the chromatogram of the y-globulin shown there is examined, it will be found, however, that the three fractions tested show a somewhat higher adsorption on rechromatography. This small increase in elntion threshold may be significant and may be caused by the effects mentioned above. Rechromatography is a very valuable test for the examination of the different fractions, but it could possibly be replaced by the following simpler procedure, which is based on the mutual displacement of the proteins. The protein solution is filtered into the column and washed with buffer as described under Cutting the columns. Then the column is cut into a number of small slabs of equal size and the amount of protein in each slab is determined. Cutting can be avoided if chromatography is performed on a column of the type shown in Fig. 13. If the protein chromatographed is homogeneous, the amount of protein should be the same in the different parts of the column. If, however, it is heterogeneous, protein concentration should be highest at the top of the column. Thus the mutual displacement of the proteins should make it possibile for the separation and test of homogeneity to be carried out in the same operation.
The displacement effect As mentioned under Cutting the columns, displacement with concentrated sodium phosphate buffer did not give satisfactory results. It is possible that a displacer should have properties that are similar to those of the displaced material. If this were true proteins displacement might be carried out with substances having molecular weights of the same order of magnitude as proteins. No experiments have yet been made with such high-molecular-weight displacers. Only a few experiments have been performed by cutting the columns (Fig. 12) or b y their sectional elntion (Fig. 13). Thus a comparison between this method of separation and the common chromatographic method would not be justified. The former method, however, was effective in isolating the heavy at-component (Fig. 15 DI) while the latter was not. If the two methods have the same resolving power for the separation of a certain protein, the former should be preferred because its use offers Re]erences p. a35.
234
s. I-IJERTI~N
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some advantages. For example the proteins emerge in much higher concentration, the capacity of the column is more effectively utilized, the experiments are much less time-consuming, and no fraction collector is necessary. It may be mentioned that a strong displacement effect is not specific of calcium phosphate as adsorbent; it seems to be characteristic of cellulose ion exchangers too, for according to SOBER et al. 2s, "the application of the yellow serum protein solution to the top of the adsorbent co]ulIm quickly produced a series of distinct colored bands". Thus desorption in the manner shown in Figs. 12 and 13 should be useful for these adsorbents also.
Qf i e f 2 0 r n / n
AI
o f f e r IOOt~in
AI
All
C
DI
AllI
Qffer ~rnln
B
DII
o f / e r 120mlm
Oll
Olll
Fig. 1 5. S e d i m e n t a t i o n d i a g r a m s . All s o l u t i o n s were dialysed a g a i n s t 0.2 M s o d i u m p h o s p h a t e buffer, p H 6.8. T h e t o t a l p r o t e i n c o n c e n t r a t i o n s v a r i e d b e t w e e n 0. 3 a n d o. 5 %. O n l y t h e fl-lipop r o t e i n h a d a h i g h e r c o n c e n t r a t i o n (o.8 %). O b s e r v e t h a t t h e v a l u e of t h e s e d i m e n t a t i o n c o n s t a n t for/~-lipoprotein is uncorrected. AI (S2o, w = 2.9, 7.3 a n d 17.1 S) c o r r e s p o n d s to fraction I in Fig. 6; AII (S2o, w = 4.2, 6.7 a n d 18.8 S) c o r r e s p o n d s to fraction I I in Fig. 6; AIII (Szo, w = 7,5 S, caeruloplasmin) c o r r e s p o n d s to fraction I I I in Fig. 6. B (S~0, w = 7.2 S) c o r r e s p o n d s to t h e second p e a k (caeruloplasmin) in Fig. 7. c (S = 2.1 S, uncorrected) c o r r e s p o n d s to t h e l a s t p e a k (~-lipoprotein) in Fig. IO. D I (S2o. to = 18.2 S) c o r r e s p o n d s to t h e c u t t i n g of c o l u m n s (uncolored solution; a~glycoprotein?) ; D I I ($20 ' w = 3.9, 5-7, 6.6 a n d 15. 7 S) c o r r e s p o n d s to t h e c u t t i n g of c o l u m n s (yellow solution); D I I I ( S 2 0 , w = 3"5 a n d 7.5 S) c o r r e s p o n d s to t h e c u t t i n g of c o l u m n s (blue solution; main component caeruloplasmin). ACKNOWLEDGEMENTS
The author wishes to thank Prof. A. TISELIUS for his kind advice and active interest in this work. This work was financially supported b y grants to the Institute of Biochemistry from the Rockefeller and Wallenberg Foundations and from the Swedish Cancer Society. ReIerences p. 235.
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REFERENCES 1 A. TISELIUS, S. HJERTI~N AND 0 . LEVIN, Arch. Biochem. Biophys., 65 (I956) 132. 2 S. MOORE AND W. H. STEIN, Advances in Protein Chem., I i (i956) I 9 I . 3 H. J. MIES, Klin. Wochschr., 31 (I953) I59. 4 j . PORATH, Biochim. Biophys. Acta, 22 (I956) 151. 5 H. I. GEDIN AND J. POEATH, Biochim. Biophys. Acta, 26 (1957) 159. e H. E. SCHULTZE, Angew. Chem., 69 (1957) 617J. A. LUETSCHER, J. Am. Chem. Soc., 6i (I939) 2888. s D. SHARP, G. COOPER, J. ERICKSON AND H. NEURATH, J. Biol. Chem., 144 (1942) I39. 9 R. A. ALBERTY, J. Phys. & Colloid Chem., 53 (I949) 114. 10 L. G. LONGSWORTH AND C. F. JAKOBSEN, J. Phys. & Colloid Chem., 53 (I949) i26. 11 A. SAIFER AND H. COREr, Proc. Soc. Exptl. Biol. Med., 86 (I954) 46. xs L. BROMAN, to be published. la G. WALLENIUS, R. TRAUTMAN, H. G. KUNKEL AND E. C. FRANKLIN, ]. Biol. Chem., 225 (1957) 253. 14 I. BRATTSTEN, Arkiv Kemi, 8 (1955) 347. 15 H. E. SCHULTZE, Z. Elektrochem., 6o (1956) 262. is B. DRAKB, Arkiv Kemi, 8 (I955) I8, case 4. 17 j . L. ONCLEY, G. SCATCHARD AND A. BROWN, J. Phys. & Colloid Chem., 51 (1947) I84. is E. J. COliN, F. R. N. GURD, D. M. SURGENOR, B. A. BARNES, R. K. BROWN, G. DEROUAUX, J. M. GILLESPIE, F. W . KAHNT, F. W. LEVER, C. H. LIU, D. MITTELMAN, R. F. MOUTON, K. SCHMID AND E. UROMA, J. Am. Chem. Soc., 72 (195o) 465 • 19 j . w . MEHL, u n p u b l i s h e d o b s e r v a t i o n s . s0 j . L. ONCLEY, F. R. N. GURD AND A. MELIN, J. Am. Chem. Soc., 72 (195o) 458. 31 N . I. KRINSKY, D. G. CORNWELL AND J. L. ONCLEY, Arch. Biochem. Biophys., 73 (1958) 233. Is N. v. BI~KI~SY, Biochem. Z., 312 (1942) IOO. 2~ S . GAULT AND CH. RANEZ, Bull. soc. chim. France, 17 (195 o) 597. 24 K. SCHL~GL AND A. SIEGEL, X. Natur/orsch. Pt. b, 9 (1954) 57 o. H. A. SOBER, F. J. GUTTER, M. M. WYCKOFF AND E. A. PETERSON, J. Am. Chem. Soc., 78 (1956) 756. s9 S. L. STEELMAN, Biochim. Biophys. Acta, 27 (1958) 4o5 . 9? F. E. KENDALL, J. Biol. Chem., 138 (1941) 97. is D. M. SURGENOR, in D. E. GREEN, Currents in Biochemical Research, I n t e r s c i e n c e P u b l i s h e r s , Inc., N e w York, 1956, p. 653. ~9 j . R. CANN AND R. A. PHELPS, J. Am. Chem. Soc., 79 (1957) 4672. 80 R. A. PHELPS AND J. R. CANN, J. Am. Chem. Soc., 79 (1957) 4677 • 31 K. SCHMID, J. Am. Chem. Soc., 79 (1957) 4679. as N. H. MARTIN, J. Am. Chem. Soc., 71 (1949) 123o. R. K. BROWN, W. H. BAKER, A. PETERKOFSKY AND D. L. KAUFFMAN, J. Am. Chem. Soc., 76 (I954) 4244 • B. R. RAY, E. O. DAVISSON AND S . L. CRESPI, J. Phys. Chem., 58 (I954) 84195 j . R. ONCLEY AND F. R. N. GURD, in J. L. TULLIS, Blood Cells and Plasma Proteins: Their State in Nature, A c a d e m i c Press, Inc., N e w York, 1953, P. 337. J. L. ONCLEY, K. W. WALTON AND D. G. CORNWELL, J. Am. Chem. Soc., 79 (1957) 4666. aT H. G. BOMAN AND L. E. WESTLUND, Arch. Biochem. Biophys., 64 (1956) 217. 9s 0 . LEVlN, Arch. Biochem. Biophys., in t h e press. 39 C. CALMON AND T . R . E . KRESSMAN, Ion Exchangers in Organic and Biochemistry, I n t e r s c i e n c e P u b l i s h e r s , Inc., N e w York, 1957. 40 B. D. POLLS AND H. W . SHMUKLER, J. Biol. Chem., 2Ol (1953) 475.
s. CHAYKIN, K. BLOCH
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REFERENCES x T. T. TCHEN AND K. BLOCH, J. Biol. Chem., 226 (1957) 921. 2 E. C. TAYLOR AND A. J. CROV'ETTI, J. Org. Chem., 19 (1954) 1633. 3 G. R. CLEMO AND H. KOENIG, J. Chem. Soc., (1949) $ 2 3 i . 4 M. L. W u CHANG AND B. C. JOHNSON, J. Biol. Chem., 226 (1957) 799. 6 K. POLLER AND W. LINNEWEH, Ber., 59 (1926) 1362. 6 G. DUNN, J. j . GALLAGHER, G. T. NRWBOLD AND F. S. SPRING, J. Chem. See., (1949) S 126. v G. R. CLEMO AND H. MclLwAIN, J. Chem. Soc., (1938) 4798 G. R. CL]EMO AND A. F. DAGLISH, ] . Chem. Soc., (195o) 1481. 9 M. S. FISH, C. C. SWXELEY, N. M. JOHNSON, E. P. LAWRENCE AND E. C. HORNING, Biochim. Biophys. Acta, 21 (1956) 196. 1o A. MAY, Enzymologia, 18 (1957) 142.
CALCIUM P H O S P H A T E C H R O M A T O G R A P H Y O F N O R M A L H U M A N S E R U M AND O F ELECTROPHORETICALLY
ISOLATED SERUM PROTEINS
STELLAN HJERTt~N
Institute o] Biochemistry, University o] Uppsala (Sweden) (Received M a y 9th, 1958)
SUMMARY
The usefulness of calcium phosphate as an adsorbent for proteins has been demonstrated by experiments with whole serum and electrophoretically isolated serum proteins. The separation of, for example, ~-lipoprotein and 7-globulin from whole serum and caeruloplasmin from as-globulin may easily be attained. A heavy a Sglobulin has been isolated b y cutting a column, utilizing the property of proteins to displace each other.
INTRODUCTION
The development of chromatographic techniques during the recent years has made it possible to achieve extensive purification of proteins without recourse to complicated and expensive equipment, usually employed for this purpose. In a previous paper 1 we described some chromatographic experiments with serum proteins o n columns of calcium phosphate. As the results obtained were interesting and promising, the chromatographic behaviour of the serum proteins has been studied in further detail. This paper deals with these investigations and also with the practical usefulness of the strong mutual displacement often shown by proteins. As model substances, electrophoretically isolated proteins of normal human serum have been used. For comparison, one experiment with whole serum has also been described. For information concerning protein chromatography on calcium phosphate as well as on other adsorbents, the reader is referred to the paper mentioned above and to a survey Re#~ences p. a35.
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CHROMATOGRAPHY OF HUMAN SERUM
2I 7
of recent progress by Moom~ AI~D STEIN2. Different adsorbents are also discussed by CALMON AND KR:ESSMANm. MATERIALS
The normal human sera used in this investigation were obtained from different blood donors at the Blood Donor Centre, Academic Hospital, Uppsala. No pooled sera were used in any of the experiments. The serum applied to columns for electrophoresis or chromatography, was one day old at the most. The proteins separated b y zone electrophoresis were chromatographed without unnecessary delay. Thus, lyophilization and freezing were omitted to avoid the risk of denaturation of the proteins. METHODS
Chromatography All chromatographic experiments were carried out on the same adsorbent, viz., hydroxylapatite. The preparation of this modified calcium phosphate was described b y TISELIUS, HJERT~N AND LEVINx in a paper in which also practical details concerning the method were treated. The columns were packed under or in the absence of pressure, depending on the flow rate desired. This corresponded to about 3oytfl]h for columns with a length of z o c m and an inner diameter of x.3 era. For the elution of the proteins, sodium phosphate buffers were mostly used. Owing to its higher solubility, potassium phosphate buffer was used when phosphate concentrations of o.3 M or higher were required for the desorption. As abbreviations for these buffers, the notations NaPB 6.8 and K P B 6.8 are introduced; the figures indicate the pH of the solution. Unless otherwise specified, the dialysis of the samples and the pretreatment of the columns was performed with the same buffer concentration as was used in the first step for the ehtion. This concentration is indicated in the corresponding chromatogram. All chromatographic experiments were carried out in the cold room ( + 4 °) with the exception of the gradient elution of a crude preparation of caeruloplasmin which was performed at room temperature ( + 2I°).
A bsorption measurements o/the e~uent The effluent was collected in an automatic time-operated fraction collector, The concentration of the proteins in the different fractions was determined b y measuring the absorption at z8o mt~ with a Beckman DU spectrophotometer, using a x-cm cell. For continuous recording of the effluent, a simple filter photometer was constructed, which will be described in a subsequent publication.
Concentration o/protein solutions The protein solutions, which were to be analysed by free electrophoresis or ultracentrifugation were concentrated b y ultraftltration through a collodion membrane according to MIES3. The collodion sack was supplied b y MembranengeseUschaft, G6ttingen. Protein concentration was determined by measurements of the refractive indices and b y using the conversion factor 1.8. xo-~. The fl-lipoprotein concentration was estimated with the same value of the factor, though this is certainly not correct. RefR~c~ p. ~35.
218
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VOL. 31 (1959)
Ultracentri[ugation Sedimentation analyses were carried out with a Spinco analytical ultracentrifuge, model E. Prior to the analyses, the protein solutions were dialysed against o.2 M sodium phosphate buffer, pH 6.8. The sedimentation constants were calculated to the density and viscosity of water at 2o °, but not extrapolated to the protein concentration zero. They are represented b y the notation $20, w.
Zone electrophoresis To obtain the main electrophoretic components of serum, zone electrophoresis on cellulose columns according to PORATH4 and GEDIN AND PORATH 5 was used. The column, which had a length of 15o cm and a diameter of 6.5 cm, was loaded with IOO ml of normal human serum, dialysed against veronal buffer, p H 8.6, ionic strength o.o3. The run was conducted in the same buffer for 7 ° h at 145 mA.
Free electrophoresis Some chromatographic fractions were analysed in a Tiselius-Svensson apparatus (type LKB), with cells requiring 12 ml of a protein solution.
Paper electrophoresis All runs were conducted in veronal buffer, p H 8.6 and ionic strength 0.05, with an apparatus of the horizontal, moist-chamber type. The electropherograms were stained with bromophenol blue. For lipoproteins Sudan-Black B was used. In the earlier experiments the concentration of the dilute chromatographic fractions, prior to the electrophoretic analyses, was achieved b y pervaporation, i.e. b y blowing air over a dialysis bag filled with the solution to be concentrated. But as pervaporation involves some disadvantages, another method was desired, preferably one which would permit comparatively large volumes to be applied directly to the paper without previous concentration. A zone-sharpening method fulfilling these requirements was worked out. It will be described in a future publication. EXPERIMENTS AND RESULTS
Whole serum I ml of undialysed serum was filtered into a column, 1. 4 × 21 cm. The first fraction in the diagram in Fig. I is characterized b y the fact that it leaves the column without any adsorption, and that it is stained b y ninhydrin but not by bromophenol blue, which indicates that this fraction contains low-molecular-weight substances. Determination of the molecular weights of these substances has not been undertaken, but it m a y be mentioned here that all of them pass through the pores of a common dialysis membrane. Paper chromatography in butanol-acetic acid and paper electrophoresis in pyridinium formate reveal several components. No further analyses of these substances have been carried out. The other chromatographic fractions have been analysed by paper electrophoresis--after concentration b y pervaporation. The result is represented in Fig. 2. It is evident from these electropherograms that a rather far-going purification is obtained already in one chromatographic experiment. According to SCHULTZE 6, m o r e than one hundred components of whole serum have been identified up to now. It is Re]erences p. 235.
VOL. 31 (I959)
CtO2M
CHROMATOGRAPHY OF HUMAN SERUM
CtO3M
~04M
~O~M
CtO6M
~08M
; CIIOM
~,15M
2I 9
n 20M
Q30M
~65M
Alb
1.0. CLS.
2bo I
•
tO0 $odiutn p h o s p h a f e buffer. OH 6.8
,
,
300 ~. l u a f e. (toO= ~( ~ofass#urn-~ phosphate buffe~ ~ £ 8
Fig. I. Chromatographic behaviour of human serum in stepwise elution with phosphate buffer, pH 6.8, on a calcium phosphate column, i. 4 × 2z cm. i ml undialysed serum was applied. expected that many of these proteins possess affinities of similar magnitude for the adsorbent, and since the number of fractions eluted from the column must necessarily be very small in comparison with the number of different protein components present in serum, it is quite plausible that many of these chromatographic fractions are of a heterogeneous nature. Some proteins, however, show a very specific adsorption, an advantage which may be utilized for chromatographic isolation directly from whole serum. This is true in the ease oi fl-lipoprotein and 7-globulin, as is evident from the corresponding chromatograms (Figs. IO and z4).
Fig. 2. Paper electropherograms of the different chromatographic serum fractions in Fig. I. The notation O refers to unfractionated serum. The presence of the great number of proteins in whole serum makes the interpretation of the chromatogram somewhat difficult. The work should be facilitated if the proteins of the whole serum were first separated into groups and these groups then chromatographed separately. Since very well defined groups are easily obtainable by zone eleetrophoresis, a separation of the serum proteins by this method was made prior to chromatography. As it is not possible to isolate al-globulin well enough b y zone electrophoresis, this component has not been studied chromatographically. Relerences p. z35.
220
VOL. 3 1 (1959)
S. HJERT~-I~I
Albumin
In a previous paper z the chromatography of bovine serum albumin has been described. The three fractions obtained were homogeneous on rechromatography. The preparation used was a freeze-dried one and contained both monomers and dimers. Some separation with regard to these was obtained. Thus the first fraction contained only monomers. Attempts to find differences in electrophoretic mobility in the three chromatographic fractions obtained were not successful. E2e
1 0,04
0.07/"/
H
ll
111
O.I114
0.65H
Potosslum phox~ohafe I.*~
buffer, p H 6.8
1.0
i
0.5.
{)
0.0
2'5 •
Sodium
phosphate
510 buffer, p H 6.0
75
Frachon number )
Fig. 3. Chromatogram of stepwise elution of h u m a n serum albumin.
With h u ma n serum albumin, however, some correlation between chromatographic and electrophoretic separation is obtained; this will now be described. 19o ml of a 0.2 % solution of human albumin, dia]ysed against 0.02 M NaPB 6.8, was applied to a column, 3.8 × 13.5 cm. The column was equilibrated for 15 h with 0.02 M NaPB 6.8. Elntion was performed with 250 ml 0.04 M N a P B 6.8, 300 ml 0.07 M NaPB 6.8, 250 ml o . I I M N a P B 6.8 and 250 ml 0.65 M K P B 6.8. The chromatogram is given in Fig. 3- Paper electrophoresis in veronal buffer, p H 8.6, ionic strength o.o5, showed that fraction I I I had a smaller mobility than I and I I (Fig. 4). With free electrophoresis in veronal buffer of the same p H but of a higher ionic strength (o.io), this difference in mobility was determined and found to be 6 %. Free electrophoresis of a mixture of the fractions I, I I and I I I resulted in one peak only. A]I three fractions contained lipid material associated with the albumin, as revealed b y paper electrophoresis and subsequent staining with Sudan-Black B. Electrophoretic analysis in acetate buffer, p H 4.1, ionic strength 0.05, was also carried out. At this lower p H two peaks were obtained ~-n. The mobilities of these were different for the three fractions. Thus fraction I I I h a d a rate of migration 50 % higher than that of I. The ratio between the amounts of the two electrophoretic components of these two chromatographic fractions was not of the same order of magnitude as shown in Fig. 5. The eiectrophoretic data are given in Table I. To test the reproducibility of these results, another experiment was performed in the way just described but albumin from another blood donor was used. The three Relerences p. 235.
VOL. 31
(I959)
CHROMATOGRAPHY OF HUMAN SERUM
22I
chromatographic fractions now showed no differences in mobility at pH 8.6. At pH 4.I, however, the three electropherograms corresponding to the three chromatographic fractions differed in appearance. However, the mobilities and the ratio between the amounts of proteins in the two peaks of the electropherogram were different from those obtained in the first experiment. A plausible reason for this low reproducibility regarding the correlation between electrophoresis and chromatography will be presented under DISCUSSION.
a
6
0
i
//
~
!
6
//I
Fig. 4. Paper electropherogram of the three albumin fractions in Fig. 3.
Fig. 5. Free electrophoresis in acetate buffer, p H 4. i, and ionic strength o.05, of the unchromatographed albumin (O) and the three chromatographic fractions (I, II, III). The peak to the left (6) represents the protein-buffer salt boundary. The photographs are of the ascending boundaries. The potential gradients were io.o, Io.5, 9.9 and 9.7 V/cm respectively. The current was in all 4 runs adjusted to 15 mA. TABLE I ELECTROPHORETIC DATA OF CHROMATOGRAPHIC FRACTIONS OF HUMAN SERUM ALBUMIN
Mobilities are given in cm]sec/V/cm • Io I and based on values from both limbs. Un~k~me~ograpl~d
Frcu$ionI
Fr~ios II
--6. 7
--6.6
Fr~ios III
Mobility in veronal buffer,, pH 8.6,/'[2
ffi o . I o
--6.6
m6.2
Mobility in acetate buffer, p H 4.I, F/2 ~ o.o 5
5.0 5-7
5.~ 6.0
4.I 5.4
7.5 9.0
The ratio between the amounts of the two electrophoretic components at p H 4.I
z.o
Difficult to determine (see Fig. 5)
z,o
o.8
~l-Glob~din Ioo ml of a o.o5 % al-globulin solution, diaiysed against o.o5 M N a P B 6.8 was added in drops to a column x.6 × 25 cm, equilibrated with the same buffer. When Re/erences ~. z 3 5 .
222
s. ~J~RT~N
voL. 31 (I959)
the whole protein solution had been filtered into the column a narrow blue band was observed at the top of the column, followed by a broad yellow zone. The lower part of the column was not colored, but from other experiments made it is clear that proteins are adsorbed there also. The chromatogram is shown in Fig. 6. Since the blue band was suspected to contain caeruloplasmin, activity tests for this enzyme were made. These measurements were performed according to BROMAN1= and the values obtained are indicated on the chromatogram. It should be mentioned that during elution with o.20 M NaPB 6.8 the blue band migrated down the column but suddenly stopped, with the result that only part of the band left the column. The rest could be eluted with a higher buffer concentration and thus two peaks with oxidase activity were obtained.
~eo
I1
2. 0
1.5. C~IO M
CXlS M
CX20 M
1.0.
0.65M Pofassiur~ phosphafe buffer, p H 6.6
0,3 0.5.
0. I 0.0
I0 20 30 <----Sodium p h o s p l ~ l e buffer, p H 6 . 8
Krocfion n u m b e r
O.O
Fig. 6. Chromatogram of stepwise elution of ai-globulin. The dashed curve refers to activity test of caeruloplasmin.
Analyses of the three main fractions were carried out with ultracentrifugation. The sedimentation constants are given in the legend to the diagrams in Fig. I5 A. For comparison it may be mentioned that WALLENIUS, TRAUTMAN, KUNKEL AND FRAI~KLINls recently reported that electrophoretically-isolated a2-globulin contains components with the sedimentation constants 4.1, 6.6, 12.1 (traces) and 18.5 S, while BRATTSTEN14 found the values 3.2, 7.0, 12.6 and 15.9 S. The 2.9 S component present in the first chromatographic fraction may be a=-seromucoid, which according to SCI~ULTZE6 is characterized b y a sedimentation constant of 2.6 S. No efforts have been made to obtain more homogeneous fractions in the ultracentrifuge by trying buffer concentrations other than those shown in the chromatogram. It is, however, quite evident that for the ehition of caeruloplasmin a phosphate concentration of 0.65 M Relerences p z35.
VOl..
31 (1959)
CHROMATOGRAPHY OF HUMAN S~ERUM
223
is unnecessarily high. It should be changed to 0.25 M. It may be of value to have caeruloplasmin available in large amounts so that one could dispense with the comparatively time-consuming zone electrophoresis as an intermediate step. Thus a crude preparation of caeruloplasmin, received from AB KABI, Stockholm, Sweden, was chromatographed. Since the main impurity in this preparation (made from the Cohn fraction IV) is albumin, and this protein is eluted with much lower phosphate concentration than caeruloplasmin, it is, of course, very easy to obtain a high degree of purification. Chromatography was carried out as gradient elution at room temperature in the following way. 4 ° mg of the crude preparation was dialysed against 0.05 M NaPB 6.8 and applied to a column 1.3 × 20 cm, treated with the same buffer. The buffer gradient was made in a simple apparatus similar to that used by DRAKEls. The mixing chamber was filled with 25 ° ml 0.05 M NaPB 6.8 and the other chamber with 13o ml 0.4 M NaPB 6.8. Continuous recording produced the chromatogram depicted in Fig. 7. The fractions corresponding to the second peak were characterized b y a blue color. Analyses of these b y paper electrophoresis (Fig. 8) and ultracentrifugation (Fig. I5 B) indicated homogeneity. The sedimentation constant was calculated to be 7.2 S, whereas SCHULTZE15 reported a value of 7.1 S. It may be observed that a concave gradient was chosen for the elution. According to the experience gathered in this laboratory, convex gradients should be avoided in protein chromatography, since they increase the risk of tailing.
Fig. 7- Continuous recording of a gradient elution of a crude preparation of caeruloplasmin. The horizontal arrow (< 7) indicates the fractions which have been combined and analysed with the aid of ultracentrifugation (Fig. I5) and paper electrophoresis (Fig. 8).
Relerences p 235.
Fig. 8. Paper electrophoresis of caeruloplasmin, obtained from the gradient elution in Fig. 7. The method of zone sharpening was used, which made it possible to apply the dilute caeruloplasmin solution to the paper without first concentrating it. The notation 0 refers to t h e unchromatographed, crude caeruloplasmin preparation.
224
s. HJERT~N
VOL. 31 (Z959)
Further information on the chromatographic behaviour of the a=-globulins will be found under Cutting the columns. How to obtain a crude preparation of caeruloplasmin directly from undialysed whole serum is described in connection with t h e discussion of the results obtained by chromatography of the a=-globulin. [3-Glob~din
The column, x.3 × z o c m was equilibrated with a comparatively high phosphate concentration, viz. o.zo M NaPB 6.8, before the application of 30 ml of a o.z % /3-globulin solution. The chromatogram (Fig. 9) shows two fractions. Pretreatment with the high buffer concentration caused the first fraction to emerge from the column without any adsorption. The second fraction, however, had a very strong affinity to the calcium phosphate. Some of the properties, characterizing the two fractions, are given in Table II, from which it is evident that the first fraction contains ill-metalcombining protein and the second fi-lipoprotein. Electrophoresis and ultracentrifugation indicated that the lipoprotein was somewhat denatured, hence its mobility and sedimentation constant were not calculated. Other indications of denaturation were the facts that the solution had lost its yellow color and that the peak at 280 m/z was very fiat, probably due to considerable light scattering, owing to condensation of lipid material. This denaturation is not caused, however, by adsorption (see DISCUSSION).
No attempts were made to separate chromatographically the two components E2ao O.lOh'l
CUSM
0.20M
t3H Po fassiurn phosp/'lafe
t.O
buffer. pPI 6 . 0
Eluafe (rnlf <'~Sodium DhosDh¢;l~ b u f f e r . ~o~'/~ 8 -'~
Fig. 9. Chromatogram of stepwise elution of H-globulin. The first fraction contains ~z-metal-
combining protein, and the last ~-lipoprotein. TABLE If
ANALYSES OF TWO CHROMATOGRAPHIC FRACTIONS OF H'GLOBULIN
Mob~Cy Colin, o! tl~ $ol'~l~
St~s~ ~ bnm~plumol b l ~
S t 4 i n ~ w~k S~dar.-B~ B
Fraction I
Reddish brown
Strong
No
Fraction II
Opalescent
Weak
Strong
R s l e r e n c e s p. z 3 5 .
im ~ o ~ b ~ t e , pH 8.6 1"1~= o.zo
- - 3.x" x o -~ cm/sec/V/cm --
Sao, w S
5.5 ( m a i n component) 7.0
VOL. 3 1
(1959)
CHROMATOGRAPHY OF HUMAN SERUM
@-25
which, according to the ultracentrifugation data, are present in the first fraction. The sedimentation constants are similar to those reported by a number of authors ~S-ls, 17,- . A comparison between the chromatograms in Fig. z and Fig. 9 shows that it would be possible to isolate the fl-lipoprotein chromatographically without previous electrophoresis. Therefore a column 1.3 × 2o cm was treated with o.I M NaPB 5.8 and loaded with 8.5 ml undialysed normal human serum. The chromatogram is given in Fig. io. The most strongly adsorbed fraction contained/~-lipoprotein. The solution was yellow and had an absorption maximum at 46o rag. As first pointed out by MEHL19 and later proved by O~cLEY", this color is due to the presence of a carotenoid pigment. The carotenoids, fl-carotene and lycopene, are transported in the body exclusively by the serum lipoproteins". That the lipoprotein is homogeneous when analysed with the aid of paper electrophoresis, is quite evident from Fig. 2. The uncorrected sedimentation constant was found to be 2.I S. The corresponding diagram is given in Fig. IsC. Since it is known 1~ that the value of the partial specific volume of the/3-1ipoprotein differs considerably from that of "ordinary" proteins, and since the author did not determine it, no correction of this sedimentation constant was made. ONCLEY, SCATCHARD AND BROWN 1~ reported a corrected value of 2.8 S for a 0.8 % solution.
E2801 O, I O M
0. 2'~ M
O;(;~M
Pofoss~Jrn p~ospkQte buffer, pl-t ~ 8
2,5.
2.0.
1.5-
1.0-
0.I 0 . 0 ~.-~ ~ " •
. . . . 50 tOO 150 Sodium ohos~'~ b u f f e r , o H (~6
~
0.0 E / u a f e (m/)
Fig. IO. Chromatography of whole serum for preparation of ~-lipoprotein. 8. 5 ml undialFsed serum was applied on a column, x.3 × 2o cm. Only the last fraction can be stained with Sudan-Black B. The dashed line refers to activity test for caeruloplasmin.
Re/eTe.ces p. 235.
226
s. HJERTI~N
VOL. 31 (I959)
y-Globulin io mg of a fast ?-globulin component, isolated by zone electrophoresis, was chromatographed on a column 1.2 × I 3 cm. Simultaneously a similar experiment with IO mg of a slow 7-globulin component was performed on another column of the same size. A comparison of the chromatograms (Fig. II) shows that the hydroxylapatite is sensitive to the differences between the two y-globulin components. However, since the elution ranges of the two y-globulins differ only rather slightly, several similar experiments were made with y-globulins from other blood donors. Buffers of lower pH ( = 6.8) were tried also. All these experiments gave chromatograms similar to that in Fig. II. In a previous paper z we described a gradient elution of 7-globulin. Only one very extended peak was obtained. Three fractions corresponding to different parts of the peak were analysed by means of paper electrophoresis and rechromatography. We reported that "there is a slight but probably significant difference in electrophoretic mobility on paper between the three v-globulin fractions--the more easily eluted fractions show somewhat faster migration". Thus these previous results agree with those presented in this paper.
E~e°I
o.o ,lo.os,o.o,io,,lo.o,io.o io.,o-
O.o~O" I
50
~
I00
,,%~ 150
E l u a t e [rn/)
Fig. H . Chromatograms showing the stepwise elution of a " f a s t " ( - O - O - O - ) and a "slow" ( - O - O - O - ) ?-globulin iraction. Elution performed with sodium phosphate buffer, pH 7.9.
Cutting the columns When filtering the solutions of colored proteins into a column, the different proteins often appear as sharply defined zones on the column already before ehition is started (see for instance the chromatographic experiment with as-globulin ) . Thus the proteins show a very strong mutual displacement. To find out whether this effect could be used as a basis for separation, the following experiments were carded out. Whole serum was applied on a column, treated with o.oz M NaPB 6.8. All serum proteins were adsorbed at this low buffer concentration. Displacement was achieved with 0.65 M K P B 6.8. This phosphate concentration was high enough to elute all protein material from the column. In the chromatogram obtained only one peak appeared. This peak was, however, extended, and analyses by paper electrophoresis of different parts of the peak indicated that a certain separation had been attained. But this separation was not efficient enough to be useful for practical purposes. It seems probable that proteins with different affinities for the calcium phosphate arranged themselves as distinct zones on the column during the application of the whole serum, but the separation thus obtained was destroyed b y the displacement brought about with the phosphate buffer. An attempt was therefore made to utilize the mutual displacement already taking place when the sample is applied to the column, and to improve the separation only by washing with a weak buffer solution. Under these conditions the zones remain on the column, and therefore a method for cutting the column was devised. References p. z35.
voL 31
(1959)
CHROMATOGRAPHY OF HUMAN S:ERUM
227
The chromatographic column was of the common type, with the exception that the glass tube was replaced by a piece of polyvinyl chloride tubing (Fig. 12). The column was pretreated with 0.02 M NaPB 6.8. I ml serum was dialysed against the same buffer. After application of the serum the column was washed with o.o2 M NaPB 6.8. Cutting was carried out with a safety-razor blade in the manner shown in Fig. 12. Each section, the length of which was about I cm was suspended in 1.5 ml 1.3 M K P B 6.8. Analysis of the supernatant, performed with paper electrophoresis, showed that the upper part of the column contained v-globulin, followed by a section containing ~-globulin and albumin. The rest of the column was covered with albumin.
G
-Y
G
8
t ]
o°%
p
Fig. I2. C u t t i n g of columns. G: Glass bulb. P : PVC-tube, o.7 × 2o cm. B: Block of perspex, I c m thick. R :
II II
T
11 []
Razor blade.
[1
Fig. 13. Method of eluting columns in sections. G: Glass bulb. T: Tube, o. 7 × 2o cm, m a d e from perspex. S: Simrit ring. I n a c h r o m a t o g r a p h i c exp e r i m e n t all t h e holes are covered with Simrit rings. Details are given in t h e text. This m e t h o d of elution is useful also for zone electrophoresis columns.
I] H
II
Fig. 12.
~]
I~
Fig. 13.
The last section, however, contained besides albumin, ax-globulin. The ~l-metalcombining protein could not be detected. Probably the column was too short for the amount of protein used, with the result that the fit-metal-combining protein was displaced from the column. Staining with Sudan-Black B showed that ~-lipoprotein was adsorbed at the top of the column. In another experiment, also aimed at investigating the possibility of using the displacement effect for the separation of proteins, the as-globulins were chosen as a2e]erences p. ~35.
228
S. HJERT~N
VOL. 31 (X959)
model substances. They were dialysed against 0.03 M NaPB 6.8 and such a large quantity was apphed to the column, equilibrated with the same buffer, that the lower part of the yellow zone (see the corresponding elution experiments with as-globulin ) migrated down to a few centimeters above the outlet of the column. The colorless protein solution that left the column was collected. It should be mentioned here that these colorless proteins are adsorbed at this phosphate concentration and thus emerge out of the column by true displacement. The column was then washed with 0.03 M NaPB 6.8. The calcium phosphate was cautiously pressed out of the glass column with the aid of water suction, and, by cutting, the yellow zone was separated from the blue one. The two zones were dispersed in 1.3 M K P B 6.8, and then the calcium phosphate was separated from the protein solution b y centrifugation. The blue, the yellow and the colorless solutions were analysed in the ultracentrifuge. The sedimentation diagrams are seen in Fig. 15D. From these it may be inferred that the blue zone consisted mainly of caeruloplasmin ($2o, w ~ 7.5 S), and the colorless zone of a component with a higher sedimentation velocity (S2o,tv = I8.2 S). The yellow zone, however, contained several components, perhaps because the yellow zone had not been cut up in spite of its covering the major part of the column. The values of the sedimentation constants of the various globuhns, as reported by other authors, are given under "a~-globuhn". From these two experiments it is evident that the displacement effect is an important factor in the chromatographic separation of proteins. Purification of proteins by displacement chromatography on columns of calcium phosphate has been reported by POLLSAND SHMUKLER40. The cutting of a column has been performed by various authors ~-24 in different ways. Instead of this procedure, which has certain drawbacks, the following can be employed. The glass tube of the column is substituted by a tube made from perspex. A number of holes are drilled in planes perpendicular to the axis of the tube. For details see Fig. 13. The column is packed, with Simrit rings covering the holes. (The Simrit rings are manufactured by C. Freudenberg, Simrit-Werke, Weinheim/Bergstr., Germany.) Each section is eluted in the following way. 0.6 M K P B 6.8 is added to the column. The volume of this buffer should be about 1. 5 times the liquid content of a section. The first upper section can now be ehited by moving the Simrit ring from the top of the column downwards, so that the proteins adsorbed in this section can be displaced b y the buffer solution and collected in the Simrit ring. When the top of the column has become dry, the protein solution is sucked up with a pipette and the Simrit ring is then placed in its original position. The same volume of 0.6 M K P B 6.8 is again added, and the second Simrit ring is moved down, and thus the proteins in the second section are ehited. In this way each section can be eluted without destroying the column. In order to utilize the advantages of the displacement effect it is essential that the packing of the column should be perfect. Ordinary elution of narrow electrophoresis columns causes considerable zone broadening. In order to avoid this broadening, the author successfully eluted these columns also in the manner shown in Fig. 13.
The e~ea o/NaCI upon adsorption The addition of sodium chloride to the buffer causes a decrease in protein adsorption to the hydroxylapatite. Of the serum proteins it is, however, only the Rsl~w~s p. z35.
VoL. 31 (I959)
CHROMATOGRAPHY OF HUMAN SERUM
229
r-globulins that show a striking effect. The result of ehromatographing 7-giobulins with and without 0.2 M NaC1 in the buffer is given in Fig. 14. The same decrease in adsorption when adding sodium chloride to the buffer is shown by hemoglobin. As v-globulin and hemoglobin are characterized by comparatively high isoelectric points (about pH 7), it may be assumed that the lowering of the elution threshold, which sodium chloride brings about, is connected with the position of the isoelectrie point.
O.Ol h'l O.O,~hl
07/'I 0 . I 0 hl ~0.15 I"1 0.20 I~1
l. I
0.6"
:
t o
o
Q
t
(~:2. 1
~
k 50 (
Sodium
too
p~ospl~afe buffer,
pH 6.8
\ Eluate (ml) >
Fig. 14. Chromatograms of 7-globulin, with ( - O - O - O -) and without ( - O - O - O - ) 0.2 M NaG1 in the phosphate buffer, pH 6.8. The other serum proteins show only a very small decrease in adsorption, when sodium chloride is added to the buffer.
The effect o/temperature upon adsorption To gain an idea as to how the adsorption changes with temperature, two chromatographic experiments with bovine serum albumin were performed at two different temperatures, viz. + 4 ° and + 2 2 °, respectively, other experimental conditions being identical. The chromatograms thus obtained showed that at + 4 ° 5o % of the total amount of protein could be eluted with o.ix M NaPB 6.8, while the corresponding figure for the experiment at -t-22 ° was 7 o %. From both the columns the rest of the protein material could be eluted with o.5 M KPB 6.8. Thus there is an increase in adsorption with decreasing temperature, as expected. The effect is, however, so slight that the temperature variations which may occur during an experiment exert no influence. Recovery Calculations of the recovery were based on determinations of the nitrogen, according to the method of Kjeldahl. Recovery was xo4 % for human serum and 99 % for bovine serum albumin. Thus no irreversible adsorption to the hydroxylapatite was detected. DISCUSSION
Whole serum The chromatography of whole serum shows that there is no clear correlation between the isoelectric points of the proteins and the order of their emergence from Re/erences p. ~ 3 5 .
230
s. I~J~-I~T~I~
voL. 31 (I959)
the column. In this respect calcium phosphate markedly differs from cellulose ionexchangers ~5. Thus these two groups of adsorbents separate proteins on the basis of different principles. This means that chromatography on calcium phosphate combined with ion-exchange chromatography or with zone electrophoresis has possibilities of bringing about extensive purification of proteins. Recently STEEL~ANz6 has purified hog pituitary FSH by chromatographing first on DEAE-cellulose and subsequently on calcium phosphate. When chromatographing undialysed whole serum (Fig. I), it was observed that the first peak contained low-molecular-weight substances without adsorption to the column. This is in agreement with the results obtained by LEVlN1 with amino acids and a number of dye-stuffs. Thus calcium phosphate seems to be a useful tool for the separation of small molecules from proteins. Albumin
Chromatographic fractionation of human serum albumin into components of different electrophoretic mobilities at PH 8.6 was reported b y SOBER et al. ~5. They also showed that a slow albumin component did not appear in the chromatogram after prolonged dialysis, which according to them may be a result of the removal of small molecules from an "albumin complex" during dialysis. This seems plausible, since one of the functions of the albumin is to transport low-molecular-weight substances in the blood. Such a transport is possible only if the protein has affinity to such substances. The binding of, for instance, fatty acids b y albumin is very strong, as was shown by KENDALL27. According to SURGENOR28, the amount of fatty acids bound seems to depend on the time elapsed between the bleeding and the fractionation. Thus it is understandable that experiments with albumin are not quite reproducible. Recently, CANN AND PHELPS29 demonstrated that at pH 4 a solution of bovine serum albumin is an equilibrium mixture of the electrophoretic components, the composition of which is influenced by the composition of the medium, particularly by the concentration of acetate ions. Amino acids appear to displace the equilibrium in one direction and acetate ions in the opposite one 8°. SCHMID31 reported on the successful isolation of a human albumin component which is homogeneous in acetate buffer, pH 4.0 and 1"/2 = o.I. Homogeneity is lost, however, after treatment with cysteine and deionization with ion-exchange resins. All these findings may account for the difficulty experienced in obtaining reproducible electrophoretic analyses at p H 4.1 of albumin fractions obtained by chromatography at pH 6.8. as-Globulin
In connection with the chromatography of as-globulin, it was mentioned that the blue caeruloplasmin zone suddenly stopped migrating down the column. When this phenomenon occurs, the result of the chromatographic experiment will depend on the length of the column. Fortunately, the phenomenon is a rare one, but one should keep this in mind when using chromatography for homogeneity tests. It may also be appropriate to point out that a chromatographic experiment should not be stopped until a migrating zone has emerged from the column. Otherwise it may happen, when the interrupted experiment is resumed, that the zone can be eluted Re[erences p. 235.
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only by increasing the buffer concentration. This is due to the fact that the adsorption increases with time. From the chromatogram in Fig. IO it will be seen that most of the activity of the caeruloplasmin is found in the second peak. Thus it is possible to separate a crude preparation of caeruloplasmin without previous zone electrophoresis. Analysis with paper electrophoresis revealed that the second peak in Fig. 1o among other substances contained ~,-globulin; however, the latter may be displaced from the column by using o.15 M NaPB 6.8 (Fig. 14). Thus to obtain caeruloplasmin in a purer state directly from whole serum the following elution scheme is recommended. (The buffers indicated in Fig. IO were chosen for the isolation of fi-lipoprotein, rather than of caeruloplasmin.) (i) Equilibrate with o.Io M NaPB 6.8. (ii) Apply undialysed serum. (iii) Elute with o.Io M NaPB 6.8. (iv) Elute with o.15 M NaPB 6.8. (v) Elute with 0.25 M NaPB 6.8. It was shown by MARTIN32 that bilirubin is bound to a=-globulin, as well as to albumin, but it was not reported to which aB-component(s) it is attached. Though the chromatographic experiments mentioned in this paper provide no satisfactory information on this point, yet it may be concluded that the pigment is not bound in great amounts to the heavy a=-component--isolated b y cutting the c o l u m n - since this component was found to be colorless. Isolation of a heavy a2-globulin was described earlier only b y BROWN et al. 38 who, starting from Cohn Fraction III-O, applied salting-out with ammonium sulphate and repeated ultracentrifugation, the product being a glycoprotein with a sedimentation constant of 14.6 S for a 2 % solution. r-Globulin
The llpoprotein of the r-globulin complex is known to be very unstable. The data given by RAY et d . ~ support the view that the denaturation is oxidative in nature. According to ONCLEYet al. 36, also the discoloration which a lipoprotein shows on storage is due to an oxidative process, in this case directed against the pigment. Thus it is very important that the isolation of this protein should be undertaken immediately, as was pointed out recently by ONCLEY~ in a paper describing a rapid method for the bulk isolation of ~-lipoproteins. The separation of the lipoprotein b y chromatography via zone electrophoresis causes denaturation, owing to the long time required for zone electrophoresis. Fortunately the purification can be achieved by chromatography of undialysed whole serum without previous zone electrophoresis. The isolation of the protein in this way is attained in a few hours and does not show any of the indications of denaturation mentioned in the description of the experiment corresponding to Fig. 9. When chromatographing labile substances it would be of some practical value to know the physical meaning of increased adsorption. It may mean an increase in adsorption force between each active site on the adsorbent and the protein molecule. On the other hand, it may involve no considerable change in this force, but instead an increase in the number of active sites per unit volume of the adsorbent. If this !',#fences p. ~35.
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were the case, the risk of denaturation would be independent of the buffer concentration used for the pretreatment of the column. It is also conceivable, however, that a decrease in buffer concentration causes a great increase in the number of active centra and in the number of bonds between the adsorbent and the protein molecule. This may force the molecule to assume a certain modified shape, with denaturation as a result. When the nature of the adsorption to an adsorbent is unknown, it is safest to use a high buffer concentration in the treatment of the columns prior to the chromatography of very unstable proteins. Accordingly, the column was equilibrated with a buffer concentration as high as o.I M in the experiment aimed at isolating the labile fl-lipoprotein.
7-Globulin Gradient elution of 7-globulin gives one flat peak. Rechromatography of fractions corresponding to different parts of the peak indicates that these fractions are characterized by different but fixed elution ranges 1. Thus it is quite evident that these fractions are different when emerging from the column. It is not quite certain, however, whether these differences also exist in the solution that is filtered into the column, even if. this appears most probable, since the adsorption of proteins to the column may cause some denaturation which might give rise to components that did not exist in the original solution. Thus it would be more satisfactory to demonstrate that the fractionation corresponded to heterogeneity present in the original solution. Therefore, an electrophoretically "fast" and an electrophoretically "slow" 7-globulin component were chromatographed and the two chromatograms compared, instead of determining the mobilities of the fractions obtained by chromatography of the whole 7-globulin. To separate v-globulin from whole serum, one can utilize the effect of sodium chloride, which is very specific inasmuch as it affects only the adsorption of y-globulin (Fig. 14). A comparison between Figs. I and 14 shows that the following elution procedure will result in the separation of 7-globulin. (i) (ii) (iii) (iv)
Equilibrate the column with 0.03 M NaPB 6.8. Apply undialysed serum. Elute with 0.o9 M NaPB 6.8. Elute with 0.07 M NaPB 6.8 + 0.2 M NaCl. The last step will desorb the y-globulin.
"False" components Though it was mentioned earlier in this paper that chromatography is a simple separation method for proteins and that it often shows a high power of resolution, the method has some disadvantages, the difficulty of interpreting the chromatograms being perhaps the most serious one. This is true for all adsorbents, and thus one is often confronted with the question, whether or not the various fractions represent different kinds of proteins. During the chromatography of a2-globulin one circumstance was met, which might lead to separation of such false components. It was mentioned (p. 222) that caeruloplasmin suddenly stopped migrating down the column. Only part of the blue band emerged from the column. An increase in the buffer concentration was required to displace the rest of the caeruloplasmin. Re#fences p. 235.
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Another procedure that may give rise to false components is the following. Let us assume that a homogeneous and stable protein is to be chromatographed on a column, small in size in comparison with the amount of protein applied. If small increments in the buffer concentration are used for the desorption, the capacity of the column is gradually decreased. This means that the protein zone will broaden but not migrate. At a certain buffer concentration the capacity of the column is exceeded and the first fraction is obtained. At the next displacement step the capacity is further decreased and the second fraction will follow and so on. This way of obtaining false components was first reported by BOMAN3~ when chromatographing proteins on Dowex-2, but it is certainly valid for all protein adsorbents. LEVlN~, using DEAE-cellulose, has made the same observations. It should be pointed out that in such cases the ehition ranges depend on the ratio between the amount of adsorbent and the amount of protein applied. However, if the column is not loaded to excess, proteins are characterized by fixed elution ranges, which are independent of the amount of substance applied. This has been verified for a large number of proteins, and is particularly evident from the rechromatographic experiments reported in a previous paper 1. If the chromatogram of the y-globulin shown there is examined, it will be found, however, that the three fractions tested show a somewhat higher adsorption on rechromatography. This small increase in elntion threshold may be significant and may be caused by the effects mentioned above. Rechromatography is a very valuable test for the examination of the different fractions, but it could possibly be replaced by the following simpler procedure, which is based on the mutual displacement of the proteins. The protein solution is filtered into the column and washed with buffer as described under Cutting the columns. Then the column is cut into a number of small slabs of equal size and the amount of protein in each slab is determined. Cutting can be avoided if chromatography is performed on a column of the type shown in Fig. 13. If the protein chromatographed is homogeneous, the amount of protein should be the same in the different parts of the column. If, however, it is heterogeneous, protein concentration should be highest at the top of the column. Thus the mutual displacement of the proteins should make it possibile for the separation and test of homogeneity to be carried out in the same operation.
The displacement effect As mentioned under Cutting the columns, displacement with concentrated sodium phosphate buffer did not give satisfactory results. It is possible that a displacer should have properties that are similar to those of the displaced material. If this were true proteins displacement might be carried out with substances having molecular weights of the same order of magnitude as proteins. No experiments have yet been made with such high-molecular-weight displacers. Only a few experiments have been performed by cutting the columns (Fig. 12) or b y their sectional elntion (Fig. 13). Thus a comparison between this method of separation and the common chromatographic method would not be justified. The former method, however, was effective in isolating the heavy at-component (Fig. 15 DI) while the latter was not. If the two methods have the same resolving power for the separation of a certain protein, the former should be preferred because its use offers Re]erences p. a35.
234
s. I-IJERTI~N
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some advantages. For example the proteins emerge in much higher concentration, the capacity of the column is more effectively utilized, the experiments are much less time-consuming, and no fraction collector is necessary. It may be mentioned that a strong displacement effect is not specific of calcium phosphate as adsorbent; it seems to be characteristic of cellulose ion exchangers too, for according to SOBER et al. 2s, "the application of the yellow serum protein solution to the top of the adsorbent co]ulIm quickly produced a series of distinct colored bands". Thus desorption in the manner shown in Figs. 12 and 13 should be useful for these adsorbents also.
Qf i e f 2 0 r n / n
AI
o f f e r IOOt~in
AI
All
C
DI
AllI
Qffer ~rnln
B
DII
o f / e r 120mlm
Oll
Olll
Fig. 1 5. S e d i m e n t a t i o n d i a g r a m s . All s o l u t i o n s were dialysed a g a i n s t 0.2 M s o d i u m p h o s p h a t e buffer, p H 6.8. T h e t o t a l p r o t e i n c o n c e n t r a t i o n s v a r i e d b e t w e e n 0. 3 a n d o. 5 %. O n l y t h e fl-lipop r o t e i n h a d a h i g h e r c o n c e n t r a t i o n (o.8 %). O b s e r v e t h a t t h e v a l u e of t h e s e d i m e n t a t i o n c o n s t a n t for/~-lipoprotein is uncorrected. AI (S2o, w = 2.9, 7.3 a n d 17.1 S) c o r r e s p o n d s to fraction I in Fig. 6; AII (S2o, w = 4.2, 6.7 a n d 18.8 S) c o r r e s p o n d s to fraction I I in Fig. 6; AIII (Szo, w = 7,5 S, caeruloplasmin) c o r r e s p o n d s to fraction I I I in Fig. 6. B (S~0, w = 7.2 S) c o r r e s p o n d s to t h e second p e a k (caeruloplasmin) in Fig. 7. c (S = 2.1 S, uncorrected) c o r r e s p o n d s to t h e l a s t p e a k (~-lipoprotein) in Fig. IO. D I (S2o. to = 18.2 S) c o r r e s p o n d s to t h e c u t t i n g of c o l u m n s (uncolored solution; a~glycoprotein?) ; D I I ($20 ' w = 3.9, 5-7, 6.6 a n d 15. 7 S) c o r r e s p o n d s to t h e c u t t i n g of c o l u m n s (yellow solution); D I I I ( S 2 0 , w = 3"5 a n d 7.5 S) c o r r e s p o n d s to t h e c u t t i n g of c o l u m n s (blue solution; main component caeruloplasmin). ACKNOWLEDGEMENTS
The author wishes to thank Prof. A. TISELIUS for his kind advice and active interest in this work. This work was financially supported b y grants to the Institute of Biochemistry from the Rockefeller and Wallenberg Foundations and from the Swedish Cancer Society. ReIerences p. 235.
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