Rapid chromatofocusing of proteins

ANALYTICAL

BIOCHEMISTRY

126, 37-43 (1982)

Rapid Chromatofocusing G. WAGNER

of Proteins

AND F. E. REGNIER

Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907 Received January 14, 1982 Chromatofocusing, a chromatographic technique whereby proteins are selectively eluted from an ion-exchange support according to their pZ values, has been adapted to high-performance liquid chromatography. It was found that chromatofocusing can detect heterogeneity in protein preparations not demonstrated by reverse-phase or size exclusion chromatography and that the resolution of chromatofocusing is comparable to ion-exchange chromatography. Although chromatofocusing may not resolve proteins as well as isoelectric focusing, it has advantages over this technique in both speed and capacity. The usefulness of chromatofocusing as an additional technique in the analysis and preparation of proteins is discussed. The rapid separation technique described here is able to resolve protein mixtures in the chromatofocusing mode in approximately 30 min.

In ion-exchange chromatography (IEC),’ charged proteins are adsorbed to an ion-exchange support and selectively eluted with either a pH or salt gradient. The gradient is formed externally and fed into the column to desorb bound species. Recently, Sluyterman and co-workers (l-4) reported a new ion-exchange technique, “chromatofocusing,” in which a pH gradient is generated within the column by making use of the natural buffering capacity of the exchanger. For example, when negatively charged proteins are adsorbed to an anion-exchange column which has been equilibrated at a high pH and a buffer of a lower pH is then pumped into the column, a pH gradient is generated that moves down the length of the column, and the proteins are selectively desorbed (when the pH I pZ) and readsorbed (when pH > pl). Thus, a protein moves along the column at or near the point in the migrating pH gradient equal to its pZ. Since the linear velocity of the mobile phase is greater than the velocity of a particular pH within the gra-

dient, the process described above will “focus” proteins of the same pZ values. The slope and length of the gradient can be manipulated by adjusting the buffer capacity and pH of the mobile phase. Ampholyte-displacement chromatography also makes use of a pH gradient established in an ion-exchange column (5). This technique differs from chromatofocusing in that elution of proteins is accomplished using carrier ampholytes instead of buffers, and the pH gradient is obtained by a continuous displacement in which more acidic ampholyte components constitute the front of the eluent (6). Most chromatographic separations based on isoelectric points have, to date, involved displacement chromatography (7- 14). However, the high cost of the ampholytes used in this technique seems to be a major disadvantage. Chromatofocusing does not have this disadvantage and would appear to be a more efficient system than displacement chromatography in that base line resolution of proteins of a 0.02 pH unit difference in their pi’s can be achieved (15). A disadvantage of open-column chromatofocusing in the analysis and preparation of

’ Abbreviations used: IEC, ion-exchange chromatography; HPLC, high-performance liquid chromatography; IEF, isoelectric focusing. 37

0003-2697/82/150037-07$02.00/O Copyright All rights

Q 1982 by Academic Press, Inc. of reproduction in any form reserved.

38

WAGNER

AND

many proteins is the 24 or more h which are required to develop and recycle a column. The purposes of this study were to determine if the principles and techniques of chromatofocusing could be applied to high-performance liquid chromatography (HPLC) making use of the rapid separation and overall efficiency of HPLC and to determine how well HPLC chromatofocusing compares in resolution with other types of HPLC techniques. MATERIALS

AND METHODS

All proteins used in this study were purchased from Sigma and were the highest grade available. Chromatofocusing columns were equilibrated with 0.025 M imidazoleacetate or -HCL, pH 7.4, and pH gradients established with a 1: 10 dilution of Polybuffer PB 96 or PB 74 (Pharmacia) adjusted to either pH 6.0 or 4.0 with glacial acetic acid or HCl, respectively. The columns used in these experiments were a 250-mm X 4.1-mm-i.d. SynChropak AX-300 and a SynChropak RP-P of the same dimension, and the instrument a Micromeritics Model 7000 high-performance liquid chromatograph equipped with a Chromonitor 785 variable wavelength detector. The pH was monitored with a Markson Model 4503 pH meter and a flow-through electrode system (Markson). Eluent temperature was monitored with the temperature probe of the pH meter, and absorbance and pH were recorded simultaneously using a Sargent Model DSRG recorder. For isoelectric focusing (IEF), 35 ~1 of a 1% conalbumin solution was suspended in 0.1 M potassium phosphate buffer, pH 7.0, and 1.0 M urea before application to a 7.5% acrylamide 10 X 15 X 0.2-cm gel in 6 M urea and 2% ampholyte, pH 5-7 (LKB). The gels were electrophoresed for 5 h at 5°C 900 V, and about 7 mA using an Isco Model 494 power supply. Gels were fixed, stained, and destained according to the method of Vesterburg ( 16).

REGNIER

RESULTS

Several different buffer systems were tested for the development of a suitable pH gradient. A pH gradient having a small and nearly constant ApH value (0.33 pH unit decrease/ml) from pH 7.0 to 4.0 was formed using 0.075 M citrate-phosphate buffer to initially equilibrate the column and to establish the pH gradient (data not shown). Linear gradients could be established with citrate buffers as long as protein samples were not applied to the column. However, when proteins were applied to the column the pH gradient was disturbed by elution of each protein component. The Polybuffer systems of Pharmacia were used in this study because they gave more constant ApH values of 0.1 and 0.03 for PB 74 and PB 96 at pH 7.0-4.0 and 7.0-6.0 gradients, respectively. Assuming a total accessible volume of 2.05 ml for this column (measured by elution of D20), the PB 74 gradient was formed over 14.6 column vol and the PB 96 gradient over 16.2 column vol. If Polybuffers were diluted more than 1: 10, or if a gradient of < 1 pH unit was used, the ApH values became somewhat erratic and were unsatisfactory. The focusing effect of HPLC chromatofocusing columns was demonstrated by injecting three 20+1 aliquots of 1% ovalbumin during a run at 0,4, and 8 min after starting. The protein profile obtained was nearly identical to that obtained when 60 ~1 of protein were injected at one time (Fig. 1). Separation of proteins with widely different pZ values (6.8-4.7) was easily achieved in less than 30 min (Fig. 2). The pH at elution for the main peaks of pyruvate kinase, conalbumin, and ovalbumin measured at 2 1 “C were 7.25, 6.80, and 5.07, respectively. The plvalues ofthese proteins are 6.75,5.88, and 4.70, respectively ( 17). Chromatofocusing of other proteins (data not shown) consistently gave elution pH values higher than pZ values reported in the literature. Decreasing the ionic strength of the buffer and flow rate did not substantially change the elution pH.

RAPID CHROMATOFGCUSING

39

OF PROTEINS

However, a decrease in flow rate resulted in an increase in resolution (&) of components of a preparation of conalbumin (Fig. 3) when R, was calculated according R = 2(T2 - Td

’

AT, + AT,’

T, and T2 are the elution times of two substances and AT, and AT2 are the respective peak widths of these compounds. A comparison of R, values at flow rates of 2 and 0.5 ml/min shows a 74% increase between peaks I and II, a 26% increase between peaks II and III and a 92% increase between peaks I and III (Table 1). Decreasing the flow rate to 0.25 ml/min did not substantially increase resolution, and at times, the ApH became irregular. Changing ionic strength of the buffer also did not appreciably affect resolution. No attempts were made to increase resolution by increasing column length or by using a different column packing material. Compared with other HPLC procedures, chromatofocusing was generally superior when based on the number of peaks resolved. Chromatofocusing partially resolved at least three components of conalbumin while re-

cm8 I I , 1 I , I I n --‘\ ‘\ - 0.07--,. ‘\ ‘\ ‘\ ‘\ ‘\ - 0.06‘. ‘\ ‘\ ‘. E ‘\ ‘\ ; 0.05‘\ ‘\ w l 0.04 -

,

o0

5

IO TIME

I5 (min)

20

25

3:

FIG. 2. Chromatofocusing profile of a mixture of proteins. Sixty microliters of a solution containing 4% each of pyruvate kinase (a), conalbumin (b), and ovalbumin (c) was injected. The pI values are 6.75, 5.88, and 4.70, respectively (17). The pH gradient (- - -) was formed as in Fig. 1. Flow rate was 1 ml/min and the temperature 21°C.

verse-phase and size exclusion chromatography indicated that the mixture was a single component (Figs. 4A-C). Using the same column, IEC (Fig. 4D) resolved peaks II and III better than chromatofocusing, but the lat-

8 B -7 -6 -5

4: y

0.03

-

5 m 0.02

-

E fij

0.01

J 0

0

’

5

’

10

’

15

’

’

20

0

TIME

lminl

’

5

’

10

’

15

’

20

10 0 2P

FIG. 1. Chromatofocusing of ovalbumin. (A) Sixty microliters of a 1% suspension of ovalbumin and (B) three 20-jd aliquots of the same preparation were injected at intervals (arrows). The pH gradient (- - -) was formed in an AX-300,250-mm X 4. I-mm-i.d. Synchropak column using a 1: 10 dilution of PB 74 Polybuffer, pH 4.0 after the column was equilibrated in 0.025 M imidazole-HCl, pH 7.4. Flow rate was 1 ml/min.

5 TIME

‘5 (mid

20

25

30

FIG. 3. Chromatofocusing profiles showing the effect of flow rate on resolution of peaks of conalbumin. Twenty microliters of a 4% suspension of conalbumin was chromatographed at (A) 2, (B) I, and (C) 0.5 ml/ min. The pH gradient (- - -) was formed using a 1:10 dilution of PB 96 Polybuffer, pH 6.0, after the column was equilibrated in 0.25 M imidazole-acetic acid, pH 7.4. I, II, and III represent the major components of conalbumin.

40

WAGNER AND REGNIER TABLE

1

A COMPARISON

OF THE RESOLUTION NENTSOFACONALBUMINPREPAJUTIONCHROMATOFOCUSEDAT DIFFERENTFLLIWRATES R = w-2 6 AT,

OF Co~po-

matofocusing. Both values are significantly higher than the value of 5.88 reported in the literature (17). DISCUSSION

- z-1)" + AT,

Between peaksb Flow rate (ml/min)

I and II

II and III

I and III

2.0 1.0 0.5

0.53 0.60 0.92

0.77 0.81 0.97

1.3 2.0 2.5

’ T, and T2 are the elution times of two substances, and AT, and AT, are the respective peak widths. *Peaks I, II, and III are the major components of conalbumin preparation and are shown in Fig. 3.

ter technique resolved peaks I and II better than IEC. Conalbumin recovery in the ion-exchange mode was approximately 93%. Chromatofocusing on the other hand showed slightly lower recovery at 89%. The relatively small difference between these two modes may not be typical of all proteins. Manipulation of some proteins at or near their isoelectric point in chromatofocusing could cause low recovery. The chromatogram of conalbumin in Fig. 4A is somewhat different than that in Fig. 3. Such differences were found between batches of conalbumin and between solutions of the same batch stored for different lengths of time. Similar results were found with ovalbumin and other proteins used in this study, and for this reason, all comparative studies were made using the same batch of material over as short a time as possible. The protein band profile on IEF gels (Fig. 5) was similar to the peak profile of chromatofocusing columns (Fig. 4A). One major and two minor bands were observed in IEF gels which is similar to the chromatofocusing profile of the same preparation. The estimated p1 of the main component from IEF was about pH 6.6 which is close to the pH 6.4 value of the p1 as measured from chro-

To establish a gradient over a specific pH range, there must be buffering species available at all points of the gradient. Thus, over a pH range of 2 or 3 units, a mixture of buffer components is necessary to supply needed buffering species. Several mixtures of “common” buffers were initially tested since easy availability and low cost would be desirable. The Pharmacia Polybuffers were chosen over the citrate-phosphate buffers because the latter occasionally gave varying ApH values both within and between gradients. The ApH values of the Polybuffer gradients were very constant and there was no pH plateau at the protein elution point as observed with citrate-phosphate buffer. The number of peaks (n) that can be resolved per pH unit of a gradient can be calculated from

where V, is the volume of the gradient, V,, is the volume of an average peak, and ZVpH is the number of gradient pH units. For the pH 7 through 4 and 7 through 6 gradients, n was 5.43 and 15.1, respectively. The term I/n will give the ApH/peak and is 0.19 and 0.07 units for the pH 7-4 and 7-6 gradients, respectively. Thus the resolution of the shallower pH 7-6 gradient is 2.7 times greater than that of the pH 7-4 gradient. Comparing 1/n of peak I in the conalbumin sample chromatographed at flow rates of 2, 1, and 0.5 ml/min shows values of 0.166, 0.102, and 0.056 pH units/peak. These calculations show that a decreased flow rate increases resolution and that maximum resolution of conalbumin is reached using the pH 7-6 gradient and a 0.5 ml/min flow rate. The 0.056 value measured with the HPLC system is about one-half the resolution obtained with

RAPID CHROMATOFOCUSING

41

OF PROTEINS

------__ ---_ A 7 --__ ---_ ------6 i i ; 0.04 It

-1 4 I

II

pII9, 5

0

,iJ: IO

I5 TIME

20 knin)

25

30

35

010

400

’

’

2

4

’

’

6 TIME

1

6 (min)

1

IO

1

12

16

14

I

-5 1.2-

-120 1-m

g l.ON + 0.6Q

-100 ,‘------

-80

.’ J 0.6 5 2 0.4 -

-60

.’

. I’ .’

- 40

/I’

5: :0.2-

,’

A s

,*’

*’ OH” 0

-7 I 1; 0 5 2 %

/ 5

’ IO

’ ’ ’ 15 20 25 TIME Imin)

1 30

1 35

;

0.05 -

.t-* 0.04 -

**

w g 0.03 8

.*

/*

HC

- 40 m 2 - 30 y

cc

d - 20 w

cc 0.02 -_/* :: fj 0.01 0

4o”

I

0

8 - IO I 5

I IO

I 15

J

TIME

I 20

I 25

I 30

I_ 35

40’

(mid

FIG. 4. Profile comparison of conalbumin chromatographed using (A) chromatofocusing, (B) size exclusion, (C) reverse-phase, and (D) ion-exchange exclusion chromatography. Chromatofocusing details are the same as in Fig. 3C. Ion-exchange chromatography on a 250-mm X 4.1-mm-i.d. AX-300 Synchropak coIumn was run at 0.5 ml/min using a 40.min, O-50% gradieat of 0.5 M Na-acetate (solvent B) in 0.02 M Tris-HCl (solvent A). Reverse-phase chromatography on a Whatman Cz 50 X 4. l-mm column was run at 0.7 ml/min using a 30-min, O-80% gradient of 2-propanol in 0.1% trifluoroacetic acid. Size exclusion chromatography was run at 0.8 ml/min using 0.2 M sodium phosphate buffer, pH 7.4, on a TSK 3000 300 X 9.0-mm column. I, II, and III represent the major peaks of conalbumin.

open-column chromatofocusing( 14), but the separation time of open-column chromatofocusing ( 14) is approximately 50 times greater than that of HPLC chromatofocusing. The focusing effect as demonstrated above will enable one to load proteins on a column in a relatively large volume and still obtain maximum resolution. This distribution volume ( Vd), the maximum volume in which a protein may initially be suspended in order to emerge as a single band(s), can be calculated from the equation Vd = v, - v, ,

where V, is the elution volume of the exeluded form of the protein and V,, is the elution volume of a neutral form of the protein (total accessible volume). For ovalbumin chromatographed in a 4.1 X 250-mm AX300 column, Vd = 1.O ml using values of 1.5 ml for V, as measured from ovalbumin chromatographed in 0.5 M salt at pH 4.0 and 0.5 ml for V. as measured from ovalbumin chromatographed at pH 4.0. Thus, ovalbumin can be suspended in 1.O ml of buffer and still emerge with maximum resolution. Such data for a protein and column can be of use in preparation of large quantities of protein.

42

WAGNER AND REGNIER

PH

FIG. 5. IEF prolile of a preparation (the same one used in Fig. 4) of conalbumin. The pH gradient was formed using 6 M urea and 2% ampholyte, pH 5-7, in 7.5% acrylamide 10 X 15 X 2-cm gels run at 7 mA and 900 V for 5 h at 5°C. A photograph (A) and diagram (B) of the gels after staining and destaining are shown as well as an estimation of the pH gradient (C).

It has been shown that ionic strength of the eluent in chromatofocusing increases throughout gradient formation and can displace bound protein (14). Proteins displaced by competing ions elute at a pH above their pZ. The effect of this phenomenon varies with the protein and can be as high as l-2 pH units above the pZ ( 14). The displacement effect may account for the apparent high pZ values found for proteins in this study; however, it is unlikely that the high pZ values can be attributed entirely to this phenomenon because displacement is less likely to occur during the early part of the gradient where the ionic strength is comparatively low and where both pyruvate kinase and conalbumin elute (Fig. 2). High-performance chromatofocusing and anion-exchange chromatography detect heterogeneity in preparations of conalbumin while high-performance size exclusion and reverse-phase chromatography do not. This heterogeneity, which is due to charge, appears to be equally well resolved by IEC and chromatofocusing, but each gives maximum

resolution of different components and neither is clearly superior. The difference in resolution is most likely due to components I and II having significantly different pZ values, but similar net charge, at a higher pH. The converse explanation could explain the difference between components II and III. Chromatofocusing thus provides another type of analysis for purity beyond that offered by IEC. Chromatofocusing yields results comparable to IEF in both the number of bands and the pZ values but is inferior in resolution. Chromatofocusing, however, offers the advantage of speed and preparative capabilities which are lacking in the IEF technique. The pZ values measured in both techniques are higher than those reported in the literature (17), and the differences are greater than can be accounted for by the different temperatures at which the pZ values were measured. This study shows that chromatofocusing can be successfully applied to the HPLC system. Chromatofocusing detected and partially resolved heterogeneous protein preparations which are not detected in other types of HPLC techniques. This technique should be a valuable tool in the continuing analysis and preparation of proteins. It would also be concluded that the value of chromatofocusing for the determination of pZ values of proteins is questionable. Further studies on HPLC chromatofocusing are needed to determine if this shortcoming can be overcome. ACKNOWLEDGMENT The authors gratefully acknowledge National Institutes of Health, U. U. S. Health Service (GM 2543 1) in is Journal Paper No. 8835 from the Agricultural Experiment Station.

the support of the S. Public Health, this research. This Purdue University

REFERENCES 1. Sluyterman, L. A. E., and Elgerima (1978) J. Chromatogr. 150, 17-30. 2. Sluyterman, L. A. E., and Wijdenes, J. (1978) J. Chromatogr. 150, 3 l-44. 3. Sluyterman, L. A. E., and Wijdenes, J. (198 1) J. Chromatogr. 206, 429-440.

RAPID CHROMATOFOCUSING 4. Sluyterman, L. A. E., and Wijdenes, J. (1981) J. Chromatogr.

206,44

them.

Biophys.

Rex

13.

Commun.

67,248-254. (1980) J. Chromatogr.

6. Emond, J. P., and Page, M. 200,57-83. 7. Page, M., and Belles-Isles, M. (1978) Canad. them.

12. Young, J. L., and Webb, B. A. (1978)

1-447.

5a. Leaback, D. H., and Robinson, H. K. (1975) Bio-

J. Bio-

14.

56, 853-856.

8. Young, J. L., and Webb, B. A. (1978) Anal. Biothem.

88,619-623.

9. Bloj, B., and Zilversmit, D. B. (1977) J. Biol. Chem. 252, 1613-1619. 10. Page, M., and Belles-Isles, M. (1978) in 9th International Symposium on Chromatography and Electrophoresis 1978. 11. Young, J. L., Webb, B. A., an Coutri, D. G. (1978) Biochem. Sot. Trans. 6, 1051-1054.

OF PROTEINS

15.

16. 17.

43 Sci. Tools 25,

54-56. Young, J. L., and Webb, B. A. (1979) in Protides of Biological Fluids, Proceedings of the 27th Colloquium (Peeters, M. ed.), pp. 739-742. Chapuis-Cellier, C., Francina, A., and Amaud, P. ( 1979) in Protides of Biological Gluids, Proceedings of the 27th Colloquium (Peeters, H. ed.), pp. 743-746. Pharmacia Fine Chemicals (1980) in Chromatofocusing with Polybuffer and PBE, Pharmacia Fine Chemicals, Uppsala. Vesterburg, O., Hassen, L., and Sjosten, A. (1977) Biochim. Biophys. Acfa 491, 160- 166. Rigletti, P. G., and Caravaggio, T. (1976) J. Chromatogr. 127, l-28.