Electrophoreris 1990, Ii, 947-952
Stability of borate-glycerol pH gradients in RIEF
troducing the sample into the separation chamber in several narrow zones, as illustrated in Fig. 1 I for the separation of ADH from cytochrome c. The injection of 4 sample streams resulted in a maximum throughput of about 20 mL sample solution per minute at a concentration factor of nearly 6, compared to the initial concentration. Purification was carried out in a prototype electrophoresis chamber after optimization with the Elphor VaP 22. All experimental parameters were the same as in the optimization experiments with the exception of voltage, which had to be multiplied by 1.5 to take into account the different chamber widths.
This work was supported by the Bundesminister f u r Forschung und TechnoIogie, FR G,Forderkennzeichen 03 18809 Al. Received May 23, 1990
4 References [ I ] Hoffstetter-Kuhn, S., Kuhn, R. and Wagner, H., Electrophoresis 1 9 9 0 , I I , 304-309. 121 Bier, M., in: Asenjo, J. A. and Hong, J. (Eds.), American Chemical Society Symposium Series 314, Washington, 1986, pp. 185-192.
Paul Todd William Elsasser Bioprocessing and Pharmaceutical Research Center, Philadelphia, PA
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131 Manzoni, R.,Ph. D. Thesis. Universitat des Saarlandes, Saarbrucken 1986. 141 Kuhn, R., Wagner, H., Mosher, R. A. and Thorrnann, W., Electrophoresis 1987,8,503-508. L5l Mosher, R. A., Thormann, W., Kuhn, R. and Wagner. H., J . Chromatogr. 1989,, 39--49. 161 Kessler, R., Manz, H. J. and Szkkely, G.,J. Chromatogr. 1989,469, 444-447. [71 Wagner, H. and Neupert, D.,J. Chromatogr. 1978,156, 219-224. [Sl Barth, F., Gruetter, M., Kessler, R. and Manz, H. J., Electrophoreyis 1986, 7,372-375. 191 Kessler, R., Manz, H. J. and Walliser, H. P.. in:3. EngineeringFoundation Conference on Recovery of Bioproducts, Uppsala 1986. l l O l Kuhn, R. and Wagner, H.,J. Chromatogr. 1989,481,343-351. I 1 11 Heydt, A., Wagner, H. and Miiller, P.,J. Virolog. Methods 1988,19, 13-22. 1121 Dickenscheid-Simon, R., Ph. D. Thesis, Universitat des Saarlandes Saarbrucken 1988. [ 131 Wagner, H., Kuhn, R. and Hoffstetter, S., in: Wagner, H. and Blasius, E. (Eds.), Praxis der elektrophoretischen Trennmethoden, Springer, Berlin, 1989, pp. 223-278. I141 Rick, W. and Stegbauer, H. P., in: Bergmeyer, H. U. (Ed.), Methoden der enzymatischen Analyse, Verlag Chemie, Weinheim, 1962, pp. 918-923. [I51 Bergmeyer, H. U., in: Bergmeyer, H. U. (Ed.), Methoden der enzymatischenAna[yse,VerlagChemie, Weinheim, 1962,pp. 392-393.
Nonamphoteric isoelectric focusing: 11. Stability of borate-glycerol pH gradients in recycling isoelectric focusing By complexing polyols with borate in recycling isoelectric focusing and by varying the ratio ofpolyol to borateovertheusefulpH range of4.0-6.0, it is possibleto control pH. Twelve solutions of 0.1 M boric acid and varying glycerol concentration were used to vary pH in a twelve-compartment commercial recycling isoelectric focusing (RIEF) system. Various concentrations of boric acid were tested as anolyte, and various Tris(hydroxymethy1amino)methane-borate buffer systems were tested as catholyte. Electroosmosis, hydrogen ion flow, and fluid balancing were characterized in two glycerol gradients; one was maintained at 0.06 pH/fraction and the other at 0.12 pH/fraction. In the latter case, ovalbumin (~14.70)migrated to the pH 4.6 1 and 4.72 compartments. It is concluded that the borate-glycerol system can be adequately stabilized in RIEF for isoelectric purification of certain proteins.
1 Introduction 1.1 Reyciing isoelectric focusing With the advent of pharmaceutical and diagnostics manufacture based on proteins from tissues, cells, or genetically engineered organisms, the need for large-scale, free-fluid purification methods has intensified. Recycling isoelectric focusing (RIEF) was introduced by Bier etal. 11-31 as a means of scaling up protein purification in free fluid. In this Correspondence: Dr. Paul Todd, National Institute of Standards and Technology, 325 Broadway 583.10, Boulder, CO 80303, USA Abbreviation: RIEF, recycling isoelectric focusing 0VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990
method isoelectric focusing takes place in a small multicompartment chamber in which each compartment corresponds to a single value of pH along the pH gradient and is (typically) separated from adjacent compartments by nylon screens. Synchronized pumps circulate the focused material from each compartment through a separate heat exchanger, and separands migrate from compartment to compartment until they reach the compartment that contains their isoelectric pH. Separands are then drained from their corresponding individual heat exchangers. Several grams of protein can be processed in a fraction of an hour by this method. Kyhse-Andersen [41 used a 32-compartment chamber (with mixing flow but not recycling) and claimed the fractionation of 10 g ofprotein in 660 ml of ampholyte solution [51. 0173-0835/90/11 11-0947 %3.50+.25/0
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1.2 Nonamphoteric buffers in isoelectric focusing Stable pH gradients can be formed without the use of amphoteric species [61. It has been suggested that the development of pH gradients in simpleionic buffers would simplify the interpretation of results and conserve biological functions in the separated material [7-9]. An artificial pH gradient was introduced experimentally by Kolin [ 101, who used nonamphoteric buffers, but since the ionic boundaries migrated in the electric field the gradients were unstable over time. Gel isoelectric focusing using organic buffers was introduced by Nguyen and Chrambach [ l 1,121, and the formation of a pH gradient on the basis of a dielectric gradient (which would not permit independent control of the dielectric constant) was introduced by Troitsky et al. [131. The use of nonamphoteric buffer pH gradients in the first step of two-dimensional electrophoresis was suggested by the work of Cuono et al. [141, but geneticists did not find this approach adequately reproducible [ 151. To prevent pH gradient migration in nonamphoteric buffered isoelectric focusing systems, including gels, the following methods have been introduced: steadystate rheoelectrophoresis IS]; buffer immobilization [ 161;and the protonating of acids and deprotonating of bases at equilibrium [17, 181, either by buffer renewal 1191 or, in the case of density gradient isoelectric focusing (described in the first paper of this series), by regenerating electrode buffers from large (150-liter) reservoirs [20]. Amphulyte materials usually used in isoelectric focusing are expensive, sometimes harmful to samples, and not suitable for very shallow pH gradients (< 0.1 pH/fraction). Isoelectric focusing in carrier ampholyte generated pH gradients results in low effective ionic strength, which can result in precipitation and/or denaturation of certain proteins. It is possible to control pH by complexing polyols with borate and by varying the ratio over the useful pH range of 4.0-6.0. Troitsky et aE. [ 2 1, 221 applied borate-polyol buffers to free “electrophoresis in a pH gradient” using simple columns and chambers with multiple outlets. In the work of Shukun 123, 241 a free-flow electrophoresis chamber with up to 42 inlets and outlets was
used; proteins electrophoresed in flowing borate-mannitol gradients in this system migrate rapidly in the electric field but may not reach their isoelectric pH.
1.3 Recycling isoelectric focusing with nonamphoteric buffers J onsson and Rilbe also introduced a multicompartment isoelectric focusing chamber [251, and Martin and Hampson [261proposed theuseofnonamphoteric buffersin amulticompartrnent system. Wenger and Javet [27],noting the desirability of replacing carrier ampholytes with nonamphoteric buffers 119, 26, 281, succeeded in stabilizing the acetic acidsodium acetate buffer system in a 5-compartment RIEF. A gradient from pH 4.05 to 4.95 was maintained for 10 h. Thus some limitations of RIEF using carrier ampholytes could be solved by using buffers. RIEF is capable of processing large volumes and sustaining stable flowing pH gradients over long periods but suffers from cost, toxicity and gradient constraints due only to the use of carrier ampholytes. Polyol-based buffer systems do not have these constraints, but they require a sustained free-fluid pH gradient; therefore, a series of investigations was designed to combine the strengths of these two methods in an attempt to eliminate some of their weaknesses. This article reports preliminary results that indicate that stable pH gradients can be produced and maintained in RIEF using an artificial borate-glycerol gradient and that this system is suitable for protein purification and could be especially useful in the purification of low-solubility proteins.
2 Materials and methods 2.1 RIEF
A commercial RIEF system, “Iso-Prep” (TM), was obtained from Ionics, Inc. (Watertown, MA) and operated with 12 buffer compartments between the anode and cathode compartments of the focusing chamber, which was 13.0 cm high, 5.60 FOCUSSING CHAMBER
U POWER SUPPLY
I HEAT EXCHANGER (14 CHANNELS)
Figure 1. Schematic diagram of RIEF as used in this study. Flow is upward in the focusing chamber and downward in the heat exchanger. Individual reservoirs and their corresponding focusing compartments are numbered. A, anode; C, cathode. Tension levers on peristaltic pump adjust individual flow rates.
Stability of borate-glycerol pH gradients in RIEF
Electrophoresis 1990, 11, 941-952
cm wide, and 0.36 cm/compartment. A schematic diagram of this RIEF is shown in Fig. 1. It differs moderately from the system described by Bier 1291. The compartments of the focusing chamber are formed by stacking, alternately, 0.325 cm polycarbonate spacers, silicone rubber gaskets, I Fm mesh nylon screen and silicone rubber gaskets so that 12 focusing chambers are formed, and the left-most chamber is the anode while the right-most chamber is the cathode - 14 chambers in all. The anode ion exchange membrane was an Tonics Inc. CR6 lCZK386 cation exchange membrane cast on a modacrylic fiber backing and using bound sulfonate groups to exchange cations. The cathode membrane was an AR anion exchange membrane cast similarly. This is one of two possible operating configurations, the other being to use the anion exchange membrane to separate the anode compartment from the focusing compartments and the cation exchange membrane to separate the cathode. This latter configuration is considered “normal”, but the former prevents separands from entering the anode and cathode compartments while permitting electrode ions to escape into adjacent focusing compartments, a situation found acceptablein buffer isoelectric focusing [20]. The peristaltic pump (Ismatec AG, FRG) has 16 individually controlled channels, two for each electrode compartment and one for each focusing compartment. These remove fluid from the top ofthe focusing chamber and deliver it to the top of the heat exchanger compartments, from which fluid is fed by gravity back into the bottom of the focusing chamber. Power was applied across the chamber using a 1000 V power supply set for constant voltage at 1000V. Interlock switches on a plexiglass door covering the focusing chamber in the cabinet prevent shock hazard. Heat exchanger cooling water was supplied at 10 OC k 1 “C from a40-liter circulating bath coupled with a cooling unit.
2.2 Solutions Anolyte solutions tested were 0.1-0.6 M boric acid, pH 4.2. The catholyte solution was Tris-borate buffer, 0.1 M in both ions, pH 8.0. A series of 12 borate-glycerol solutions was made by dissolving 0.01, 0.04, 0.09, 0.18, 0.27, 0.38, 0.46, 0.55,0.64,0.73,0.83, and 0.92 % w/v glycerol in 0.1 M boric acid for “high pH” experiments (Figs. 2 and 3) and 0.0, 0.0, 0.25,0.5,1.0,2.5,5.0,7.5,10,15,25,and30 %w/vglycerolin 0.05 M boric acid for “low pH” experiments (Figs. 4 and 5 ) . The low pH end of the RIEF system (reservoir #I) always contained the solution with the highest glycerol content. Ovalbumin was dissolved at 10 mg/mL in Tris-borate buffer as starting sample.
2.3 Procedure The RIEF system was operated without power for several hours before each experiment to balance the peristaltic pumps, using 10 % aqueous ethanol as test fluid. All 14 exchange reservoirs were drained, and the anode reservoir was filled with 100 mL boric acid, 0.1,0.3, or 0.6 M (depending on experiment); the cathode reservoir was filled with Tris-borate buffer, 0.1 M in both ions, pH 8.00, or modifications of this buffer as described below, and the individual reservoirs 1 through 12 were each filled with 50 mL of the different borateglycerol solutions specified above. The previously balanced peristaltic pumps were turned on at a setting of 60 (Ismatic Scale) and adjusted manually, individually, throughout the subsequent run time, depending upon particular experiment
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protocols: electroosmotic flow balancing, no balancing, or flow-through. Maximum field strength was maintained by operating the power supply at 1000 V constant voltage; current was recorded every 10 min. A 10 mL sample was collected for analysis from each reservoir at 0.5-1.0 h intervals. The pH was measured using acommercial pH meter with an Ag/AgCl glass pH electrode calibrated at pH 4.0 and 7.0, and conductivity was measured with a Yellow Springs (Yellow Springs, OH) calibrated conductivity cell or the conductometric system of a Pen Kem (Bedford Hills, NY) System 3000 Automated Electrokinetic Analyzer.
3 Results 3.1 Current profile and pH gradient stabilization The buffer gradient system depends on the formation of borated polyols utilizing the reaction H,B03 + HOCHZCHOHCHZOH P HOCH,CHOCH,OB(OH); + H,O + H’ to produce dissociable acids 1211. The pH depends upon the concentration of glycerol at the time of the reaction. Once the reagents are mixed, the pH is stable, as was indicated by a pair of pH readings performed on a series of solutions before and after three months of storage at 2 OC (datanot shown). The application of 1000 V across the 0.1 M borate-glycerol gradient resulted in the rapid movement of hydrogen ions out of the 0.3 M borate anolyte into the left (low pH) compartments of the chamber. The buffering capacity of the intermediate compartments prevented further pH drop, and the excess H+ions carried current across the chamber for a short period during the first hour of operation. Thus, the current, typically 15 mA, more than doubled during the first 10-30 min. After this period the expected drop in current followed the equilibration of H’ions with the glycerol borate gradient. This early rise in current does not occur in natural pH gradients formed with carrier ampholytes.
3.2 Measured pH gradients The immediate appearance of excess H’ ions in the low pH compartments is also seen in the pH profiles, as in Fig. 2, which shows the pH gradients in a “high pH” experiment as a function of time from 0 to 2.5 h of applied current. The pH in compartments 1 and 2 dropped from 4.85 k 0.5 and remained low throughout the run (Figs. 2 and 3A). This result suggests that, when using buffers to form artificial pH gradients in RIEF, the anolyte should match the pH at the low end of the gradient as closely as possible. A shallow pH gradient can be maintained over a limited number of compartments. Even during the period of early drastic current fluctuation the pH gradient over compartments 3-8 was stable and nearlylinear, as can be seen in Fig. 2.
3.3 Electroosmotic flow The contribution of electroosmotic flow to fluid motion in RIEF is difficult to assess quantitatively [31; however, because the pumping loop of the IsoPrep fills each reservoir from the top and because each reservoir has headspace, the volume of buffer in each reservoir is indicative of elec-
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Electrophoresis 1990.11, 941-952
PH
-I
O
L
COMPARTMENT NUMBER Figure 2. pH Gradient in the “high pH” borate-glycerol system as a func tion of time of current application at 1000 V. 0, pH readings on buffers before addition to the system (these are close to predicted values I2 1 I); 0 , 1 / 2 h ; A , 1 h;and 0 , 2 h. Thebargraphrepresentsthevolume(height)of fluid in each numbered reservoir in the heat exchanger.
troosmotic transport. An “acid notch” developed near the cathode in Fig. 2 and 3A, whichdescribe thesameexperiment. Figure 2 also shows the fluid height in each reservoir at the end of the 3 h period, and the profile indicates that more than 112 of the fluid in the low pH reservoirs was transported to the high pH reservoirs. It was therefore postulated that H ions were being transported cathodally across the focusing chamber and that measures which counteract electroosmotic flow and/ or reduce the source ofprotons would also suppress the formation of an “acid notch”.
In the experiment of Fig. 3A the tops of the reservoirs were left open, thereby permitting free flow of fluid from one focusing compartment to the other, opposed only by the hydrostatic head in each reservoir. When the tops of the reservoirs were sealed, pressure could develop within them and further oppose lateral fluid flow in the focusing chamber. In the experiment represented in Fig. 3B, this was done and the acid concentration in the anode was reduced; these measures eliminated the “acid notch”. Further reduction of the boric acid concentration in the anode without sealing the reservoirs had a similar effect (Fig. 3C) while at the same time preventing the early rise in pH in the middle compartments, as seen in Fig. 2. An alternative means of dealing with electroosmotic flow is to permit it to be unidirectional by providing an outlet; this has the undesirable effect of removing fluid from the system, so an inlet must also be provided. In the experiment described in Fig. 3D, reservoir #11 was allowed to drain while the lost fluid volume was continuously replaced in reservoir #l. This procedure eliminated the pH gradient distortion caused by electroosmotic flow, but it also gradually eliminated the glycerol gradient because only one concentration (0.92 %)of glycerol was used as replacement fluid. The overall higher pH (presumably due to the draining of H +suppliedby the anolyte) and flatter gradient are seen in Fig. 3D. It is concluded that electroosmotic flow distorts the “artificial” pH gradient formed by borate-glycerol in RIEF and that combinations of anolyte and flow adjustments can be found that counteract it. 3.4 Effect of glycerol concentration
+
The glycerol concentration range used in the experiments of Figs. 2 and 3 was 0.01-0.92 % w/v - the “high pH” range (from about 4.9 to 5.4). The resulting gradients were very flat, about 0.06 pH unitdfraction. A “low p H ’ borate-glycerol
C = TRIS/BORATE
2-
,
,
I-
0
8
2.0 HR 3.0 HR
,
9
EXP 1753 8 - A = O.IM H,BO, 7 - C = TRISIBORATE OPEN
:
0 Anode
2
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8
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C
65PH
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32I -
0
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1
’
1
1
1
1
’
1
1
1
1
1
I
I
I
I
I
I
I
I
I
Figure 3. pH Gradients in the“high pH” borate-glycerol system as a function of time of current application at 1000 V in experiments designed to characterize electroosmotic flow and to stabilize pH gradients. In (A) plotting symbols represent pH readings on buffers as for Fig. 2. In (B), (C), and (D) plotting characters correspond to those in panel (A). Triangles (A)correspond to 4 h.
Electrophoresis 1990. 11, 9477952
Stability of borate-glycerol pH gradients in RIEF
gradient was also studied (see Section 2.3), and this system stabilized in a pH range below that of the unelectrophoresed individual buffers (dots in Fig. 4). The pH droppedrapidly during the first 0.5 h, after which is dropped slowly, producing a pH gradient of 0.12-0.15 p H units/fraction over 8 fractions. While experiments were not performed that successfully eliminated the cathodal “acid notch” in this system a more usable p H range was produced by making the catholyte more basic by mixing 4 parts of the Tris-borate buffer with 1 part of 0.1 M NaOH. This measure resulted in a p H gradient stabilized over 8 fractions between p H 3.8 and 4.8 as shown in Fig. 5, thereby overcoming the early decrease in pH seen in Fig. 4. This measure also introduced OH- ions into the chamber resulting in a steady increase in current from 5-8 mA to 15-20 mA over a 2-h period, during which time the conductivity typically rose from 3 to 7 mS/cm.
95 1
p H gradient (dots, also nearly the same as predicted I 2 I ] ) except in the uppermost fractions. At the end of 1 h most of the protein, as determined by the method of Bradford I301 and shown as A (595 nm) in Fig. 5, was found in compartments 7 and 8, corresponding to pH 4.6 I and pH 4.72, respectively.
3.6 Hydraulic adjustments It was possible to independently adjust flow rates in individual channels using the multichannel peristaltic pump (Fig. 1). It was noted in the course of performing this research that adjustments in real time were often required to maintain reasonable fluid volume distributions among the reservoirs or to prevent low pH compartments of the chamber from draining completely. Such adjustments obviously draw fluid from one loop to an adjacent loop, thereby also locally modifying the pH gradient.
3.5 Protein isoelectric focusing Ovalbumin (PI 4.70) was introduced into reservoir #12 at time = 10 mi, at p H 5.2, at which pH the molecule is anionic. The“low pH” system just described was used. Figure 5 shows that the pH gradient (open circles) closely resembled the input 7
1
1
1
1
1
1
1
1
1
1
1
EXP 1761 HsB03-GLYCEROL
PH
1
2
4 6 8 COMPARTMENT NUMBER
12
10
Figure 4. p H Gradient in the “low pH” borate-glycerol system a s a function of time of current application. Dots represent pH readings on buffers before addition to the systems (these are close to predicted values (21I); plotting characters for each time are shown on the graph. Broad horizontal bars and vertical arrows indicate beginning and final pH in anode and cathode buffers. 0.3
0.2 A(595 nm)
3.1
31
I I r n I Ill I I IIIII I I 2 3 4 5 6 7 8 9 1 0 1 1 1 2 0.0
COMPARTMENT NUMBER
Figure 5. Isoelectric focusing of ovalbumin (pl 4.70) in the “low pH” borate-glycerol system for 1.0 h. Starting sample (100 mg) was added to reservoir #12. The bar graph represents protein concentration, in absorbance units, measured by the Bradford method [33 I.
4 Discussion The results of this investigation are preliminary in the sense that considerable further refinements are possible. For example, pH gradients can be further controlled by the regulation of pumpingrates in individual channels, and a wider variety of p H ranges can be achieved by choosing the nature and concentration of polyols [211. In discussing their study of RIEF of interferon, Nagabhushan and Trotta [ 3 11 alluded to several desired improvements of the method. Some of these desiderata should become reality as a result of using nontoxic, nonamphoteric buffers in RIEF. The usually low ionic strength (
Stability of borate-glycerol pH gradients in RIEF
troducing the sample into the separation chamber in several narrow zones, as illustrated in Fig. 1 I for the separation of ADH from cytochrome c. The injection of 4 sample streams resulted in a maximum throughput of about 20 mL sample solution per minute at a concentration factor of nearly 6, compared to the initial concentration. Purification was carried out in a prototype electrophoresis chamber after optimization with the Elphor VaP 22. All experimental parameters were the same as in the optimization experiments with the exception of voltage, which had to be multiplied by 1.5 to take into account the different chamber widths.
This work was supported by the Bundesminister f u r Forschung und TechnoIogie, FR G,Forderkennzeichen 03 18809 Al. Received May 23, 1990
4 References [ I ] Hoffstetter-Kuhn, S., Kuhn, R. and Wagner, H., Electrophoresis 1 9 9 0 , I I , 304-309. 121 Bier, M., in: Asenjo, J. A. and Hong, J. (Eds.), American Chemical Society Symposium Series 314, Washington, 1986, pp. 185-192.
Paul Todd William Elsasser Bioprocessing and Pharmaceutical Research Center, Philadelphia, PA
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131 Manzoni, R.,Ph. D. Thesis. Universitat des Saarlandes, Saarbrucken 1986. 141 Kuhn, R., Wagner, H., Mosher, R. A. and Thorrnann, W., Electrophoresis 1987,8,503-508. L5l Mosher, R. A., Thormann, W., Kuhn, R. and Wagner. H., J . Chromatogr. 1989,, 39--49. 161 Kessler, R., Manz, H. J. and Szkkely, G.,J. Chromatogr. 1989,469, 444-447. [71 Wagner, H. and Neupert, D.,J. Chromatogr. 1978,156, 219-224. [Sl Barth, F., Gruetter, M., Kessler, R. and Manz, H. J., Electrophoreyis 1986, 7,372-375. 191 Kessler, R., Manz, H. J. and Walliser, H. P.. in:3. EngineeringFoundation Conference on Recovery of Bioproducts, Uppsala 1986. l l O l Kuhn, R. and Wagner, H.,J. Chromatogr. 1989,481,343-351. I 1 11 Heydt, A., Wagner, H. and Miiller, P.,J. Virolog. Methods 1988,19, 13-22. 1121 Dickenscheid-Simon, R., Ph. D. Thesis, Universitat des Saarlandes Saarbrucken 1988. [ 131 Wagner, H., Kuhn, R. and Hoffstetter, S., in: Wagner, H. and Blasius, E. (Eds.), Praxis der elektrophoretischen Trennmethoden, Springer, Berlin, 1989, pp. 223-278. I141 Rick, W. and Stegbauer, H. P., in: Bergmeyer, H. U. (Ed.), Methoden der enzymatischen Analyse, Verlag Chemie, Weinheim, 1962, pp. 918-923. [I51 Bergmeyer, H. U., in: Bergmeyer, H. U. (Ed.), Methoden der enzymatischenAna[yse,VerlagChemie, Weinheim, 1962,pp. 392-393.
Nonamphoteric isoelectric focusing: 11. Stability of borate-glycerol pH gradients in recycling isoelectric focusing By complexing polyols with borate in recycling isoelectric focusing and by varying the ratio ofpolyol to borateovertheusefulpH range of4.0-6.0, it is possibleto control pH. Twelve solutions of 0.1 M boric acid and varying glycerol concentration were used to vary pH in a twelve-compartment commercial recycling isoelectric focusing (RIEF) system. Various concentrations of boric acid were tested as anolyte, and various Tris(hydroxymethy1amino)methane-borate buffer systems were tested as catholyte. Electroosmosis, hydrogen ion flow, and fluid balancing were characterized in two glycerol gradients; one was maintained at 0.06 pH/fraction and the other at 0.12 pH/fraction. In the latter case, ovalbumin (~14.70)migrated to the pH 4.6 1 and 4.72 compartments. It is concluded that the borate-glycerol system can be adequately stabilized in RIEF for isoelectric purification of certain proteins.
1 Introduction 1.1 Reyciing isoelectric focusing With the advent of pharmaceutical and diagnostics manufacture based on proteins from tissues, cells, or genetically engineered organisms, the need for large-scale, free-fluid purification methods has intensified. Recycling isoelectric focusing (RIEF) was introduced by Bier etal. 11-31 as a means of scaling up protein purification in free fluid. In this Correspondence: Dr. Paul Todd, National Institute of Standards and Technology, 325 Broadway 583.10, Boulder, CO 80303, USA Abbreviation: RIEF, recycling isoelectric focusing 0VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990
method isoelectric focusing takes place in a small multicompartment chamber in which each compartment corresponds to a single value of pH along the pH gradient and is (typically) separated from adjacent compartments by nylon screens. Synchronized pumps circulate the focused material from each compartment through a separate heat exchanger, and separands migrate from compartment to compartment until they reach the compartment that contains their isoelectric pH. Separands are then drained from their corresponding individual heat exchangers. Several grams of protein can be processed in a fraction of an hour by this method. Kyhse-Andersen [41 used a 32-compartment chamber (with mixing flow but not recycling) and claimed the fractionation of 10 g ofprotein in 660 ml of ampholyte solution [51. 0173-0835/90/11 11-0947 %3.50+.25/0
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1.2 Nonamphoteric buffers in isoelectric focusing Stable pH gradients can be formed without the use of amphoteric species [61. It has been suggested that the development of pH gradients in simpleionic buffers would simplify the interpretation of results and conserve biological functions in the separated material [7-9]. An artificial pH gradient was introduced experimentally by Kolin [ 101, who used nonamphoteric buffers, but since the ionic boundaries migrated in the electric field the gradients were unstable over time. Gel isoelectric focusing using organic buffers was introduced by Nguyen and Chrambach [ l 1,121, and the formation of a pH gradient on the basis of a dielectric gradient (which would not permit independent control of the dielectric constant) was introduced by Troitsky et al. [131. The use of nonamphoteric buffer pH gradients in the first step of two-dimensional electrophoresis was suggested by the work of Cuono et al. [141, but geneticists did not find this approach adequately reproducible [ 151. To prevent pH gradient migration in nonamphoteric buffered isoelectric focusing systems, including gels, the following methods have been introduced: steadystate rheoelectrophoresis IS]; buffer immobilization [ 161;and the protonating of acids and deprotonating of bases at equilibrium [17, 181, either by buffer renewal 1191 or, in the case of density gradient isoelectric focusing (described in the first paper of this series), by regenerating electrode buffers from large (150-liter) reservoirs [20]. Amphulyte materials usually used in isoelectric focusing are expensive, sometimes harmful to samples, and not suitable for very shallow pH gradients (< 0.1 pH/fraction). Isoelectric focusing in carrier ampholyte generated pH gradients results in low effective ionic strength, which can result in precipitation and/or denaturation of certain proteins. It is possible to control pH by complexing polyols with borate and by varying the ratio over the useful pH range of 4.0-6.0. Troitsky et aE. [ 2 1, 221 applied borate-polyol buffers to free “electrophoresis in a pH gradient” using simple columns and chambers with multiple outlets. In the work of Shukun 123, 241 a free-flow electrophoresis chamber with up to 42 inlets and outlets was
used; proteins electrophoresed in flowing borate-mannitol gradients in this system migrate rapidly in the electric field but may not reach their isoelectric pH.
1.3 Recycling isoelectric focusing with nonamphoteric buffers J onsson and Rilbe also introduced a multicompartment isoelectric focusing chamber [251, and Martin and Hampson [261proposed theuseofnonamphoteric buffersin amulticompartrnent system. Wenger and Javet [27],noting the desirability of replacing carrier ampholytes with nonamphoteric buffers 119, 26, 281, succeeded in stabilizing the acetic acidsodium acetate buffer system in a 5-compartment RIEF. A gradient from pH 4.05 to 4.95 was maintained for 10 h. Thus some limitations of RIEF using carrier ampholytes could be solved by using buffers. RIEF is capable of processing large volumes and sustaining stable flowing pH gradients over long periods but suffers from cost, toxicity and gradient constraints due only to the use of carrier ampholytes. Polyol-based buffer systems do not have these constraints, but they require a sustained free-fluid pH gradient; therefore, a series of investigations was designed to combine the strengths of these two methods in an attempt to eliminate some of their weaknesses. This article reports preliminary results that indicate that stable pH gradients can be produced and maintained in RIEF using an artificial borate-glycerol gradient and that this system is suitable for protein purification and could be especially useful in the purification of low-solubility proteins.
2 Materials and methods 2.1 RIEF
A commercial RIEF system, “Iso-Prep” (TM), was obtained from Ionics, Inc. (Watertown, MA) and operated with 12 buffer compartments between the anode and cathode compartments of the focusing chamber, which was 13.0 cm high, 5.60 FOCUSSING CHAMBER
U POWER SUPPLY
I HEAT EXCHANGER (14 CHANNELS)
Figure 1. Schematic diagram of RIEF as used in this study. Flow is upward in the focusing chamber and downward in the heat exchanger. Individual reservoirs and their corresponding focusing compartments are numbered. A, anode; C, cathode. Tension levers on peristaltic pump adjust individual flow rates.
Stability of borate-glycerol pH gradients in RIEF
Electrophoresis 1990, 11, 941-952
cm wide, and 0.36 cm/compartment. A schematic diagram of this RIEF is shown in Fig. 1. It differs moderately from the system described by Bier 1291. The compartments of the focusing chamber are formed by stacking, alternately, 0.325 cm polycarbonate spacers, silicone rubber gaskets, I Fm mesh nylon screen and silicone rubber gaskets so that 12 focusing chambers are formed, and the left-most chamber is the anode while the right-most chamber is the cathode - 14 chambers in all. The anode ion exchange membrane was an Tonics Inc. CR6 lCZK386 cation exchange membrane cast on a modacrylic fiber backing and using bound sulfonate groups to exchange cations. The cathode membrane was an AR anion exchange membrane cast similarly. This is one of two possible operating configurations, the other being to use the anion exchange membrane to separate the anode compartment from the focusing compartments and the cation exchange membrane to separate the cathode. This latter configuration is considered “normal”, but the former prevents separands from entering the anode and cathode compartments while permitting electrode ions to escape into adjacent focusing compartments, a situation found acceptablein buffer isoelectric focusing [20]. The peristaltic pump (Ismatec AG, FRG) has 16 individually controlled channels, two for each electrode compartment and one for each focusing compartment. These remove fluid from the top ofthe focusing chamber and deliver it to the top of the heat exchanger compartments, from which fluid is fed by gravity back into the bottom of the focusing chamber. Power was applied across the chamber using a 1000 V power supply set for constant voltage at 1000V. Interlock switches on a plexiglass door covering the focusing chamber in the cabinet prevent shock hazard. Heat exchanger cooling water was supplied at 10 OC k 1 “C from a40-liter circulating bath coupled with a cooling unit.
2.2 Solutions Anolyte solutions tested were 0.1-0.6 M boric acid, pH 4.2. The catholyte solution was Tris-borate buffer, 0.1 M in both ions, pH 8.0. A series of 12 borate-glycerol solutions was made by dissolving 0.01, 0.04, 0.09, 0.18, 0.27, 0.38, 0.46, 0.55,0.64,0.73,0.83, and 0.92 % w/v glycerol in 0.1 M boric acid for “high pH” experiments (Figs. 2 and 3) and 0.0, 0.0, 0.25,0.5,1.0,2.5,5.0,7.5,10,15,25,and30 %w/vglycerolin 0.05 M boric acid for “low pH” experiments (Figs. 4 and 5 ) . The low pH end of the RIEF system (reservoir #I) always contained the solution with the highest glycerol content. Ovalbumin was dissolved at 10 mg/mL in Tris-borate buffer as starting sample.
2.3 Procedure The RIEF system was operated without power for several hours before each experiment to balance the peristaltic pumps, using 10 % aqueous ethanol as test fluid. All 14 exchange reservoirs were drained, and the anode reservoir was filled with 100 mL boric acid, 0.1,0.3, or 0.6 M (depending on experiment); the cathode reservoir was filled with Tris-borate buffer, 0.1 M in both ions, pH 8.00, or modifications of this buffer as described below, and the individual reservoirs 1 through 12 were each filled with 50 mL of the different borateglycerol solutions specified above. The previously balanced peristaltic pumps were turned on at a setting of 60 (Ismatic Scale) and adjusted manually, individually, throughout the subsequent run time, depending upon particular experiment
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protocols: electroosmotic flow balancing, no balancing, or flow-through. Maximum field strength was maintained by operating the power supply at 1000 V constant voltage; current was recorded every 10 min. A 10 mL sample was collected for analysis from each reservoir at 0.5-1.0 h intervals. The pH was measured using acommercial pH meter with an Ag/AgCl glass pH electrode calibrated at pH 4.0 and 7.0, and conductivity was measured with a Yellow Springs (Yellow Springs, OH) calibrated conductivity cell or the conductometric system of a Pen Kem (Bedford Hills, NY) System 3000 Automated Electrokinetic Analyzer.
3 Results 3.1 Current profile and pH gradient stabilization The buffer gradient system depends on the formation of borated polyols utilizing the reaction H,B03 + HOCHZCHOHCHZOH P HOCH,CHOCH,OB(OH); + H,O + H’ to produce dissociable acids 1211. The pH depends upon the concentration of glycerol at the time of the reaction. Once the reagents are mixed, the pH is stable, as was indicated by a pair of pH readings performed on a series of solutions before and after three months of storage at 2 OC (datanot shown). The application of 1000 V across the 0.1 M borate-glycerol gradient resulted in the rapid movement of hydrogen ions out of the 0.3 M borate anolyte into the left (low pH) compartments of the chamber. The buffering capacity of the intermediate compartments prevented further pH drop, and the excess H+ions carried current across the chamber for a short period during the first hour of operation. Thus, the current, typically 15 mA, more than doubled during the first 10-30 min. After this period the expected drop in current followed the equilibration of H’ions with the glycerol borate gradient. This early rise in current does not occur in natural pH gradients formed with carrier ampholytes.
3.2 Measured pH gradients The immediate appearance of excess H’ ions in the low pH compartments is also seen in the pH profiles, as in Fig. 2, which shows the pH gradients in a “high pH” experiment as a function of time from 0 to 2.5 h of applied current. The pH in compartments 1 and 2 dropped from 4.85 k 0.5 and remained low throughout the run (Figs. 2 and 3A). This result suggests that, when using buffers to form artificial pH gradients in RIEF, the anolyte should match the pH at the low end of the gradient as closely as possible. A shallow pH gradient can be maintained over a limited number of compartments. Even during the period of early drastic current fluctuation the pH gradient over compartments 3-8 was stable and nearlylinear, as can be seen in Fig. 2.
3.3 Electroosmotic flow The contribution of electroosmotic flow to fluid motion in RIEF is difficult to assess quantitatively [31; however, because the pumping loop of the IsoPrep fills each reservoir from the top and because each reservoir has headspace, the volume of buffer in each reservoir is indicative of elec-
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Electrophoresis 1990.11, 941-952
PH
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COMPARTMENT NUMBER Figure 2. pH Gradient in the “high pH” borate-glycerol system as a func tion of time of current application at 1000 V. 0, pH readings on buffers before addition to the system (these are close to predicted values I2 1 I); 0 , 1 / 2 h ; A , 1 h;and 0 , 2 h. Thebargraphrepresentsthevolume(height)of fluid in each numbered reservoir in the heat exchanger.
troosmotic transport. An “acid notch” developed near the cathode in Fig. 2 and 3A, whichdescribe thesameexperiment. Figure 2 also shows the fluid height in each reservoir at the end of the 3 h period, and the profile indicates that more than 112 of the fluid in the low pH reservoirs was transported to the high pH reservoirs. It was therefore postulated that H ions were being transported cathodally across the focusing chamber and that measures which counteract electroosmotic flow and/ or reduce the source ofprotons would also suppress the formation of an “acid notch”.
In the experiment of Fig. 3A the tops of the reservoirs were left open, thereby permitting free flow of fluid from one focusing compartment to the other, opposed only by the hydrostatic head in each reservoir. When the tops of the reservoirs were sealed, pressure could develop within them and further oppose lateral fluid flow in the focusing chamber. In the experiment represented in Fig. 3B, this was done and the acid concentration in the anode was reduced; these measures eliminated the “acid notch”. Further reduction of the boric acid concentration in the anode without sealing the reservoirs had a similar effect (Fig. 3C) while at the same time preventing the early rise in pH in the middle compartments, as seen in Fig. 2. An alternative means of dealing with electroosmotic flow is to permit it to be unidirectional by providing an outlet; this has the undesirable effect of removing fluid from the system, so an inlet must also be provided. In the experiment described in Fig. 3D, reservoir #11 was allowed to drain while the lost fluid volume was continuously replaced in reservoir #l. This procedure eliminated the pH gradient distortion caused by electroosmotic flow, but it also gradually eliminated the glycerol gradient because only one concentration (0.92 %)of glycerol was used as replacement fluid. The overall higher pH (presumably due to the draining of H +suppliedby the anolyte) and flatter gradient are seen in Fig. 3D. It is concluded that electroosmotic flow distorts the “artificial” pH gradient formed by borate-glycerol in RIEF and that combinations of anolyte and flow adjustments can be found that counteract it. 3.4 Effect of glycerol concentration
+
The glycerol concentration range used in the experiments of Figs. 2 and 3 was 0.01-0.92 % w/v - the “high pH” range (from about 4.9 to 5.4). The resulting gradients were very flat, about 0.06 pH unitdfraction. A “low p H ’ borate-glycerol
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Figure 3. pH Gradients in the“high pH” borate-glycerol system as a function of time of current application at 1000 V in experiments designed to characterize electroosmotic flow and to stabilize pH gradients. In (A) plotting symbols represent pH readings on buffers as for Fig. 2. In (B), (C), and (D) plotting characters correspond to those in panel (A). Triangles (A)correspond to 4 h.
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Stability of borate-glycerol pH gradients in RIEF
gradient was also studied (see Section 2.3), and this system stabilized in a pH range below that of the unelectrophoresed individual buffers (dots in Fig. 4). The pH droppedrapidly during the first 0.5 h, after which is dropped slowly, producing a pH gradient of 0.12-0.15 p H units/fraction over 8 fractions. While experiments were not performed that successfully eliminated the cathodal “acid notch” in this system a more usable p H range was produced by making the catholyte more basic by mixing 4 parts of the Tris-borate buffer with 1 part of 0.1 M NaOH. This measure resulted in a p H gradient stabilized over 8 fractions between p H 3.8 and 4.8 as shown in Fig. 5, thereby overcoming the early decrease in pH seen in Fig. 4. This measure also introduced OH- ions into the chamber resulting in a steady increase in current from 5-8 mA to 15-20 mA over a 2-h period, during which time the conductivity typically rose from 3 to 7 mS/cm.
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p H gradient (dots, also nearly the same as predicted I 2 I ] ) except in the uppermost fractions. At the end of 1 h most of the protein, as determined by the method of Bradford I301 and shown as A (595 nm) in Fig. 5, was found in compartments 7 and 8, corresponding to pH 4.6 I and pH 4.72, respectively.
3.6 Hydraulic adjustments It was possible to independently adjust flow rates in individual channels using the multichannel peristaltic pump (Fig. 1). It was noted in the course of performing this research that adjustments in real time were often required to maintain reasonable fluid volume distributions among the reservoirs or to prevent low pH compartments of the chamber from draining completely. Such adjustments obviously draw fluid from one loop to an adjacent loop, thereby also locally modifying the pH gradient.
3.5 Protein isoelectric focusing Ovalbumin (PI 4.70) was introduced into reservoir #12 at time = 10 mi, at p H 5.2, at which pH the molecule is anionic. The“low pH” system just described was used. Figure 5 shows that the pH gradient (open circles) closely resembled the input 7
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Figure 4. p H Gradient in the “low pH” borate-glycerol system a s a function of time of current application. Dots represent pH readings on buffers before addition to the systems (these are close to predicted values (21I); plotting characters for each time are shown on the graph. Broad horizontal bars and vertical arrows indicate beginning and final pH in anode and cathode buffers. 0.3
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Figure 5. Isoelectric focusing of ovalbumin (pl 4.70) in the “low pH” borate-glycerol system for 1.0 h. Starting sample (100 mg) was added to reservoir #12. The bar graph represents protein concentration, in absorbance units, measured by the Bradford method [33 I.
4 Discussion The results of this investigation are preliminary in the sense that considerable further refinements are possible. For example, pH gradients can be further controlled by the regulation of pumpingrates in individual channels, and a wider variety of p H ranges can be achieved by choosing the nature and concentration of polyols [211. In discussing their study of RIEF of interferon, Nagabhushan and Trotta [ 3 11 alluded to several desired improvements of the method. Some of these desiderata should become reality as a result of using nontoxic, nonamphoteric buffers in RIEF. The usually low ionic strength (
