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Preparative separation of hemoglobins A and S by gel electrofocusing, using selective zone elution by gel transposition between suitable anolytes and catholytes
ANALYTICAL
BIOCHEMISTRY
85,
209-218 (1978)
Preparative Separation of Hemoglobins A and S by Gel Electrofocusing, Using Selective Zone Elution by Gel Transposition between Suitable Anolytes and Cat holytes ALICE
G. MCCORMICK,~
HELENA ANDANDREASCHRAMBACH~
WACHSLICHT,
Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20014 Received April 6, 1977; accepted October 6, 1977 Preparative electrofocusing on polyacrylamide gels has been limited, until recently, to excision of gel slices, diffusion, and collection ofthe slice diffisates. An advance was made by the introduction of a method of selective electrophoretic zone recovery by specific changes of anolyte (A. McCormick, L. E. M. Miles, and A. Chrambach, 1976, Anal. Biochem. 75, 314-324). It was shown (a) that selective zone recovery could be achieved by transposition of the gels into either isoelectric ampholytes or charged buffers, (b) that it could be applied to the gram scale, and (c) that zone elution could proceed either continuously or discontinuously. The early study was, however, limited to a trivial model problem, the separation of hemoglobin from bovine serum albumin (BSA). The present study was an attempt to apply a similar selective zone recovery method to a more demanding separation problem, the separation of hemoglobin A from hemoglobin S as well as from other minor components contained in a sickle-trait human hemolysate. The study shows that selective electrophoretic zone elution from an electrofocusing gel 18 mm in diameter is capable of yielding hemoglobin A, separated from hemoglobin S, differing by only 0.2 pH units in isoelectric point. The recovery of hemoglobin A was 70%. with a load of 32 mg of hemoglobin mixture per gel, using discontinuous zone elution into a collection cup.
Preparative electrofocusing on gels is important since its performance tolerates, and may be improved by, protein precipitation at the isoelectric point (pZ) which occurs routinely at elevated milligramor grampreparative loads. Three techniques of preparative gel electrofocusing have been described: (I) slicing of wide diameter polyacrylamide gel cylinders (2) or slabs (3) followed by diffusion of the protein from the gel slice; (II) slicing and extraction of flat Sephadex beds (4); and (III) selective electrophoretic zone elution brought about by choice of suitable anolytes (1). Method I has the defect of being excessively laborious and ill suited to ’ Guest worker. * To whom requests for reprints should be addressed. 209
0003-2697/78/0851-0209$02.00/O Copyright All rights
0 1978 by Academic F’ress. Inc. of reproduction in any form reserved.
210
MCCORMICK,
WACHSLICHT,
AND CHRAMBACH
scaling upward. Method II is difficult to apply reproducibly, since it depends on a constant moisture content of the Sephadex bed; it also gives rise easily to nonlinear pH gradients across the gel bed. Like the first method, it produces numerous fractions which must be analyzed. Method III is conceptually attractive, but had not previously been applied to any serious separation problem. Suitable procedures and apparatus remain rudimentary and in need of further development. The present study was designed to test and develop Method III by application to a representative demanding fractionation problem: the separation of hemoglobins A and S, differing by two net charges per molecule and by approximately 0.2 pH units in pZ. MATERIALS
AND METHODS
(i) Hemoglobin. Human hemolysates from patients with sickle-cell anemia were obtained from Ms. Minna Feld (Clinical Pathology Department, Clinical Center, NIH) at concentrations of 64 and 36 mg/ml. The hemoglobin content of the hemolysate saturated with CO gas was determined spectrophotometrically, using a 1 mM extinction coefficient at 3.5- 10 GABA SEPARATOR KOH
6-8 LYS
6-8 HIS
pH 6.2
FIG. 1. Comparison between the resolution of hemoglobins A and S in three electrofocusing systems. Gel concentration: 5% T, 15% C&n. Numbers to the right of the gels designate protein zone positions relative to gel length. Numbers to the left designate pH values at which the protein zones are found. Center: pZ-range 6-8 Ampholine; catholyte, 0.01 M lysine (pH 9.5); anolyte, 0.01 M aspartic acid (pH 3.0); 200 V; 8 hr. Left: pZ-range 3.5- 10 Ampholine; catholyte, 0.2 N KOH; anolyte, 0.2 N H,SO,; 40 mg of y-aminobutyric acid (GABA) per 6-mm-diameter gel; 200 v; 14 hr. Right: pZ-range 6-8 Ampholine; catholyte, 0.01 M histidine (pH 7.6); anolyte, 0.01 M glycine (pH 6.2); 1000 v; 6 hr.
PREPARATIVE
ELECTROFOCUSING
OF HEMOGLOBIN
S
211
538 nm of 15.0 (5a). In the experiment described in section 5 of Results, hemoglobin was determined by the method of Beau (5b). Relative amounts of hemoglobin A in the hemolysate were estimated by gel densitometry at 540 nm, using a Gilford gel densitometer, assuming equal extinction coefficients for all hemoglobin components. (ii) Gel elecrrojiicusing. Electrofocusing on polyacrylamide gel was carried out as described previously (6) except that (a) gel concentration was 5% T, 15% C,,,, (7); (b) pZ-range 6-8 Ampholine (LKB) was used; (c) buffer anolytes and catholytes were used (8) as indicated under Results; (d) the gels were mechanically supported by dialysis membrane held at the anodic gel end by a Tygon sleeve. The analytical apparatus was the all-Pyrex construction recently described (9). The preparative devices were a bag of dialysis membrane fastened to the end of a 6-mm gel tube (10) by a Tygon sleeve; a gel tube 18 mm in diameter fitted in the same way with a dialysis bag and held in an upper electrolyte reservoir of suitable grommet dimensions (9); a funnel apparatus of the type previously described (1,ll) except that the porous glass membrane of the elution cup was replaced by a
RELATIVE GEL LENGTH
PH
7
TIME (HOURS1
FIG. 2. Time course of pH gradients, pH values at zones of hemoglobins A and S, relative positions of the hemoglobins, and pHs of anoIyte and catholyte in the optimized electrofocusing system. (A) pH gradients as a function of time of electrofocusing. (B) pH values at which hemoglobin A and S are found as a function of time of ekctrofocusing. (C) Positions of hemoglobins A and S relative to gel length. (D) pHs of catholyte and anolyte. The arrows designate changes of electrolyte.
212
MCCORMICK,
WACHSLICHT,
AND CHRAMBACH
stretched dialysis membrane held in place at the bottom of the cup by a Teflon ring seated in a groove and exerting a press fit against the perimeter of the stretched membrane (Fig. 7). The funnel apparatus was cleaned with methanolic KOH and coated with 1% Gelamide 250 (12). Determination of pH gradients was carried out on identical gels for which relative protein positions were measured, using the automated pH gradient measuring device recently described (13). Hemoglobin fractions isolated either in the dialysis bags attached to the bottom of preparative elution focusing gels or in the elution cup of such gels were subjected to reanalysis on electrofocusing gels at the analytical scale, using an electrolyte system identical to that previously used at the preparative scale (prior to the change to “elution anolyte and catholyte”). RESULTS (i) Optimization of pH gradient for the resolution of hemoglobins A and
S. A representative separation of hemoglobins in the pZ range 6-8 in conjunction with a lysine catholyte and aspartic acid anolyte (8) is shown in Fig. 1 (center). To improve resolution by pH gradient flattening, a
/ PY;i\ FIG. 3. Comparison between two electrolyte systems designed to elute hemoglobin A selectively from the optimized electrofocusing system. The gel on the left depicts a representative fractionation of the hemolysate by the method described in the legend to Fig. 1 (right). On the upper right, the gradual elution of hemoglobin A into the anolyte reservoir, effected by an anolyte change to 0.01 M histidine and a catholyte change to 0.01 M lysine, is depicted. Numbers to the right of the gels designate relative zone positions. To the lower right the analogous elution effected by a change of anolyte to 0.01 M pyridine is shown.
PREPARATIVE
ELECTROFOCUSING
OF HEMOGLOBIN
S
213
y-aminobutyric acid “separator” (40 mg per gel of 6-mmdiameter) was applied as described by Caspers et al. using 0.2 N H2S04 and KOH as anolyte and catholyte (14) (Fig. 1, left). A different pH gradient flattening device used consisted of limiting the pH gradient termini to 6.2 and 7.8, respectively, by selecting an anolyte and a catholyte at these pH values, viz., 0.01 M glycine and histidine (Fig. 1, right). Figure 1 shows, that by comparison with the two other types of electrofocusing tested, the pZ range 6-8 in conjunction with glycine anolyte and histidine catholyte provided optimal resolution of hemoglobins A and S. (ii) Resolution of hemoglobins A and S in the optimized electrofocusing system as a function ofpHgradient changes, and of anolyte -catholyte pH changes, with’time. Figure 2A shows the pH gradients in the pZ 6-8
Ampholine system with glycine and histidine as anolyte and catholyte, function of the duration of electrofocusing. The pH gradients
RELATlVE 7.5
as a are
GEL LENGTH B
RELATIVE Mb POSITION
FIG. 4. Time course of the selective electrophoretic elution of hemoglobin A from electrofocusing gels. (A) pH gradients after the change of electrolytes from 0.01 M histidine catholyte) and 0.01 M glycine (anolyte) to 0.01 M lysine (catholyte) and 0.01 M histidine (anolyte). (B) pH values at which zones of hemoglobins A and S are found. (C) Positions of hemoglobins relative to each other (expressed in terms of positions relative to gel length). (D) pH changes in catholyte and anolyte prior to (left of the dotted line) and after (right of the dotted line) transposition of the gels between lysine (catholyte) and histidine (anolyte).
214
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WACHSLICHT,
AND
CHRAMBACH
reasonably stable in the pH range 6.5-7.5. This shallow pH gradient extends over the entire gel. Hemoglobins A and S appear to approach their isolectric positions gradually up to 6 hr of electrofocusing, but are displaced to slightly more alkaline positions in the further course of electrofocusing, possibly in losing competition with the intermittent anodic pH gradient drift (15) observed for this system (Fig. 2B). Figure 2C shows that the positions of hemoglobins A and S are maximally separated at 6 hr of electrofocusing. This figure also illustrates the relative inertia with which the proteins follow the changing pH gradient, due to their limited net charge close to the ~1. Figure 2D illustrates the relative constancy of pH of the catholyte, in contrast to the progressive acidification of the anolyte, presumably due to Ampholine migration into the electrolyte chamber (6). The figure illustrates the need for several changes of electrolyte buffers in this system. (iii) Recovery of hemoglobin A separated from hemoglobin S. Using the anodic drift (15) and the principle of electrophoretic elution of proteins in gel electrofocusing previously described (l), hemoglobin A was allowed to migrate into a dialysis bag attached to the anodic end of the gel tube (10). Two combinations of catholyte and anolyte capable of producing this migration were used: a lysine (pH 9.45) catholyte in conjunction with a histidine (pH 7.6) anolyte (Fig. 3, top) and a histidine catholyte (pH 7.6) in conjunction with a pyridine anolyte (pH 7.2) (Fig. 3, bottom). Figure 3 shows the hemoglobin electrofocusing patterns before (left) and after 7.50
7.30
HbX - --
rn.. .
PH 7.10
6.90
FIG. 5. pH values ofthe hemoglobin components ofa human hemolysate froma patient with sickle-cell anemia before (left) and after (right) separation and recovery of hemoglobins A and X from a mixture of hemoglobins A, X, and S. The hemolysate and conditions of electrofocusing were those described in the legend to Fig. 4. Zone positions marked by an event marker on the automated pH gradient scans are displaced due to gel stretching in the groove of the scanning device. The hemoglobin fraction analyzed as shown on the right, containing hemoglobins A and X, was recovered in a dialysis bag attached to the anodic end of the gel. It appears free of hemoglobin S.
PREPARATIVE
ELECTROFOCUSING
18
OF HEMOGLOBIN
S
215
27
FIG. 6. Load capacity of preparative electrofocusing: 9, 18, and 27 mg of hemolysate were fractionated by electrofocusing as described in the legend to Fig. 4, except that 18mm-diameter gels were used. The gel patterns show that resolution is decreased as loads exceed 4 mgkm2 of gel surface area.
(right) the change from the original electrolyte system (histidine catholyte, glycine anolyte) to each of the two systems designed to give rise to hemoglobin A elution. Relative protein positions along the gel are indicated on this figure. It is seen that both systemsare capable of giving rise to the selective electrophoretic recovery of hemoglobin A, but that band dispersion is less in the case of the lysine-histidine electrolyte system. (iv) Time course of the electrophoretic recovery of hemoglobin A. Figure 4 shows the pH gradients formed after transposition of the electrofocusing gel (after 6 hr between histidine catholyte and glycine anolyte) to the elution system of electrolytes (lysine catholyte, histidine anolyte). While the pH gradient becomes progressively more alkaline, the pHs of the zones of hemoglobins A and S, as well as the pH at the minor hemoglobin band designated as x3 between the two, remain constant (Fig. 4B). .Four hours after transposition of the gels into the “elution system,” hemoglobin A has reached the anodic end of the gel (Fig. 4C); after 5 hr it has eluted, together with minor componentx. Catholyte and anolyte solutions were changed as shown by the arrows in Fig. 4D, but remained reasonably constant with time. Figure 5 shows the pH values at which the hemoglobin components of a human hemolysate from a patient with sickle-cell anemia are found before and after separation by electrofocusing and elution under the conditions of the experiment described by Fig. 4. (v) Resolution as a function of protein loads. A comparison was made between protein loads of 9,18, and 27mg/gel of 2.54cmz surface area (18 mm in diameter), using a hemolysate approximately equally divided into 3 Presumably “hemoglobin
x”
is fetal hemoglobin (hemoglobin
F).
216
MCCORMICK,
WACHSLICHT,
AND CHRAMBACH
FIG. 7. Elution cup for protein recovery. The elution cup was attached to a funnel-shaped gel tube as previously described (1,ll). The lower surface of the cup consists of stretched dialysis membrane. A: Funnel containing electrofocusing gel. B: Plexiglas flange. C: Plexiglas elution cup. D: Silicone rubber gasket. E: Dialysis membrane. F: Pressure release tube. G: Groove and fitting Kel-F ring for stretching of the membrane. Dimensions are in millimeter.
hemoglobins A and S, with a minor third hemoglobin component (X). Figure 6 shows that resolution between hemoglobins A and S was maintained even with the highest load of hemolysate used, but that it was optimal at the lowest load. (vi) Collection system and yield. Figure 7 shows the construction of the elution cup. Dialysis membrane forms the bottom of the cup. The cup is attached to the stem of the funnel-shaped gel tube apparatus described previously (1,ll). With a load of 32 mg of hemolysate and using this cup, hemoglobin A separated from hemoglobin S was obtained in 70% yield based on spectrophotometric analysis. Figure 8 shows the gel patterns obtained in electrofocusing on the hemolysate and the separated hemoglobin A. A higher yield than 70%, viz., 85%, was obtained upon prolonged electrophoresis into the cup so that not only hemoglobin A but also its nearest protein zone, designated as hemoglobin X,3 migrated into the chamber. Upon rerun at the analytical scale, both A and X (identified by pH 7.1 and 7.3, respectively, as compared to pH 7.5 for hemoglobin S which remained on the preparative gel and was discarded), were found in the concentrate. DISCUSSION
The present study demonstrates that preparative gel electrofocusing with selective zone recovery by electrophoretic migration into a small-volume elution cup is feasible in the application to a relatively
PREPARATIVE
ELECTROFOCUSING
ORIGINAL MIXTURE
OF HEMOGLOBIN
S
217
ISOLATED HEMOGLOBIN
’ A
FIG. 8. Electrofocusing analysis of 32 mg of hemolysate prior (left) and subsequent to (right) electrofocusing fractionation using the apparatus described in the legend to Fig. 7. Conditions of reanalysis were the same as those of the preparative fractionation described in the legend to Fig. 4.
demanding separation problem involving the separation of hemoglobins A and S differing by approximately 0.2 pH units in isoelectric point. Successful application of the method depends on previous optimization of separation in electrofocusing by flattening of the pH gradient. Such flattening can be achieved either according to Caspers et al. (14), by introduction of a “separator” into the electrofocusing gel, or by selection of anolyte and catholyte with pH values as closely adjacent as required by the pls of the proteins to be separated. The latter approach was followed in this study, since it appears more amenable to rational manipulation than the first. Collection of the protein in an improvised apparatus consisting of a dialysis bag fastened to the end of the gel tube (10) is possible, but collection into a cup of constant dimensions (Fig. 7) attached to a large capacity gel
218
MCCORMICK,
WACHSLICHT,
AND CHRAMBACH
tube (funnel) (1 ,l 1) seems preferable. The method lends itself to the recovery of hemoglobin A from 32-mg loads of hemolysate, estimated by gel densitometry to contain 16 mg of hemoglobin A per gel tube. Considering that six 18-mm gel tubes can be run simultaneously in the apparatus recently described [Fig. 13 of Ref. (9)], the method is capable of handling several hundred milligram loads of protein. Recovery of hemoglobin A, calculated by spectrophotometry and densitometric estimation of the original content in hemoglobin A of the hemolysate, is 70%, which appears satisfactory. A remaining limitation of the method in application to noncolored proteins is the need for frequent changes of the contents of the elution cup for assay which is laborious and may lead to eluate dilution. The solution to this problem is the introduction of a protein detection device (e.g., uv detector) into the elution cup. Development of such a device is presently under investigation. ACKNOWLEDGEMENT We thank Ms. Helen Orem (Graphics Section, MAPB, DRS, NIH) for expert preparation of the figures.
REFERENCES 1. 2. 3. 4.
McCormick, A., Miles, L. E. M., and Chrambach, A. (1976)Anal. Biochem. 75,314-324. Finlayson, R., and Chrambach, A. (1971)Anal. Biochem. 40, 292-311. Graesslin, D., Weise, H. C., and Rick, M. (1976) Anal. Biochem. 71, 492-499. Radola, B. J. (1976) in Isoelectric Focusing (Catsimpoolas, N., ed.), pp. 119-167, Academic Press, New York. 5a. Lemberg, R., Legge, J. W. (1949) in Hematin Compounds and Bile Pigments: Their Constitution, Metabolism and Function, pp. 229-230, Interscience, New York. Sb. Beau, A. F. (1%2) Tech. Bull. Regis. Med. Technol. 32, 11l- 112. 6. Baumann, G., and Chrambach, A. (1975) in Progress in Isoelectric Focusing and Isotachophoresis (Righetti, P. G., ed.), pp. 13-23, Elsevier, Amsterdam. 7. Baumann, G., and Chrambach, A. (1976) Anal. Biochem. 70, 32-38. 8. Nguyen, N. Y., and Chrambach, A. (1977) Anal. Biochem. 79, 462-469. 9. Chrambach, A., Jovin, T. M., Svendsen, P. J., and Rodbard, D. (1976) in Methods of Protein Separation (Catsimpoolas, N., ed.), pp. 27-144, Plenum Press, New York. 10. Suzuki, T., Benesch, R. E., Yung, S., and Benesch, R. (1973) Anal. Biochem. 55, 249-254. 11. Wachslicht, H., and Chrambach, A. (1977) Anal. Biochem. 84, 533-538. 12. Nguyen, N. Y., and Chrambach, A. (1977) Anal. Biochem. 82, 54-62. 13. Chidakel, B. E., Nguyen, N. Y., and Chrambach, A. (1976)Anal. Biochem. 77,216-225. 14. Caspers, M. L., Posey, Y., and Brown, R. K. (1977) Anal. Biochem. 79, 166-180. 15. Nguyen, N. Y., McCormick, A. G., and Chrambach, A. (1978)Anal. Biochem., submitted.
BIOCHEMISTRY
85,
209-218 (1978)
Preparative Separation of Hemoglobins A and S by Gel Electrofocusing, Using Selective Zone Elution by Gel Transposition between Suitable Anolytes and Cat holytes ALICE
G. MCCORMICK,~
HELENA ANDANDREASCHRAMBACH~
WACHSLICHT,
Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20014 Received April 6, 1977; accepted October 6, 1977 Preparative electrofocusing on polyacrylamide gels has been limited, until recently, to excision of gel slices, diffusion, and collection ofthe slice diffisates. An advance was made by the introduction of a method of selective electrophoretic zone recovery by specific changes of anolyte (A. McCormick, L. E. M. Miles, and A. Chrambach, 1976, Anal. Biochem. 75, 314-324). It was shown (a) that selective zone recovery could be achieved by transposition of the gels into either isoelectric ampholytes or charged buffers, (b) that it could be applied to the gram scale, and (c) that zone elution could proceed either continuously or discontinuously. The early study was, however, limited to a trivial model problem, the separation of hemoglobin from bovine serum albumin (BSA). The present study was an attempt to apply a similar selective zone recovery method to a more demanding separation problem, the separation of hemoglobin A from hemoglobin S as well as from other minor components contained in a sickle-trait human hemolysate. The study shows that selective electrophoretic zone elution from an electrofocusing gel 18 mm in diameter is capable of yielding hemoglobin A, separated from hemoglobin S, differing by only 0.2 pH units in isoelectric point. The recovery of hemoglobin A was 70%. with a load of 32 mg of hemoglobin mixture per gel, using discontinuous zone elution into a collection cup.
Preparative electrofocusing on gels is important since its performance tolerates, and may be improved by, protein precipitation at the isoelectric point (pZ) which occurs routinely at elevated milligramor grampreparative loads. Three techniques of preparative gel electrofocusing have been described: (I) slicing of wide diameter polyacrylamide gel cylinders (2) or slabs (3) followed by diffusion of the protein from the gel slice; (II) slicing and extraction of flat Sephadex beds (4); and (III) selective electrophoretic zone elution brought about by choice of suitable anolytes (1). Method I has the defect of being excessively laborious and ill suited to ’ Guest worker. * To whom requests for reprints should be addressed. 209
0003-2697/78/0851-0209$02.00/O Copyright All rights
0 1978 by Academic F’ress. Inc. of reproduction in any form reserved.
210
MCCORMICK,
WACHSLICHT,
AND CHRAMBACH
scaling upward. Method II is difficult to apply reproducibly, since it depends on a constant moisture content of the Sephadex bed; it also gives rise easily to nonlinear pH gradients across the gel bed. Like the first method, it produces numerous fractions which must be analyzed. Method III is conceptually attractive, but had not previously been applied to any serious separation problem. Suitable procedures and apparatus remain rudimentary and in need of further development. The present study was designed to test and develop Method III by application to a representative demanding fractionation problem: the separation of hemoglobins A and S, differing by two net charges per molecule and by approximately 0.2 pH units in pZ. MATERIALS
AND METHODS
(i) Hemoglobin. Human hemolysates from patients with sickle-cell anemia were obtained from Ms. Minna Feld (Clinical Pathology Department, Clinical Center, NIH) at concentrations of 64 and 36 mg/ml. The hemoglobin content of the hemolysate saturated with CO gas was determined spectrophotometrically, using a 1 mM extinction coefficient at 3.5- 10 GABA SEPARATOR KOH
6-8 LYS
6-8 HIS
pH 6.2
FIG. 1. Comparison between the resolution of hemoglobins A and S in three electrofocusing systems. Gel concentration: 5% T, 15% C&n. Numbers to the right of the gels designate protein zone positions relative to gel length. Numbers to the left designate pH values at which the protein zones are found. Center: pZ-range 6-8 Ampholine; catholyte, 0.01 M lysine (pH 9.5); anolyte, 0.01 M aspartic acid (pH 3.0); 200 V; 8 hr. Left: pZ-range 3.5- 10 Ampholine; catholyte, 0.2 N KOH; anolyte, 0.2 N H,SO,; 40 mg of y-aminobutyric acid (GABA) per 6-mm-diameter gel; 200 v; 14 hr. Right: pZ-range 6-8 Ampholine; catholyte, 0.01 M histidine (pH 7.6); anolyte, 0.01 M glycine (pH 6.2); 1000 v; 6 hr.
PREPARATIVE
ELECTROFOCUSING
OF HEMOGLOBIN
S
211
538 nm of 15.0 (5a). In the experiment described in section 5 of Results, hemoglobin was determined by the method of Beau (5b). Relative amounts of hemoglobin A in the hemolysate were estimated by gel densitometry at 540 nm, using a Gilford gel densitometer, assuming equal extinction coefficients for all hemoglobin components. (ii) Gel elecrrojiicusing. Electrofocusing on polyacrylamide gel was carried out as described previously (6) except that (a) gel concentration was 5% T, 15% C,,,, (7); (b) pZ-range 6-8 Ampholine (LKB) was used; (c) buffer anolytes and catholytes were used (8) as indicated under Results; (d) the gels were mechanically supported by dialysis membrane held at the anodic gel end by a Tygon sleeve. The analytical apparatus was the all-Pyrex construction recently described (9). The preparative devices were a bag of dialysis membrane fastened to the end of a 6-mm gel tube (10) by a Tygon sleeve; a gel tube 18 mm in diameter fitted in the same way with a dialysis bag and held in an upper electrolyte reservoir of suitable grommet dimensions (9); a funnel apparatus of the type previously described (1,ll) except that the porous glass membrane of the elution cup was replaced by a
RELATIVE GEL LENGTH
PH
7
TIME (HOURS1
FIG. 2. Time course of pH gradients, pH values at zones of hemoglobins A and S, relative positions of the hemoglobins, and pHs of anoIyte and catholyte in the optimized electrofocusing system. (A) pH gradients as a function of time of electrofocusing. (B) pH values at which hemoglobin A and S are found as a function of time of ekctrofocusing. (C) Positions of hemoglobins A and S relative to gel length. (D) pHs of catholyte and anolyte. The arrows designate changes of electrolyte.
212
MCCORMICK,
WACHSLICHT,
AND CHRAMBACH
stretched dialysis membrane held in place at the bottom of the cup by a Teflon ring seated in a groove and exerting a press fit against the perimeter of the stretched membrane (Fig. 7). The funnel apparatus was cleaned with methanolic KOH and coated with 1% Gelamide 250 (12). Determination of pH gradients was carried out on identical gels for which relative protein positions were measured, using the automated pH gradient measuring device recently described (13). Hemoglobin fractions isolated either in the dialysis bags attached to the bottom of preparative elution focusing gels or in the elution cup of such gels were subjected to reanalysis on electrofocusing gels at the analytical scale, using an electrolyte system identical to that previously used at the preparative scale (prior to the change to “elution anolyte and catholyte”). RESULTS (i) Optimization of pH gradient for the resolution of hemoglobins A and
S. A representative separation of hemoglobins in the pZ range 6-8 in conjunction with a lysine catholyte and aspartic acid anolyte (8) is shown in Fig. 1 (center). To improve resolution by pH gradient flattening, a
/ PY;i\ FIG. 3. Comparison between two electrolyte systems designed to elute hemoglobin A selectively from the optimized electrofocusing system. The gel on the left depicts a representative fractionation of the hemolysate by the method described in the legend to Fig. 1 (right). On the upper right, the gradual elution of hemoglobin A into the anolyte reservoir, effected by an anolyte change to 0.01 M histidine and a catholyte change to 0.01 M lysine, is depicted. Numbers to the right of the gels designate relative zone positions. To the lower right the analogous elution effected by a change of anolyte to 0.01 M pyridine is shown.
PREPARATIVE
ELECTROFOCUSING
OF HEMOGLOBIN
S
213
y-aminobutyric acid “separator” (40 mg per gel of 6-mmdiameter) was applied as described by Caspers et al. using 0.2 N H2S04 and KOH as anolyte and catholyte (14) (Fig. 1, left). A different pH gradient flattening device used consisted of limiting the pH gradient termini to 6.2 and 7.8, respectively, by selecting an anolyte and a catholyte at these pH values, viz., 0.01 M glycine and histidine (Fig. 1, right). Figure 1 shows, that by comparison with the two other types of electrofocusing tested, the pZ range 6-8 in conjunction with glycine anolyte and histidine catholyte provided optimal resolution of hemoglobins A and S. (ii) Resolution of hemoglobins A and S in the optimized electrofocusing system as a function ofpHgradient changes, and of anolyte -catholyte pH changes, with’time. Figure 2A shows the pH gradients in the pZ 6-8
Ampholine system with glycine and histidine as anolyte and catholyte, function of the duration of electrofocusing. The pH gradients
RELATlVE 7.5
as a are
GEL LENGTH B
RELATIVE Mb POSITION
FIG. 4. Time course of the selective electrophoretic elution of hemoglobin A from electrofocusing gels. (A) pH gradients after the change of electrolytes from 0.01 M histidine catholyte) and 0.01 M glycine (anolyte) to 0.01 M lysine (catholyte) and 0.01 M histidine (anolyte). (B) pH values at which zones of hemoglobins A and S are found. (C) Positions of hemoglobins relative to each other (expressed in terms of positions relative to gel length). (D) pH changes in catholyte and anolyte prior to (left of the dotted line) and after (right of the dotted line) transposition of the gels between lysine (catholyte) and histidine (anolyte).
214
MCCORMICK,
WACHSLICHT,
AND
CHRAMBACH
reasonably stable in the pH range 6.5-7.5. This shallow pH gradient extends over the entire gel. Hemoglobins A and S appear to approach their isolectric positions gradually up to 6 hr of electrofocusing, but are displaced to slightly more alkaline positions in the further course of electrofocusing, possibly in losing competition with the intermittent anodic pH gradient drift (15) observed for this system (Fig. 2B). Figure 2C shows that the positions of hemoglobins A and S are maximally separated at 6 hr of electrofocusing. This figure also illustrates the relative inertia with which the proteins follow the changing pH gradient, due to their limited net charge close to the ~1. Figure 2D illustrates the relative constancy of pH of the catholyte, in contrast to the progressive acidification of the anolyte, presumably due to Ampholine migration into the electrolyte chamber (6). The figure illustrates the need for several changes of electrolyte buffers in this system. (iii) Recovery of hemoglobin A separated from hemoglobin S. Using the anodic drift (15) and the principle of electrophoretic elution of proteins in gel electrofocusing previously described (l), hemoglobin A was allowed to migrate into a dialysis bag attached to the anodic end of the gel tube (10). Two combinations of catholyte and anolyte capable of producing this migration were used: a lysine (pH 9.45) catholyte in conjunction with a histidine (pH 7.6) anolyte (Fig. 3, top) and a histidine catholyte (pH 7.6) in conjunction with a pyridine anolyte (pH 7.2) (Fig. 3, bottom). Figure 3 shows the hemoglobin electrofocusing patterns before (left) and after 7.50
7.30
HbX - --
rn.. .
PH 7.10
6.90
FIG. 5. pH values ofthe hemoglobin components ofa human hemolysate froma patient with sickle-cell anemia before (left) and after (right) separation and recovery of hemoglobins A and X from a mixture of hemoglobins A, X, and S. The hemolysate and conditions of electrofocusing were those described in the legend to Fig. 4. Zone positions marked by an event marker on the automated pH gradient scans are displaced due to gel stretching in the groove of the scanning device. The hemoglobin fraction analyzed as shown on the right, containing hemoglobins A and X, was recovered in a dialysis bag attached to the anodic end of the gel. It appears free of hemoglobin S.
PREPARATIVE
ELECTROFOCUSING
18
OF HEMOGLOBIN
S
215
27
FIG. 6. Load capacity of preparative electrofocusing: 9, 18, and 27 mg of hemolysate were fractionated by electrofocusing as described in the legend to Fig. 4, except that 18mm-diameter gels were used. The gel patterns show that resolution is decreased as loads exceed 4 mgkm2 of gel surface area.
(right) the change from the original electrolyte system (histidine catholyte, glycine anolyte) to each of the two systems designed to give rise to hemoglobin A elution. Relative protein positions along the gel are indicated on this figure. It is seen that both systemsare capable of giving rise to the selective electrophoretic recovery of hemoglobin A, but that band dispersion is less in the case of the lysine-histidine electrolyte system. (iv) Time course of the electrophoretic recovery of hemoglobin A. Figure 4 shows the pH gradients formed after transposition of the electrofocusing gel (after 6 hr between histidine catholyte and glycine anolyte) to the elution system of electrolytes (lysine catholyte, histidine anolyte). While the pH gradient becomes progressively more alkaline, the pHs of the zones of hemoglobins A and S, as well as the pH at the minor hemoglobin band designated as x3 between the two, remain constant (Fig. 4B). .Four hours after transposition of the gels into the “elution system,” hemoglobin A has reached the anodic end of the gel (Fig. 4C); after 5 hr it has eluted, together with minor componentx. Catholyte and anolyte solutions were changed as shown by the arrows in Fig. 4D, but remained reasonably constant with time. Figure 5 shows the pH values at which the hemoglobin components of a human hemolysate from a patient with sickle-cell anemia are found before and after separation by electrofocusing and elution under the conditions of the experiment described by Fig. 4. (v) Resolution as a function of protein loads. A comparison was made between protein loads of 9,18, and 27mg/gel of 2.54cmz surface area (18 mm in diameter), using a hemolysate approximately equally divided into 3 Presumably “hemoglobin
x”
is fetal hemoglobin (hemoglobin
F).
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AND CHRAMBACH
FIG. 7. Elution cup for protein recovery. The elution cup was attached to a funnel-shaped gel tube as previously described (1,ll). The lower surface of the cup consists of stretched dialysis membrane. A: Funnel containing electrofocusing gel. B: Plexiglas flange. C: Plexiglas elution cup. D: Silicone rubber gasket. E: Dialysis membrane. F: Pressure release tube. G: Groove and fitting Kel-F ring for stretching of the membrane. Dimensions are in millimeter.
hemoglobins A and S, with a minor third hemoglobin component (X). Figure 6 shows that resolution between hemoglobins A and S was maintained even with the highest load of hemolysate used, but that it was optimal at the lowest load. (vi) Collection system and yield. Figure 7 shows the construction of the elution cup. Dialysis membrane forms the bottom of the cup. The cup is attached to the stem of the funnel-shaped gel tube apparatus described previously (1,ll). With a load of 32 mg of hemolysate and using this cup, hemoglobin A separated from hemoglobin S was obtained in 70% yield based on spectrophotometric analysis. Figure 8 shows the gel patterns obtained in electrofocusing on the hemolysate and the separated hemoglobin A. A higher yield than 70%, viz., 85%, was obtained upon prolonged electrophoresis into the cup so that not only hemoglobin A but also its nearest protein zone, designated as hemoglobin X,3 migrated into the chamber. Upon rerun at the analytical scale, both A and X (identified by pH 7.1 and 7.3, respectively, as compared to pH 7.5 for hemoglobin S which remained on the preparative gel and was discarded), were found in the concentrate. DISCUSSION
The present study demonstrates that preparative gel electrofocusing with selective zone recovery by electrophoretic migration into a small-volume elution cup is feasible in the application to a relatively
PREPARATIVE
ELECTROFOCUSING
ORIGINAL MIXTURE
OF HEMOGLOBIN
S
217
ISOLATED HEMOGLOBIN
’ A
FIG. 8. Electrofocusing analysis of 32 mg of hemolysate prior (left) and subsequent to (right) electrofocusing fractionation using the apparatus described in the legend to Fig. 7. Conditions of reanalysis were the same as those of the preparative fractionation described in the legend to Fig. 4.
demanding separation problem involving the separation of hemoglobins A and S differing by approximately 0.2 pH units in isoelectric point. Successful application of the method depends on previous optimization of separation in electrofocusing by flattening of the pH gradient. Such flattening can be achieved either according to Caspers et al. (14), by introduction of a “separator” into the electrofocusing gel, or by selection of anolyte and catholyte with pH values as closely adjacent as required by the pls of the proteins to be separated. The latter approach was followed in this study, since it appears more amenable to rational manipulation than the first. Collection of the protein in an improvised apparatus consisting of a dialysis bag fastened to the end of the gel tube (10) is possible, but collection into a cup of constant dimensions (Fig. 7) attached to a large capacity gel
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AND CHRAMBACH
tube (funnel) (1 ,l 1) seems preferable. The method lends itself to the recovery of hemoglobin A from 32-mg loads of hemolysate, estimated by gel densitometry to contain 16 mg of hemoglobin A per gel tube. Considering that six 18-mm gel tubes can be run simultaneously in the apparatus recently described [Fig. 13 of Ref. (9)], the method is capable of handling several hundred milligram loads of protein. Recovery of hemoglobin A, calculated by spectrophotometry and densitometric estimation of the original content in hemoglobin A of the hemolysate, is 70%, which appears satisfactory. A remaining limitation of the method in application to noncolored proteins is the need for frequent changes of the contents of the elution cup for assay which is laborious and may lead to eluate dilution. The solution to this problem is the introduction of a protein detection device (e.g., uv detector) into the elution cup. Development of such a device is presently under investigation. ACKNOWLEDGEMENT We thank Ms. Helen Orem (Graphics Section, MAPB, DRS, NIH) for expert preparation of the figures.
REFERENCES 1. 2. 3. 4.
McCormick, A., Miles, L. E. M., and Chrambach, A. (1976)Anal. Biochem. 75,314-324. Finlayson, R., and Chrambach, A. (1971)Anal. Biochem. 40, 292-311. Graesslin, D., Weise, H. C., and Rick, M. (1976) Anal. Biochem. 71, 492-499. Radola, B. J. (1976) in Isoelectric Focusing (Catsimpoolas, N., ed.), pp. 119-167, Academic Press, New York. 5a. Lemberg, R., Legge, J. W. (1949) in Hematin Compounds and Bile Pigments: Their Constitution, Metabolism and Function, pp. 229-230, Interscience, New York. Sb. Beau, A. F. (1%2) Tech. Bull. Regis. Med. Technol. 32, 11l- 112. 6. Baumann, G., and Chrambach, A. (1975) in Progress in Isoelectric Focusing and Isotachophoresis (Righetti, P. G., ed.), pp. 13-23, Elsevier, Amsterdam. 7. Baumann, G., and Chrambach, A. (1976) Anal. Biochem. 70, 32-38. 8. Nguyen, N. Y., and Chrambach, A. (1977) Anal. Biochem. 79, 462-469. 9. Chrambach, A., Jovin, T. M., Svendsen, P. J., and Rodbard, D. (1976) in Methods of Protein Separation (Catsimpoolas, N., ed.), pp. 27-144, Plenum Press, New York. 10. Suzuki, T., Benesch, R. E., Yung, S., and Benesch, R. (1973) Anal. Biochem. 55, 249-254. 11. Wachslicht, H., and Chrambach, A. (1977) Anal. Biochem. 84, 533-538. 12. Nguyen, N. Y., and Chrambach, A. (1977) Anal. Biochem. 82, 54-62. 13. Chidakel, B. E., Nguyen, N. Y., and Chrambach, A. (1976)Anal. Biochem. 77,216-225. 14. Caspers, M. L., Posey, Y., and Brown, R. K. (1977) Anal. Biochem. 79, 166-180. 15. Nguyen, N. Y., McCormick, A. G., and Chrambach, A. (1978)Anal. Biochem., submitted.