Some recent developments in preparative electrophoresis and gel filtration

Some Recent Developments in Preparative Electrophoresis and Gel Filtration By JERKER PORATH.

,\S PHOF. TISELIUS, Dr. Albertsson and I recently painted out;' studies in

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fractionation methodology ought to be performed with a particular objective connected with some actual scientific or technological problem, which requires puriflcation or isolation of one or several substances. My collaborators and I have recently started a long-term project aiming at a better understanding of the relationship between chemical structure and biological function of immunoglobulins. This field of immunochemistry seems now to be explorable but still there arc many obstacles to be overcome, particularly concerning the isolation of well-defined y-globulins. \Ve have felt it necessary to further improve familiar methods as well as to explore new technics and more selective means. Today, I will treat some of the former methods, viz, zone electrophoresis and gel filtration as applied by us presently Jat the Biochemical Institute of Uppsala, I intend to discuss some of their merits and drawbacks and will touch upon the question of the physical background for the molecular sieving in' gels. Zone Electrophoresis

From a theoretical viewpoint, frec zone electrophoresis, viz. electrophoresis in a medium without anti convection agents, is the most satisfactory form of preparative electrophoresis, To my knowledge, three ways of free zone electrophoresis have been advanced: In one of them the test solution and buffer solution arc distributed as a streaming thin layer on a cooled support with the electric field. perpendicular to the buffer How." In another, introduced by Dobry and ·Finn,:1 the test solution and buffer solution are distributed from adjacent baffles from the bottom of a column to form a stream of upwards flowing liquid. To place an electric field perpendicular to the flow, the electrode compartments are separated from the column by semipermeable membranes along the vertical side of the column'. The substances in the test solution will thus be clcctrophoretically separated on their way upwards and are removed through separate outlet ducts at the top of the column. The third method has been suggested by Kolin and independently by Hjerten.! A horizontal tube serves as the separation chamber. Convection is eliminated by rotating the tube at a constant speed of revolution. The difficulty of keeping the field of flow stable in the two cases first mentioned has limited their success so far. The third method, however, appears to be very useful, particularly for analytical. purposes where great precision is desired and also for rapid screening of buffer systems suitable for preparative electrophoresis. Hjerten has also shown' that his method is applicable to From the Institute of Biochemistrij, Uppsala, Sicctlen,

1004 ~IETAnOLlS:\I, VOL.

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ELECffiOPllOHESlS A!':D GEL FILTRATION

1005

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Fig. l.-EIectropherogram of pancreatic homogenate from starved obese-hyperglycemic mice obtained by free zone electrophoresis. Conditions: Inner diameter of tho revolving tube 0.3 cm.; current 10 ~IA; buller 0.067 ~I sodium phosphate, pH 7.3; temperature +3°. The recordings of ultraviolet absorption were made 0, 5, 12, 19, 25 and 47 minutes after start. The starting position is indicated by an arrow (Hjerten, 19G3)~

the fractionation of particles, for example, cell fragments from pancreatic insulae (fig. I). Furthermore, the rotating tube is an excellent instrument for determining electrophoretic homogeneity of scarce material. Dy means of a scanning UV-absorptiometer, such a test can be made with ·100 I-'g. or less. Separation can be checked while electrophoretic fractionation is still in progress and samples may be withdrawn at any time and from any part of the tube. For preparative zone electrophoresis, where amounts of test substances greater than 10 mg. are to be fractionated, severe disturbances due to convec tion must be eliminated by addin'g some antieonvection agent to the electrolyte solution. One way of accomplishing this is to distribute an uncharged, inert substance in the bufler-solutlonfn' such a way that a stable density gradient is created. Density gradient electrophoresis, however, has certain limitations which arc usually not pointed out by the supporters of this method. The most serious weakness is the low capacity. Slow convection currents are likely to be created after some time when ordinary buffer systems are used. As a result of these disturbances the originally continuous density gradient will be transformed into a step-wise gradient. I have tried to use a density gradient formed by heavy and light water in short columns. By introducing a zone of a dye in such a column, it was readily demonstrated that when a voltage was applied, a central upward flow of liquid developed which at some distance from the starting position distributed towards the wall of the tube in a fountainlike manner. It appeared very difficult to make long-term runs in such a system and I had a similar experience with sugar gradients. Although dyes of high mobilities could be easily separated, attempts to fractionate protein mixtures were often' unsuccessful.

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JERKER PORATII

Continuous flow zone electrophoresis by density gradient stabilization was introduced by Philpot and has recently been studied by MeP Stabilization' of the liquid medium can be achieved by dispersing a gel formed with agar or agarose,? Introduction of small amounts of solid material is necessary. Unfortunately, however, agarose and agar in' particular, are negatively charged. This fact results in a slow movement of the gel column in the electric field, and because of the low tensile strength the column may crack. Another disadvantage should be mentioned: the test substance is likely to be contaminated by soluble carbohydrates from the agar or agarose. A cross-linked gcl of the dextran type, on the other hand, is practically insoluble and uncharged. Irreversible adsorption is seldom encountered. As a consequence, these gels arc very good convection depressors. TIleY too, however, II ave a weakness which they share in common with starch and similar gels, namely, to allow the buffer ion to pass through the grains. The proteins will not enter the gel if it is strongly cross-linked, as is for example Sephadex G25. Joule heat is thus formed in regions Inaccessible to the proteins during fractionation. If, on the other hand, the gel is permeable to proteins, molecular sieving will occur. This may sometimes be an advantage, but not if pure electrophoresis is desired. Aside from homogenous gels, we have found cellulose powder to yield the best stabilization. Very compact beds may be obtained by sedimenting the dry powder in a solution of 10 per cent acetic anhydride in acetone, pouring the column and washing with pure water and finally with the buffer to be used. Unfortunately the adsorption of many proteins to cellulose cannot be disregarded. This is particularly true at low ionic strength and low pH. Plastic powders of the Geon or Pevikon type yield low adsorption but are inferior stabilizers when compared to the materials already mentioned. Furthermore, the heat conductance is low and the effective cross-section is small. For these reasons, plastic columns have low capacity. Zone electrophoresis in powder-stabilized media can be performed in troughs, in horizontal or vertical column tubes. \Ve prefer the last type because it makes it easy to prevent conductivity and pH gradients to develop, thus making possible extremely long periods of operation with perfect control of temperature and liquid movements. The most versatile column' construction that has been published so far is probably that or Hochstrasser et al," It permits migration-elution" and countercurrent elution," \Ve have recently constructed a similar apparatus but with a more effective cooling system and with certain other improvements. To allow larger quantities of test mixtures to be fractionated, we perform the electrophoresis in an annular separation chamber internally as well as externally cooled (fig. 2).10 This apparatus is now commercially available (LKBProdukter, Stockholm). A diagram from a fractionation of lipoprotein' depleted human serum is shown in figure 3. The experiment was performed in veronal buffer of pH 8.4 and ionic strength 0.1, with cellulose powder as the stabilizer. Evidently, the classical pattern is obtained with satisfactory resolution of the components in spite of the high load on the column. We usually

ELEcrROPIIOHESIS A!'\D GEL

FILTRATIO~

IOll7

Fig. 2.-Schematic drawing of the large-scale electrophoresis apparatus used for the separations referred to in flgurcs.S, 4 and 5.

make exploratory analytical experiments in small jacketed columns'! if the electrophoretical behavior of the material in the buffer system to be used is unkown. From the data thus obtained, it is easy to calculate the number of ampere-hours necessary for a satisfactory experiment in the large apparatus. The electrolyte medium, of course, plays a decisive role for the outcome of the operation. In this fact resides the great flexibility of the electrophoretic method and it is, therefore, surprising that so few systematic searches have been made to find electrolyte systems to fit particular problems. Most workers in the protein field prefer to utilize only a few conventional systems and then spend a great deal of time in devising precise conditions for adsorption chromatography on ion exchangers, although the latter task in my opinion is more difficult.

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JERIo:En rORATII

AbsorbancC/ (280rnJlJ

2 ,5

20

15

10

05

1000

2000

3000

";000 D;splac~d volum~(ml)

Fig. 3.-20ne clcctrophcrogrnm of lipoproteins depleted human serum. Conditions: convection depressor, cellulose powder; bed height 68 inches; sample volume 88 ml., buffer 0.1 11 veronal pH 8.6; voltage 500 V; current around 400 rna; 40 amp hours.

I shall now show two examples of unconventional electrolyte systems useful for purification of human serum. As before, lipoproteins had been removed by flotation in the ultracentrifuge cmploying a dense medium. This prcfractionation is advisable; otherwi se the lipoproteins may precipitate on the supportin'g powder. The pattern shown in figure 4 was obtained in a verona I buffcr of the same pH as in the previous example, hut of a much higher ionic strength due to the prcscnce of molar lithium sulfate. The purpose of using a high concentration of lithium sulfate was to decrease adsorption and to improve the separation between albumin and the a-globulins. The high conductivity of the buffer makes it necessary to extend the electrophoresis over a long pcriod of time but this is not serious since the high solubility of lithium sulfate (as compared with the sodium salt) in the cold permits an operating temperature close to zero. If we compare the pattern's,of figures 3 and 4, we find that in the latter case the at-globulin peak has disappeared and that the fJ- and y.globulins have been better separated. A closer examination of the fractions reveal that at-globulin which is not easily separated from albumin in the veronal buffer of low ionic strength is mixed with CI:!-globulins but well behind the albumin. It is, therefore, evident that two separations in scqucncc in either buffcr system should yield an cfficicnt purification. This example demonstrates the usefulness of consecutive electrophoretic runs in dlflercnt buffcr systems where a few components shift their position relative to the main components of the original mixture. \Ve have recently bcgun to investigatc the electrophoretic behavior of proteins in' the highly polar solvent media, particularly in strong amino acid so[utions.P Unexpected results arc often encountered. For example, previously unknown human y-globulins were recently discovered by' Prof. Ui and myself when we used glycine-rich buffer systems for serum fractionation. Figure 5 shows the pattern obtained with such a system composed of 1.5 M glycin'e and

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1.0

0 .5

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Fig. 4.-Zone clectropherogram of lipoprotein-depleted human serum. Conditions: convection depressor, cellulose powder; bed height 68 inches; sample volume 112 ml.; buffer 0.05 11 in veronal and 0.3 11 in Li2S04 • pH 8.6; voltage 250 V; current around 1500 ma; 220 amp hours. A b eor b anc q (280 m}-l ) 1.5

1..0.

(J

0.5

2

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5 titers

Displaced volume

Fig. 5.-Zone clectropherogram of lipoprotein-depleted human serum. Condilions: convection depressor, cellulose powder; bed height 69 inches; sample volume 350 ml.; buffer, upper part 0.02,5 1\1 tris + 1.5 11 glycine pH 7.6; lower part 0.1 1\1 tris + 1.51\1 glycine pH 7.6; voltage 500 V; current 300 rna; 20 amp hours.

0.1 M tris sulfate, pH 7.6. Albumin, together with al and a2 globulins, move together in the Fast-moving zone followed by ,B-globulins and the y-globulins, Although a poor system for the fractionation of the fast-moving protein'S, it is highly efficient for the slow-moving y-globuIins. In fact, the y-globulins arc obtained as two main groups. We have utilized this behavior to demonstrate the presence of subgroups of the 7S-type of y-globulins. The characteristic effects obtained by the glycine systems can at least partly be explained by the weak protein'.protein interaction due to the stronger polarity as compared with the conventional aqueous systems. Low molecular weight species of y-globulins were revealed to be present

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JERKEn FORATIt

in serum by subjecting the electrophorctically obtained 'Y-globulin fraction' to gel filtration on Sephadex G200. GEL FILTTIATIO:-l

When a solution is brought into contact with a gel, a segregation of solutes take place between the solution imbibing the gel matrix and the surrounding liquid. This segregation' is determined by the rate of diffusion and the extent of exclusion. In several cases, adsorption may cause a preferential enrichment in the gel phase. In finely granulated gels, the diffusion is rapid and equilibria are quickly approached. In gel filtration, therefore, molecular exclusion and adsorption are the most important factors that determine the different migration rates in the beds. TIle molecular sieving in gels formed by swelling Sephadex, cross-linked polyacrylamide, starch, agar, etc., is now a well-recognized phenomenon. Its merits as a basis for fractionation according to molecular size has often been emphasized. I don't believe it will discourage the users of the gel filtration method if I now discuss a number of complication's and drawbacks often encountered and how they can be utilized respectively, be minimized or avoided. The adsorption may be caused by ion interaction if the gel matrix contains axed ionic groups. Also, in the absence of charged groups, a mutual attraction between the gel proper and certain' substances might occur. Cross-linked dextran (Sephadex), for example, exhibits an obvious affinity for aromatics. Aromatic adsorption is not limited to dextran and dextran gels; it is encountered in all kinds of hydrophilic gels we have tested; for example, in gels formed from cross-linked polyvinylalcohol, cellulose, starch, polyacrylamide and others. The adsorption may be more or less pronounced; on Sephadex it seems to be particularly favorable for practical applications. Aromatic adsorption can be modified and also extinguished (in strong urea solution). It seems in some way to be dependent on the hydrophilic character of the gel. Gelotte (personal communication) has studied certain organophilic gel·formers and found that they do not exhibit any preferential adsorption for aromatics in organic solvents. On cross-linked dextrans, most aromatics travel in the column' with a speed independent of their concentration. This fact makes these gels particularly useful as an adsorbent. Dr. Determann has told me that in Prof. Th. Wieland's laboratory in Frankfurt the organic chemists make use of Sephadex more extensively than the biochemists. They utilize adsorption for purification of peptides from. natural sources as well as for cleaning up synthetic mixtures. In the routine work, Sephadex chromatography has been almost as indispensible as recrystallization. Aromatic adsorption has also been' utilized for purification of iodothyronines in blood, isoflavones in red clover, tannins, etc. However; the potentialities have not as yet been widely appreciated. Obviously, the adsorption capacity increases with the matrix density of the gel. Fractionation of solutes of molecular weight below 1000 should preferably be performed in denser gels than in those now commercially available. Such

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ELECrROl'1I0RESIS AJ'D GEL FILTRATION

Na CONC.

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li

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THO

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Fig. B.-Separation of Li, Na and K in a 70-inch long column of cross-linked dextran (W.H. 0.9) in 0.05 1[ tris-lIe!. "D ex 500 " and THO were included as reference substances (M arsden, 1963) .

dextran gels have now been produced in' the laboratories of Pharmacia, Crosslinked dextran of water regain 1 gig will soon be industrially produced under the trade name Sephadex GIO. Dr. N. V. B. Marsd en has al ready investigated Sephadex GlO and has made some interesting discoveries which reveal other hitherto unknown adsorption effects.P For example, he was able to achieve a partial separation of hexoses. Dr. Marsden has also studied the behavior of alkali ion's in Sephadex columns. In methanol-water mixtures, quite good fractionations can be obtained on Sephadex G25. In this case, liquid-liquid partition plays the most important role for the separation since the heavier alkali ions are strongly retarded on the columns. El cctroneutrality must be preserved and this also wiII strongly influence the behavior of the cations particularly at low ionic strength. Li, Na and K can' be separated from each other in aqueous buffers on short. columns of Sephadex GlO (fig. 6). Polyisornaltose "Dex 500" isolated from hydrolysate. of dextran travels totally excluded from the gel. "Dex 500" has a mean molecular weight of 500. He also observed that tritiated water, THO, migrates as a well-defined zone. Dr. Marsden has also told me tliat butanol is more retarded on Sephadex GlO than is methanol. This surprising finding indicates that unknown phenomena also are important. A comparison of the adsorption properties of strongly cross-linked gels of different types is likely to be rewnrding. Convenient procedures for desalting solutions containing small peptides as well as the removal of reaction products from reagents could probably be de vised. Of particular interest in studies concerning glycoprotein structure has been the apparently low recovery from Sephadex of sialic acid and gIycopeptides which contain sialic acid. This has now been shown to be rather a conforma-

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JERKER POUATII

Fig. 7.-De\'iCe for osmotic concentration of efficient from columns.

tional change in structure, presumably catalyzed by the gel matrix rather than an irreversible binding. However, this alteration of structure can be averted by incorporating EDTA in the eluting buffer during Fractionntion.I! Fractionation of proteins and other polymers in gel beds has been the subject of several reviews." Rather than further exemplify the use of gel filtration methods for these purposes, I would like to mention some weaknesses of the procedure and give some remedies. When dealing with pure molecular sicving, the theoretical limit for the retentiuu volume can' certainly not exceed the volume of the gel phase. In gel filtration, the unabsorbed test substances must be separatcd within rather a limited efHuent volume in contrast to the usual case in liquid-liquid partition or adsorption chromatography. In order to, allow separation on' the basis of minor differences, the substance must pass through considerable bed heights. Two undesired effects are then encountered: (1) The gel particles in the lower part of a long column deform, thus impairing the flow properties, and (2) the solution containing the test substances is excessively diluted because of zone spreading. The introduction of spherical gel particles has significantly reduced the resistance to flow in' the columns. Furthermore, excessive compression of the gel grains can be counteracted by both directing the flow upwards and by dividing the bed into a number of smaller columns connected in tandem. The recycling technic permits extensive filtration in a short column and has the advantage of permitting easy continuous control of the progressing fractionation. The second major weakness is the problem of dilution. Dr. Bennich and I

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ELECI1l0PHOHESIS AND GEL FILTRATIO:-;


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20 .30 ~O

50 60 70 80

90 100

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Fig. B.-Gel filtration diagrams of six times diluted serum (25 ml. applied) fractionated Oil a cross-linked dextran ('V.R. 12.4). The lower curve was recorded directly on the effluent with a UVICORD-absorptiometer (at 2.5,1 mI.). The upper curve was obtained with a similar instrument with the absorption cell connected to the outlet of the "concentration screw" seen in figure 7.

have' constructed some simple concentration devices. to counteract problems caused by dilution. Figure 7 shows a concentration' apparatus that consists of a central cylinder with a helical groove covered by a dialysis tubing. The effluent from the chromatographic column is conducted through the helical groove. As osmotically active polymer solution Is circulnted on the outside of the membrane. By this technic water is continuously withdrawn from the effluent. By proper adjustment of the dimensions of the apparatus, the rate of flow and the concentration of the polymer, it is possible to obtain any desired ratio of concentration' without remixing the substances just separated. The material distribution of serum proteins from a gel filtration experiment at the entrance and the outlet of the "concentration screw" is seen in figure 8. Gel filtration has been most frequently used for preparative purposes. It may also be applied for estimation of molecular size, for example, when small amounts of an easily detectable material are available or when the substance has not yet been isolated. Lathe and Ruthven emphasized this kind of application in their paper on molecular sievin'g in starch beds. Recently, some papers have been published on the use of gel columns for the determination of molecular weights. The calibration is based on the retention of standard substances. \Vhen' utilizing a separation method for physical characterization, it is highly desirable to understand the underlying principles. Attempts are in progress to formulate a theory for molecular sieving in gels. Two models have so far been advanced both on the assumption that the partition of a solute between' gel and surrounding liquid is determined by the fraction of the gel volume accessible to the solute. One of them assumes the presence of density gradients in the matrix.!" In order to develop a relationship between the distribution co-efficients and the molecular size of the solutes, sieving has been assumed to take place in conical alveolar spaces and to be influenced solely by steric factors. Furthermore, the cffective radii of the solute molecules have been assumed to be proportional to the square root of the molecular weight. Based on these premises, a simple formula is derived:

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JEllKER !'ORATII

where Kd = distribution co-efficient, 1( = molecular wcight.. S, solvent regain and k, k 1 and a are constants.

=

Good agreement between experimental and predicted Kd-values were obtained from the fractionation of high molecular weight dextran's using three kinds of Sephadex Gels. Andrews'" and Wieland et alY' have found that the predicted linear relationship between KIf:! and },Pf~ also holds for proteins. Another model has been' proposed by T. Laurent and J. Killander!" in which the gel is considered to be approximately described as a suspension of randomly distributed straight fibers. The extent of exclusion of spherical nonelastic particles from such a polymer solution has been theoretically investigated by Ogston. The space accessible to the particles can be expressed as an exponential function containing the concentration and radius of the fibers and the radius of the particles. This space c~n be calculated by making reasonable estimations of the fiber parameters and assuming the radii of the particles to be equivalent to those of spheres calculated from diffusion data. The calculated values for a number of substances have been compared with those determined by experiments in columns. Corresponding values arc in good agreement. The close agreement between predicted and experimental values may be a weak support for the theories since it may merely reflect the insensitivity of the measurements. Nevertheless, it inspires the hope of formulating a theory for molecular sieving in' terms of volumes accessible to the solutes. An adequate theory should take into consideration the combined effects of variations in density and structural heterogeneity of the gel. CO~CLUD!KG HDIAHKS

Electrophoresis and gel filtration are a very efficient pair of purification methods because they are based on entirely different physical principles. TIley have in common a high reproducibility and both methods may be applied on any preparative scale in the laboratory. REFERENCES

1. Tiselius, A., Porath, J., and Albertsson, P. A.: Science 141:13, 1963 2. Barrollier, J. E., Watzke, E., and Gibian; II.: Ztschr. Naturforsch. 13b: 754, 1958; Hannich, K., Ztschr. anal. Chern. 181:244, 1961. 3, Dobry, R., and Finn, R. K.: Science 127:697, 1958. 4. Hjerten, S.: Proteides of the Biological Fluids. Amsterdam, Elsevier 1960, pp. 28-30.

5. 1(c1: Science 127:697, 1958. 6. Hjertcn, S.: J. Chromatog. 12:510, 1963. 7. Hochstrasser, H., Skeggs, L. T., Jr-. Lentz, K. E., and Kahn, J. R.: Anal. Biochcm. 6:13, 1963. 8. Porath, J., Lindner, E. B., and jerstedt, S.: Nature, London. 9. Eliassen, R., Hammnrsten, E., Lindahl, T., and Palmsticrna, H.: Acta chcm, scandinav. 15:570, 1961.

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ELEcrnOPHonESIS AND GEL FILTRATION'

10. Porath, ].: Science Tools. In press . 11. - : Arkiv for Kcmi, 11, nr. 28: 1957. 12. - : and Vi, K : Biochem. Biophys. Acta in press. 13. Marsden, N, V. n.: Science. In pres s. 14. Marshall, W., and Porath, J.: To he published. 15. Porath, J.: Advances Protein Chern. 17: 209 , 1962; Flodin, P.: Dextran Gels and Their Appli cation in Gel Filtration. Dissertation, Uppsala, H)62; Crunath, K: New bio chemical scpa-

16. 17. 18.

19.

ratlons. In press; Colette, n.: New bio chemical sep.irations. In press; Boardman, N. K. : Modern Methods of Plan Analysis, Vol. 5. Berlin, Springer, H)62, pp. 205--213. - : ]. Pure & Appl. Chern, 6:233, 1963. Andrews: j. BioI. Chern. 91:222, 1964. Wi eland, T ., Ducsbcrg, P., and Determann, H.: Bioch cm, Ztschr, 337 :303 , 1963. Laurent, T., and Killundcr, ].: To he published.