selective elution of zones from preparative isoelectric focusing gels by ampholytes or buffers

ANALYTICAL BIOCHEMISTRY 75, 314-324 (1976)

Selective Elution of Zones from Preparative Isoelectric Focusing Gels by Ampholytes or Buffers

Protein zones formed by isoelectric focusing on polyacrylamide gels (IFPA) can be eluted without mechanical disruption of the gels. Specific elution is achieved by replacement of the original anolyte, a strong acid, with an ampholyte of a pI higher than that of the protein which is to be eluted. Alternatively, the anolyte may be a buffer of a pH higher than the pZ of the focused protein zone. A rudimentary apparatus and procedures for the application of this method of zone elution are described but are not as yet sufficiently developed to provide a ready- to-use preparative IFPA procedure.

The cardinal defect of preparative isoelectric focusing in density gradients (1) are the load limitation and loss of resolution due to the isoelectric precipitation of proteins at high loads with subsequent sedimentation of precipitated proteins, either of the protein of interest or of contaminants. Isoelectric focusing on Sephadex (2) is free of this problem, but it raises its own problems of irregularity of pH across the width of the Sephadex bed and problems of pH gradient reproducibility, presumably due to the difficulty of achieving bed uniformity and a constant degree of bed hydra- tion. Also, both methods suffer from convection prob1ems.l Isoelectric focusing on polyacrylamide gels (IFPA) is free of the above problems, but previously it had been impossible to overcome the problem of recovery of the protein from the isoelectric zone by means other than slicing and diffusion from slices (3,4). Slicing of the “nonrestrictive” gels required for IFPA (5) is less reproducible than slicing of the harder gels in polyacrylamide gel electrophoresis (PAGE) (6). Accurate slicing is particularly difficult with N,N’-diallyltartardiamide (DATD)-cross-linked gels which are useful in IFPA because of their relatively good wall- adherence properties (7). This method is also laborious and introduces possibly more soluble contaminants derived from polyacrylamide than continuous elution (8). Because of limited heat dissipation (both heat of polymerization and Joule heat) in presently’available apparatus, the gel diameter of cylindrical gels is limited to IO- 15 mm, i.e., a cross-sectional area of 0.8 to 1.8 cm2, making it necessary to slice and elute many gels, when substantial preparative capacity is required. For preparative gel slabs (blocks), slicing devices exist (9) but have not found wide application to date. Therefore, it appeared of interest to develop a method by which isoelectric protein zones could be eluted from gels in IFPA without excision of gel slices and which utilizes gels with cross-sectional areas of

i A. Chrambach, unpublished results

314 Copyright 0 1976 by Academic Ress, Inc. All rights of reproduction in any form reserved.

SHORT COMMUNICATIONS 315

lo-20 cm2 (as used in preparative PAGE) rather than l-2 cm2, as hitherto used in preparative IFPA. A suitable manner of doing this has been pro- posed previously (5,lO). It consists of replacing one of the electrolytes, after attainment of the steady-state (lo), by an ampholyte at its isoelectric point (pZ). The pZ of this compound is selected slightly above (or below) the isoelectric point of the protein zone which is to be eluted from the gel, depending on whether the elution is to proceed into the anolyte or catholyte. Carrier ampholytes with pZ less (or more) than that of the protein will thereby migrate out of the gel, followed by the protein zone and the carrier ampholytes of a pZ identical to that of the amphoteric compound in the electrolyte chamber, at which point elution ceases. The process can be repeated for successive protein zones by substitution of amphoteric compounds of increasing (or decreasing) pZ in the electrolyte chamber. This rationale has been tested and verified by the work reported here.

MATERIALS AND METHODS

(i) Proteins. Bovine serum albumin (BSA) (Sigma No. A-4378) and human hemoglobin (Worthington No. 53E321 or a hemolysate of 50 mg/ml) were dissolved at 4-20 mg/ml in Ampholine (pZ range, 8- 10) just before use, to give a final concentration of 1%. Bromphenol blue was added to 0.005% and sucrose to 20-40% final concentration (the symbol“%” designates “% (w/v)” throughout).

(ii) Analytical ZFPA. The all-glass apparatus recently described (I 1>2 was used with the 6-mm tube reservoir. Gel concentration was 5%T, 150JOCDATD,3 and gel volume was 1.7 ml/tube. Gels contained 1% Ampholine (pZ range, 3.5- 10). Polymerization was effected, after a 5-min evacuation of the polymerization mixture, at 10 mm Hg, using final concentrations of 0.015% K-persulfate, 5 x 10v4% riboflavin, 1 ~1 of TEMED/ml of gel (1 l- 13). Protein loads consisted of 0.2- 1.0 mg of BSA-hemoglobin mixtures. IFPA was carried out at 0-5°C with 0.2 N KOH as the (upper) catholyte and 0.2 N H2SO4 as the anolyte, at 1 mA/tube, until 200 V was reached, and was then regulated at 200 V. Focusing was terminated when the current showed no appreciable decrease over a period of 1 hr, usually after 4 hr. The gels were photographed, and focusing was continued either with 0.2 N H2S04, or with 0.02 M glycine or Na-cacodylate, respectively, pH 6.2, 0.01 ionic strength, in the anode reservoir. After an additional 5 hr of focusing, the gels were rephotographed, and the anodic electrolyte was changed from glycine to 0.02 M arginine (free base, pZ = 10.5) or from Na-cacodylate to Na-glycinate, pH 10.5, 0.01 ionic

’ Available upon request. 3 %T = (acryalmide (grams) + cross-linking agent (grams))/100 ml; %C = cross-linking

agent (gram) x lOO/%T, where the nature of the cross-linking agent is defined by a subscript.

316 SHORT COMMUNICATIONS

strength. Focusing was then continued for another 5 hr, when gels were again photographed. Replicate gels were removed at each of the three stages of focusing, and pH gradients were determined by sectioning of the gels into l-mm slices (4,6), suspension of the slices in 0.5 ml of 0.025 M

KCl, removal of COz from the suspension by overnight storage in vacua over NaOH pellets, and use of a Metrohm EAX 125 microcombination electrode (Brinkman).

(iii) Preparative IFPA with continuous elution. A preparative PAGE apparatus with 10 cm2 cross-sectional area (Buchler, Polyprep 100) was used. The gel (50 ml, 5%T, 2%C, 1% Ampholine, pZ range, 3.5- 10) was prepared using the procedure previously described for preparative PAGE (Appendix III of Ref. (14)). The top reservoir on the column contained the catholyte, 0.2 N KOH; it was recirculated from a 2-liter reservoir. The anolyte, 0.2 N HzS04, was contained in a thermostated lower buffer reservoir as used for analytical PAGE (3,ll). After attainment of the steady-state of isoelectric focusing, the gel column was connected to the lower buffer reservoir of the Polyprep apparatus, containing 0.02 M

glycine. Isoelectric focusing was then continued, using 0.02 M glycine as the elution buffer, until the eluate maintained a pH of 6.20. The elution buffer was then changed to 0.02 M lysine. Lysine at the same concentration was also pumped into the lower buffer reservoir at a rate of 3 ml/min, gradually replacing glycine.

At all steps, great care was taken to maintain hydrostatic equilibrium of the gel by intermittent monitoring of the liquid level in the manometer tube connected to the elution chamber and adjustment of the height of the Mariotte-type elution buffer bottles to maintain identical liquid levels between manometer tube and liquid surface in the column (Appendix III of Ref. (14)). The eluate was analyzed for AzSO, and elution peaks were analyzed by subjecting fractions to IFPA.

Band elution was also carried out as above with 0.02 M Na-cacodylate, pH 6.20, replacing glycine and 0.02 M Na-glycinate, pH 9.45, replacing lysine.

(iv) Preparative IFPA with discontinuous elution. IFPA was carried out in conventional fashion, using the conical bottom and stem of a funnel (see Fig. 3) to hold the gel. The gel concentration was 5%T, 5%CDATD; the pZ range, 3-10. After attainment of the isoelectric end point the gel funnel was transferred into a lower buffer reservoir containing 0.02 M

glycine, and the elution cup (Fig. 3) was attached to the funnel. After elution of bands with pZ equal to or less than pH 6.20 and emptying of the contents of the cup, the anolyte and the cup contents were changed to 0.02 M lysine. After elution of all bands with pZ equal to or less than pH 9.45, the contents of the cup were collected and analyzed by IFPA and stained for protein (15).

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H2S04

Glvcine

Arginine

H2SO4

FIG. 1. pH gradients in IFPA (pl range. 3.5-10: 5%T. 15%CnATD, 0°C) as a function of time and the nature of the anodic electrolyte. The positions of BSA (~1 = 4.8) and hemoglobin (pZ = 7.0) detected by their blue and red colors are indicated by the arrows. The anodic electrolyte is: in I. II, IV. 0.2 N H,SO,; in III, 0.02 M glycine (~1 = 6.2); in V, 0.02 M

arginine (~1 = 10.5). Duration of experiments I, II, III, IV. and V was 4. 9. 5. 14, and 5 hr respectively. A corresponding series of experiments, using 0.01 M Na-cacodylate. pH 6.2 (VIII). and 0.01 M Na-glycinate, pH 10.5 (Xl, as consecutive eluents. is shown in the bottom panel. The control experiments, using acid anolyte for a duration identical to that used in the elution steps. are shown in VI, VII, and IX.


RESULTS

(A) Displacement of Isoelectrically Focused Zones of Protein from the Gel in Analytical IFPA as a Function of the pl of Amphoteric Compounds in the Anodic Reservoir

BSA stained with bromphenol blue and hemoglobin were fractionated by analytical IFPA to the “isoelectric end point” (Fig. 1, I). After change of the anolyte to 0.02 M glycine (pZ = 6.2), BSA migrated into the anode reservoir. Concurrently, the pH gradient in the gel changed from its original value, 3.5- 10.5, to a range from 6.2 to 11.2 (Fig. 1, III). The control experiment, carried out for an equal length of time but without the substitution of H,SO, by glycine, showed no such shift in pH gradient and no migration of BSA into the anode reservoir (Fig. 1, II).

Replacement of the glycine contained in the anode reservoir by arginine (pZ = 10.5) largely abolished the pH gradient, except in the region from 8.0 to 9.5, and caused the migration of hemoglobin into the anode chamber (Fig. 1, V). A control experiment carried out for the same amount of time but with H,SO, in the anode reservoir failed to show any significant altera- tion of the pH gradient or a migration of hemoglobin into the anode chamber (Fig. 1, IV). At each stage, the positions of BSA and hemoglobin were detected by their blue and red color, respectively (indicated by the arrows in Fig. 1).

That the migration of protein zones into the anode reservoir, under the conditions used, occurs while the proteins remain at their isoelectric pH, was documented by analogous experiments on a preparative scale, reported in Section C.

(B) Displacement of Isoelectric Zones in IFPA by Electrophoretic Migration, brought about by Replacement of the Anodic Acid with Buffer of the Appropriate pH

BSA and hemoglobin separated by IFPA were eluted from the gel in the same fashion as described in Section A, using sodium cacodylate buffer, pH = 6.20 (Fig. 1, VIII), and sodium glycinate, pH = 10.45 (Fig. 1, X), as the consecutive eluents in the anolyte reservoir. The control experiments, using acid anolyte for the identical time periods as used for the elution, are depicted in Fig. 1, VI, VII, and IX. Protein displacement was detected as described in Section A.

(C) Displacement of Isoelectric Protein Zones in Preparative IFPA by Specific Amphoteric Compounds

BSA and hemoglobin (50 mg each) were fractionated by IFPA (pZ range, 3.5-10, 5%T, 2%C) on a preparative PAGE apparatus of lo-cm2 gel surface area.


After attainment of the steady-state and separation of protein zones, the anodic H,SO, was replaced by 0.02 M glycine, and continuous elution with 0.02 M glycine was begun at 1 ml/hr. Thereafter, BSA eluted in a peak at pH 4.8 (Fig. 2, top panel). When the eluate had reached a pH of 6.2 (the pl of glycine), the anodic electrolyte was changed from glycine to lysine (free base). Thereafter, hemoglobin eluted starting at pH 7.5, very close to the pZ of hemoglobin of 7.0 (Fig. 2, top panel). The yields, based on recovery ofA2*,,, were 99% in the case of BSA and 37% in the case of hemoglobin. (The consistent low yield of hemoglobin may possibly be a general peculiarity of basic proteins.) Analytical IFPA of the eluate peaks, with de- tection of protein bands by staining, showed the absence of crosscontamination in the eluate between BSA and hemoglobin.

(D) Displacement of Isoelectric Protein Zones in Preparative IFPA by a Single Amphoteric Compound

The experiment described in Section C was repeated, except that the anodic acid was replaced only once, viz., with lysine, pZ = 9.5, after ap- proximation to the isoelectric end point and separation had taken place. Figure 2 (bottom panel) shows the resulting elution pattern and separation of BSA and hemoglobin. Recoveries, based on Azso, were 8% for BSA and 46% for hemoglobin. Analytical IFPA of the peak tubes of BSA and hemoglobin showed the absence of crosscontamination in these tubes, due to disparity in concentration ratios between the two proteins. However, there is considerable distribution overlap between the elution peaks. The causes of the bimodal elution of BSA in the experiment shown are not known.

(E) Displacement of Isoelectric Proteins in Preparatilve IFPA by Specijic Buffers or by a Single Buffer

The preparative experiment described in Section C was repeated, using as eluents sodium cacodylate, pH 6.2, and sodium glycinate, replacing glycine and arginine.

The elution of BSA and hemoglobin was also achieved by Na- glycinate, pH 10.5, alone, in analogy to the results reported in Section D.

(F) Discontinuous Band Elution in IFPA

BSA and hemoglobin (10 mg each) were loaded onto an IFPA gel (pZ range, 3-10, 5%T. 15%CohTo) contained in the funnel tube apparatus shown in Fig. 3. After conventional focusing between 0.2 N KOH (catholyte) and 0.2 NH,SO, (anolyte) to the isoelectric end point, with formation of sharp zones of the two proteins, the anolyte contained in a lower buffer reservoir (of the type used for analytical PAGE) was changed


FIG. 2. Elution patterns of preparative IFPA with continuous elution: 50 mg of BSA (hatched) and hemoglobin (cross-hatched) separated by IFPA on the Polyprep- apparatus were eluted (top panel) by 0.02 M glycine (pZ = 6.2), followed at the time indicated by the arrow by 0.02 M lysine (pl = 9.5). BSA elutes at pH 4.6-6.0, hemoglobin at pH 9.2. The bottom panel shows elution by use of lysine as a single eluent. Both proteins elute at pH 9.2. Gel concentration, S%T, 2%C; pZ range, 3.5 10; eluent flow, 1 ml/min; regulated voltage, 200 V.

to 0.02 M glycine, and the elution cup was attached to the funnel. The cup also contained 0.02 M glycine. Elution at 200 V was allowed to proceed for a period of 2 days. Then the cup contents were withdrawn and analyzed by IFPA, and both anolyte and cup contents were replaced by 0.02 M lysine. Elution at 200 V was continued for another day. Recoveries of BSA and hemoglobin, based on AzBO, were 28 and 48%, respectively. The BSA fraction was homogeneous on rerun in analytical IFPA. How- ever, the hemoglobin fraction was contaminated with BSA under the conditions used.

DISCUSSION

The feasibility of eluting specific isoelectric zones formed in IFPA electrophoretically has been demonstrated. However, a number of problems remains to be solved before the new elution technique can become a widely applicable preparative method in biochemistry.

(i) Resolving power. The separation used to demonstrate the elution method is a very simple one, employing two proteins with widely differing pls. It remains to be established by further work what the limits in pZ differences of proteins are which can be resolved by the method.

(ii) Ampholytes vs buffers as eluents. The method of elution of proteins


FIG. 3. Apparatus for discontinuous elution of zones from preparative IFPA: ( 1) jacketed lower buffer reservoir: (2) Pyrex funnel; (3) anolyte: (4) catholyte; (5) IFPA gel (6) threaded collar; (7) elution cup; (8) silicone rubber gasket: (9) glass (Corning 7930) membrane: (10) air inlet into elution cup.

by ampholytes, with maintenance of the order of zones during elution and of the isolectric pH for the component of interest throughout the elution process, depends on the availability of amphoteric compounds of suitable pH. At this time, their selection is rather limited. Some are listed in the early work of Rilbe (16) preceding the synthesis of Ampholine. Homogene- ous components of Ampholine or ranges of well-defined Ampholine components could serve that purpose, as recently provided by the frac- tionation of carrier ampholytes and by the synthesis of mixtures of only a few components of ampholytes (17).

The use of buffers for elution of isoelectric bands in IFPA has the great advantage that the buffers are available at any pH. However, in this case the bands may not elute at their isoelectric pHs, and the electrophoretic migration of the zones may entail band spreading in proportion to the length of migration path, increase in Joule heating, possibly a change in the order of eluting bands, which may lead to impairment of resolution.

(iii) Choice of efuent pZ OY pH. There may be applications where the


use of a selective pZ or pH, to remove a single component from the gel selectively, is inappropriate and where it is desirable to apply an eluent at a high pZ (or low pZ, if polarity is reversed). However, this approach is likely to lead to decreased resolution (Fig. 2) in analogy to application of a steep buffer gradient for elution in chromatography, as compared to a shallow gradient or use of “equilibrium elution” by a single buffer. To date, gradient elution has not as yet been applied to IFPA, although it ap- pears promising.

(iv) Continuous vs discontinuous elution. Continuous elution leads to extreme dilution of eluate and is therefore undesirable. In this work, it has been used only to demonstrate the method. In practice, recycling of elution buffer should be preferable. Recycling allows one to limit to a few milliliters the eluent volume by which the eluting sample is diluted. How- ever, since ampholytes elute continuously, the pH of the eluent would change during recycling, unless the recycled eluent is also continuously dialyzed or unless it is replaced by fresh eluent at intervals. Optimally, it would be replaced discontinuously, when pH or absorbance detectors built into the flow line indicate the elution of the material of interest.

The simplest solution to specific elution of zones is a static cup of the type shown in Fig. 3. However, it presents problems of membrane adsorp- tion which are aggravated compared to those encountered with membranes in the elution chambers of eluent flow devices. Also, the membrane in a static device has to be freely permeable to Ampholine components which are continuously entering the cup and which tend, if not removed, to alter the pH within the cup and thus oppose the elution of the component of interest. Dialysis membrane is more suitable than the relatively impermeable 7930 glass membrane used in this work.

(v) Apparatus. An inherent problem in all preparative IFPA methods is pH-dependent gel swelling or shrinking causing problems in wall adherence, current leakage, and complete severance of the gel. The occurrence and degree of severity of these problems will depend on the choice of carrier ampholyte pZ range and of eluent and duration of elution. Remedies are: the use of DATD-cross-linked gels with improved wall-adherence properties and greater elasticity than Bis-cross-linked gels (7), careful maintenance of hydrostatic equilibrium of the gel of all stages subsequent to polymerization, possibly attachment of mechanical supports to the gel bottom (see 1 l), and, possibly, the use of plastic apparatus which allows the gel to extend, coupled with mechanical supports (18). Our limited ex- perience to date indicated that nylon mesh supports are not very helpful and that it is very difficult to avoid mechanical stability problems under the conditions of elution-IFPA when other than 2%C,, gels are used in the Polyprep apparatus. Such gels have the disadvantage of being more restrictive than the correspondingly low ‘ST gels with high %C (7). In contrast, in the funnel apparatus, where the gel is supported by the


conical part of the upper electrolyte reservoir, gels of 5%T, 15%C are perfectly stable for many days of operation at 200 V. The price to pay for this advantage is reduction of load capacity in the ratio of cross-sectional gel areas of funnel apparatus (3.1 cmz) and Polyprep apparatus (10 or 20 cm2). Nonetheless, the load capacity of IFPA being of the order of 10 mgkm2 of gel, and considering that the geometry of a funnel allows one to apply very large sample volumes, even the funnel apparatus has substantial milligram-preparative capacity.

The large elution buffer reservoirs and one-way elution buffer how in present preparative PAGE apparatus may have to be supplemented by small reservoirs and an easy way for shunting elution flow to recycling and collection. It is also to be expected that the optimized preparative IFPA apparatus with continuous elution should be scaled down to a much smaller gel than the preparative PAGE apparatus uses, since the load capacity of IFPA (3) exceeds that of PAGE (11) by possibly as much as two orders of magnitude.

CONCLUSIONS

It is concluded that elution of protein zones from preparative IFPA gels by use of amphoteric compounds or by buffers is feasible. The elution can be made specific for each zone by proper selection of pZ or pH of the eluent. Optimal apparatus and procedures remain to be developed.

ACKNOWLEDGMENT

We thank Dr. G. Baumann for a critical review of the manuscript.

REFERENCES4

1. Vesterberg, O., and Svensson, H. (1%6) Acta Chem. Stand. 20, 820. 2. Radola, B. J. (1973) Biochim. Biophys. Acta 295, 412; (1973) Ann. N.Y. Acad.

Sci. 209, 127. 3. Finlayson, R., and Chrambach, A. (1971) Anal. Biochem. 40, 292. 4. Tipton, H., Rumen. N. M., and Chrambach. A. (1975) Anal. Biochem. 69, 323. 5. Chrambach, A., and Baumann, G. (1975) in Isoelectric Focusing (Catsimpoolas, N.,

ed.), Academic Press, New York, p. 77. 6. Peterson, J. I., Tipton, H. W.. and Chrambach, A. (1974)Anal. Biochem. 62, 274. 7. Baumann, G., and Chrambach, A. (1976) Anal. Biochem. 70, 32. 8. Baumann, G.. and Chrambach, A., in preparation. 9. Bell, P. H., McClintock, D. R., and Snedeker. E. H. (197.5) Anal. Biochem. 65, 586.

10. Chrambach, A., Doerr. P., Finlayson, R., Miles, L. E. M., Rodbard, D.. Sherins, R.. and Rodbard. D. (1973) Ann. N. Y. Acad. Sci. 209, 44.

1 I. Chrambach, A., Jovin, T. M., Svendsen, P. J.. and Rodbard, D. (1976) In Methods of Protein Separation (Catsimpoolas, N.. ed.). vol. 2. Plenum, New York, p. 27.

12. Rodbard. D., and Chrambach. A. (1971) Anal. Biochem. 40, 95. 13. Doer-r, P., and Chrambach. A. (1971)AnaL Biochem. 42, 96.

a References (5). (8) and (11) are available upon request.


14. Kapadia, G., and Chrambach, A. (1972) Anal. Biochem. 48, 90. 15. Reisner, A. H., Nemes, P., and Bucholtz, C. (1975) Anal. Biochem. 64, 509.

16. Svensson, H. (1961) Acta Chem. Stand. 15, 325; (1962) 16, 456. 17. Brown, R. K., Lull, J. M., Bagshaw, J. C.. Lowenkron, S., and Vinogradov, S. N.

(1975) Fed. Proc. 34, 625; (1976) Anal. Biochem. 71, 325. 18. Svendsen, P. J. (1973) Sci. Tools 20, 1.

ALICE MCCORMICK~ LAUGHTON E.M. MILE@ ANDREAS~HRAMBACH

Reproduction Research Branch National Institute of Child Health and Human Development National Institutes of Health

Bethesda, Maryland 20014. Received January 7, 1976; accepted April 20, 1976

5 Guest worker. 6 Present address: Department of Medicine, Stanford University, Palo Alto, Calif.

selective elution of zones from preparative isoelectric focusing gels by ampholytes or buffers

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