Interacting Macromolecules (gel filtration/sievorptive chromatography/adsorption chromatography/ribosomes/ protein-nucleic acid interactions)

Transcription

1 Proc. Nat. Acad. Sci. USA Vol. 70, No. 8, pp , August 1973 Intervent Dilution Chromatography: Concept for Separation of Strongly Interacting Macromolecules (gel filtration/sievorptive chromatography/adsorption chromatography/ribosomes/ protein-nucleic acid interactions) LESLIE KIRKEGAARD* AND C. COE AGEEt School of Chemical Sciences, Departments of * Chemistry and tbiochemistry, University of Illinois, Urbana, Ill Communicated by N. J. Leonard, May 18, 1978 ABSTRACT Intervent dilution chromatography separates interacting macromolecules by subjecting them to a dynamic environment in which the association constant is continuously varied. The dynamic environment is produced by using the sieving properties of a gel to repeatedly propel the molecular complex across an intervent boundary. Behind the boundary, at high inteirvent concentrations, the complex dissociates;'ahead of the boundary, the component molecules are separated by adsorption processes. By selective adjustment of the intervent composition of the sample and the conditions of column equilibration, the process is adapted to a particular need. This report describes the chromiatographic' concept and shows how parameters are adjusted to obtain the desired separation. In this study a particularly difficult separation, i.e., the separation of ribosomal proteins from ribosomal RNA, is chosen to illustrate the power of the procedure. Separation of macromolecules that interact strongly with each other is one of the most persistent problems of biochemistry. Although chromatography has been used to measure the amount of association between molecules (1), current chromatographic procedures have found only limited use in separation of molecules that significantly interact with each other. This limitation results from the fact that the basic chromatographic process demands that molecules behave independently. It is sometimes possible to alter the chromatographic environment to permit the molecules to behave independently while retaining sufficient interaction with the stationary phase to cause separation; however, such changes usually reduce the interactions necesary for separation to a point where resolution becomes impossible. Intervent dilution chromatography is a combination of molecular sieve (gel filtration) and adsorption chromatography that effectively circumvents the stated limitation in classical chromatography. Because molecular sieve processes may be combined with adsorption chromatography in several ways to optimize for various needs, we suggest'some systematic nomenclature. Chromatographic systems in general that use any active combination of molecular sieve and adsorption processes are called sievorptive chromatography. Intervent dilution chromatography, as well as the recently defined ion filtration chromatography (2), are specific modes of the general process of sievorptive chromatography. Although ion filtration and intervent dilution chromatography are conceptually distinct, in practice both processes may occur within the same column, as happens when ion filtration chromatography is used to "desalt" an enzyme sample. The chromatographic system that is introduced has broad utility and may be adjusted for a selective or complete dissociation of biological complexes. To illustrate the power of the new chromatographic approach, conditions were found to separate ribosomal proteins from ribosomal RNA, one of the strongest macromolecular complexes encountered in biochemistry. As a historical framework for this study, the separation of ribosomal proteins from ribosomal RNA (rrna) has required harsh conditions in which the unwanted component is destroyed. rrnas have been obtained by repeated phenol extractions (3), and ribosomal proteins have been isolated by extraction in 66% glacial acetic acid, by degradation of ribosomes in 3 M LiCl-4 M urea (4), or by extraction with 80% 2-chloroethanol acidified with HC1 to 0.06 M (5). CONCEPT OF INTERVENT DILUTION CHROMATOGRAPHY Biological macromolecules are usually polyfunctional compounds containing many ionic and nonionic groups. If the functional moieties of one molecule complement those of another molecule and if the groups interact poorly with the solvent, strong intermolecular adhesion results. This adhesion is reduced by changing the qualities of the solvent to make it interact more effectively with the functional groups of the macromolecules. If the attraction between macromolecules is ionic, the solvent is improved by increasing its ionic strength. This is true up to the point where high salt concentrations cause the interacting macromolecules to precipitate. When macromolecular adhesion is nonionic in character, the aqueous solvent is improved by addition of certain monofunctional substances that complement the functional groups of the macromolecules, such as urea, glycerol, sucrose, glycine, or dimethylsulfoxide. For the purpose of describing chromatographic phenomena, low-molecular-weight agents that are added to a macromolecular solution to reduce intermolecular binding are called intervents, a contraction of the coined expression "intervenants," meaning molecules that are added to intervene in the interactions between other molecules4 (6-8). t Any solute in high concentration influences the inter- and intramolecular behavior of macromolecules. The term "intervent" focuses attention on the ability of the solutes to reduce intermolecular interaction in contrast to "denaturants," which refer to the ability of the solutes to manipulate intramolecular forces (6, 7). Solutes have been used as intervents in ultracentrifugation, electrophoresis, counter-current distribution, dialysis, and chromatography for many years (8). 2424

2 Proc. Nat. Acad. Sci. USA 70 (1973) To understand the chromatographic separation of interacting macromolecules, it is useful to consider the ideal case of two adhering molecules, a and (3. In this case, four basic interactions must be considered. These include the interactions of the two solutes with each other and with the chromatographic resin, R. Equations for these interactions are listed in order of decreasing association constant. a + ( =- a(3 ( + R =3,R a + R =a(r a + R ar [1] [2] [3] [4] If a and ( are ionic, an increase in the ionic strength of the system drives each of these reactions to the left. Additives that reduce nonionic forces have a similar effect. In other words, addition of intervents to the system lowers the association constants of the listed reactions. The process of intervent dilution chromatography uses a gel column to repeatedly drive the interacting macromolecules across an intervent concentration boundary to cause a repeated shift in the equilibria of the above reactions. Under appropriate conditions, this continual change in the equilibrium constant leads to the separation of a and (3. To accomplish the process of intervent dilution chromatography, the intervent concentration in the sample is raised to a level such that all four reactions are predominantly in the left-hand form. A gel column is equilibrated at a lower intervent level such that reactions 1-3 are predominantly in the right-hand direction while reaction 4 remains on the lefthand side, i.e., molecular species (3 and a(3 stick to the gel, whereas a has no affinity for the resin. Because the intervent concentration of the sample is high, as the sample is applied to the column, none of the macromolecules adsorbs on the resin and the sieving properties of the gel cause a, (3, and a(3 to outrun the sample intervents into a region of low intervent concentration. Here the migration rates of molecular species ( and a(3 are slowed by increased interaction with the resin to a rate less than that of the intervent boundary formed at the leading edge of the sample, whereas a continues to move rapidly. Eventually, the intervent front overtakes (3 and a(, and the complex a(3 has another opportunity to dissociate. Again, the macromolecular species are projected ahead of the sample intervents into a region of low intervent concentration where dissociated a continues to move ahead of (3 and ao. This recurring process eventually leads to complete separation of a and (. Fig. 1 shows how intervent dilution chromatography is related to conventional gel filtration and adsorption chromatography. Plots a and b show where macromolecules a and (3 would migrate on an adsorption column when chromatographed separately under the conditions used to equilibrate the intervent dilution column. Plot c shows the behavior of a,,and a(3 when a and (3 are chromatographed together on the above column (9, 10), and illustrates the inability of conventional adsorption chromatography to separate the interacting molecules. Plot d illustrates how a, (3, and a( would elute with reference to sample intervents if chromatographed on a gel in the absence of adsorptive processes. Plot e shows how a and (3 would migrate when the adsorptive column of c is combined with the gel column of d under optimum conditions for intervent dilution chromatography. A property =ii a a Intervent Dilution Chromatography 2425 b G c d e... I... IN:ERVE:! FRONT Cag -.:-.. -:.'. FIG. 1. Relationship of intervent dilution chromatography to adsorption and molecular sieve chromatography. of intervent dilution chromatography is that solute a is always eluted ahead of the sample intervents whereas (3 is usually eluted at the leading edge of the intervent front, although it may happen that (3 is eluted well after the front or not at all. A careful consideration of the presented model suggests some important aspects of the chromatographic system. First, the gel used in the separation must be sufficiently crosslinked to exclude the macromolecules, while adequately porous to permit penetration of the sample intervents. Second, actual separation of a and (3 depends upon a unique adsorptive interaction of each of these molecules with the gel. The success of the method depends upon the availability of gels of appropriate porosity and adsorption characteristics. The highly cross-linked ion-exchange dextran gels commercially available from Sephadex have suitable characteristics of adsorption and permeation for separation of most watersoluble biological compounds. Third, separation of complex a(3 by intervent dilution chromatography requires a sample solvent system that permits a and ( to spend a finite amount of time as independent, monomolecular species. The intervent composition of the sample otherwise is relatively unimportant, provided that it keeps a and (3 sufficiently independent, since the actual separation of a and ( occurs ahead of the sample intervents. Fourth, in order for separation to occur by the intervent dilution process, the gel column must be equilibrated with a solvent such that macromolecule a travels faster than the intervent front, whereas molecule (3 and complex a(3 migrate slower than the front. An important aspect of the equilibrating solvent is that it must be compatible with purified a. It may happen that as a is removed from (3 and the sample intervents are lowered, the solubility properties of a may sharply change causing a to precipitate. This problem is illustrated later. In summary, the problem of adjusting intervent dilution chromatography to a particular separation is one of identifying the appropriate sample solvent and properly equilibrating the gel column. MATERIALS AND METHODS DEAE-Sephadex A-25 (Pharmacia Inc., meq/g of dry resin) was used for intervent dilution chromatography. Before use, the gel was washed with volumes of M KOH, followed by sufficient M HCl to make the chloride form of the resin. After excess acid was removed with

3 2426 Biochemistry: Kirkegaard and Agee Proc. Nat. Acad. Sci. USA 70 (1973) composition described in Fig. 2, was applied to the above column. The size and homogeneity of the RNAs recovered from the columns was estimated by electrophoresis at ph 7.7 in 7.5% polyacrylamide gels. FRACTlON NUMBER FIG. 2. Inwervent dilution chromatography of ribobomes under various conditions. DEAE-Sephadex A-25 columns were prepared and developed at 25. The solid line is absorbance at 280 nm, and the dashed line is absorbance at 260 nm. Equilibrating Sample Column solution solvent A B C D E M KC M MgCl M I*1HCI, ph 7.0* 0.40 M KC1 4 M Urea 0.1 M I-HCI 1 M KCI 0.01 M MgCl M I1HC10 Same as B M K1( 7 M Urea M MgCl2 0.1 M I.101 Column eluant 1 M KCI 0.01 M MgC2 0.1 M I HCl 0.80 M KC1; other components same as E M KCI 7 M Urea M MgC1l 0.1 M I.101 The ph is defined in terms of the acid and base forms of the buffer (2). A 0.1 M I*HCl buffer (ph 7.0) contains 0.05 M imidazole plus 0.05 M imidazole HCl, I/I.1HC1 = H20, the ion-exchange gel was packed into a column and equilibrated with appropriate buffer until the ph of the column effluent was the same as the input buffer. (Usually equilibration requires 'about 10 column volumes of buffer when the buffer is used at its pk. at a concentration of 0.1 M.) Columns 30 cm high and 0.8 cm in diameter were eluted at 7 ml/hr (14 ml/hr per cm2 cross-sectional area) with the solutions listed in the figures. Cell-free extracts (S-30) of Escherichia coli MRE 600 were prepared by the method of Clark et al. (11). Ribosomnes were isolated by overlaying the S-30 extract on a cushion of 10 mm Tris' HCl (ph 7.4)-10 mm MgCl2-1 mm dithiothreitol-10% glycerol and centrifuging at 150,000 X for 2 hr. The ribosomal' pellet was rinsed with the cushion solution and then suspended at a concentration of 500 A200 units/ml in the cushion solution and stored frozen. A sample of 0.4 ml at a concentration of 250 A2m0 units/ml, having the RESULTS AND DISCUSSION Ribosomes are an aggregate of proteins and RNAs that is very stable and does not readily dissociate under conditions appropriate for ion-exchange chromatography. In fact, ribosomes have been washed free of loosely' bound factors by chromatography on DEAE-cellulose (12). Ribosomes contain many proteins and at least three RNAs, and are sufficiently complex to permit us to illustrate the flexibility and properties of intervent dilution chromatography. Moreover, under appropriate conditions, it is possible to make the ribosomal proteins and RNAs behave' as a pseudo twocomponent system with proteins behaving as molecule a and'rna as molecule,. Fig. 2 is a progression of intervent dilution columns that starts with a gentle system useful for removing loosely bound proteins from ribosomes and ends with a powerful system capable of separating all of the proteins from the rrnas. In this figure, proteins (8olid line) elute at the excluded front and early within the sieving range [that region of the chromatogram between the excluded front and the column liquid volume (2)1, and RNA-containing compounds (dashed line) elute late in the sieving range with the intervent front. A mild intervent dilution system that may be adapted for washing ribosomes is shown in chromatogram A, Fig. 2. In this experiment, the ribosome sample is dissolved in a Mg++-containing buffer at 1.0 M KC1, and the column is equilibrated in the same buffer at 0.30 M KC1. A small peak of weakly bound ribosomal proteins elutes at the excluded volume, and ribosomal particles, principally 30S and 50S subunits, elute with the intervent front. Whether one obtains 70S ribosomes or 30S and 50S subunits depends upon the exact conditions of chromatography. Chromatogram E, Fig. 2, shows a powerful system that separates virtually all of the proteins from the RNA. The sample is dissolved in sufficient intervents to completely disaggregate the ribosome. The column is equilibrated with a solution that maintains the solubility of the ribosomal proteins while providing sufficient intervents to permit the proteins to move along the column at a higher rate than the front produced by the sample intervents. The column is developed with the sample solvent with the KCI concentration increased to 1.2 M. Proteins elute at the excluded volume in a sharp peak. The small peak (dashed line) at tube 15 contains RNA, predominantly of low molecular weight. High-molecular-weight rrnas cannot be eluted from the column after the intervent dilution process has removed all the protein. Apparently, the RNA is left unfolded and extended over the surface of the ion-exchange gel. By the time intervents in the eluant are raised to a level sufficient to disrupt the RNAresin interaction, the RNA becomes insoluble. A summary of the results of about 25 experiments shows that no combinations of KCI (0-2 M), urea (0-7 M), MgCl2 (04.5 M), sucrose (0-0.5 M), sodium citrate (0-0.5 M), and guanidine-hcl (0-3 M) are able to elute the bound RNA. Fortunately, most of the large RNAs can be recovered by methods described later.

4 Proc. Nat. Acad. Sci. USA 70 (1978) In a system as complex as the ribosome, it is difficult to establish the complete separation of proteins from RNAs. The characteristic UV spectra with a peak at 277 nm and a minimum at 250 nm of the protein fractions from many columns run under various circumstances indicate that the protein peak is virtually free of nucleic acids even though the Ame:A ratio is 1.44, which is somewhat lower than for cellular proteins. Protein could not be detected in the RNA fractions by standard colorimetric methods. Further, it is unlikely that significant protein remains in the RNA fractions since remaining protein would have to be insoluble in the equilibration buffer, bound to the ion-exchange resin more strongly than the eluted RNAs, and/or bound to the high-molecularweight RNA in 7 M urea and 1.2 M KCl. Chromatograms B, C, and D, Fig. 2, are intermediates between the gentle system in A and the powerful system in E and show some important properties of intervent dilution chromatography. In chromatogram B, the sample is dissolved in 7 M urea and the column is eluted as in experiment E, but the column is equilibrated without urea, as in A. Although the intervent concentration of the sample is sufficiently high to permit the ribosomal components to behave independently, the solution used to equilibrate the column would not maintain the solubility of the ribosomal proteins. As the proteins are projected ahead of the sample intervents, they precipitate on the column. This protein precipitation prevents separation of the proteins from the rrnas by the intervent dilution process. In the presence of ribosomal proteins, most of the ribosomal RNAs elute from the column with 1.2 M KCl in 7 M urea, hence the large peak of material absorbing at 260 nm. In chromatogram C the column is equilibrated with 0.4 M KCl-4 M urea as in E, but the sample solution, which is the same as in A, does not favor dissociation. Under these conditions, a large portion of the ribosomal proteins elutes at the excluded volume. Although column C is eluted with the same eluant as in column A, the remaining RNA-containing particles could not be eluted with 1 M - KCl-0.01 M MgCI2 in 0.1 M imidazole HCl, hence the small peak absorbing at 260 nm, probably containing 5S RNA and RNA fragments. The sharpness of the peak of protein at the excluded front depends upon the effectiveness of the sample solvent and the column eluant in maintaining the independent behavior of the sample components. A comparison of chromatograms D and E, Fig. 2, shows the improvement obtained by increasing the KCl concentration in the column eluant from 0.8 to 1.2 M. Even though the area under the protein peak is the same in both columns, the additional KCl in the eluant significantly sharpens the peak. In experiments where the sample volume is relatively small (less than 2-4% of column volume), the intervent front may be established or highly modified by the composition of the column eluant. Most of the time, however, it is preferable to develop the intervent dilution column with the same solution used to dissolve the sample. An important aspect of intervent dilution chromatography that is not systematically shown in Fig. 2 is the effect of changing the ionic strength of column equilibration. To simplify the presentation of Fig. 2, conditions were chosen to cause the proteins and RNAs to behave as classes. The columns were equilibrated at the highest ionic strength that would maintain the RNA-containing components moving Intervent Dilution Chromatography FRACTION NUMBER FIG. 3. Separation and recovery of ribosomnal proteins and RNAs. 0.5 ml of unwashed E. coli ribosomes, 260 A260 units/ml, in 1.2 M KC1-7 M urea-0.04 M MgC M imidazole-hcl (ph 7.0) were chromatographed on a DEAE-Sephadex A-25 column equilibrated in 0.4 M KCI-4 M urea-0.1 M imidazole- HC1 (ph 7.0) at 250. The column was initially eluted with the sample solvent. At the point marked by arrow I, the column eluant was changed to 1 M KC1. At arrow II, 1 M Tris base in 0.5 M KOH was applied to the column. The solid line is absorbance at 254 nm, and the dashed line is ph. slower than the intervent front. If the column is equilibrated at lower ionic strength, only the more positively charged proteins elute at the excluded volume. The remaining proteins elute at the leading edge of the intervent front. If the ionic strength of equilibration is increased, low-molecularweight RNAs also elute in the sieving range with the proteins. It is well known that Mg++ is necessary for maintenance of ribosome integrity; thus it was unexpected to find Mg++ required for the complete dissociation of protein from rrna. If Mg++ is omitted from the denaturing solution used to dissolve the ribosomes in experiment E, Fig. 2, the eluted protein is smeared throughout the sieving range. The addition of EDTA to the sample results in the liberation of only a small fraction of the ribosomal proteins. Ideally for the complete separation of RNA from proteins, the MIg++ concentration of the sample should be between 0.02 and 0.04 M when the ribosome concentration is between 200 and 400 A260 units/ml. The problem of recovering the high-molecular-weight RNA from the ion-exchange gel was solved in the experiment shown in Fig. 3. In this experiment, proteins are separated from the RNA by conditions similar to those of experiment E in Fig. 2. Although the solution used to dissolve the ribosomes and to elute the column is excellent for minimizing the interactions between ribosomal proteins and rrna, the proteinfree, high-molecular-weight RNAs are relatively insoluble in this solution and cannot be eluted from the column using it. Therefore, after the intervent dilution process has separated the proteins and RNA, the urea-containing eluant is replaced with 1 M KCl. After the column has equilibrated with 1 M KCl, a solution of 1 M Tris base in 0.5 M KOH is applied to the column to set up a series of complex actions that result in the recovery of most of the high-molecular-weight RNA. As the strongly basic solution enters the top of the column, the weakly basic ion-exchange gel is neutralized. After the resin is discharged, the RNA is free to migrate, and the sieving properties of the gel project the RNA ahead of the basic front. The high-molecular-weight RNA at the very top of the column is probably hydrolyzed in a few places as it outruns

5 2428 Biochemistry: Kirkegaard and Agee the base; nevertheless, this "nicked" RNA establishes a cascade of RNA that effectively elutes the intact RNA that is further down the column. The 1 M Tris in the basic eluant establishes a ph gradient ahead of the KOH front. Once the ph gradient is formed, most of the bound high-molecularweight rrna elutes from the resin before the ph becomes excessively high, thus reducing RNA hydrolysis. From acrylamide gel analysis, the leading edge of the first RNA peak contains predominantly low-molecular-weight RNAs, probably 5S RNA, trna, and rrna fragments. The trailing edge of the first peak, as well as the large peak resulting from the alkaline treatment, consists of -mainly high-molecularweight RNAs of equivalent size to those isolated by a phenol extraction of the ribosomes. Some resolution of the highmolecular-weight rrnas occurs within the large peak. Fractions in the leading edge are rich in the smaller highmolecular-weight component. Intervent dilution chromatography is an important mode of sievorptive chromatography that rapidly separates strongly interacting macromolecules. It therefore complements ion filtration chromatography (2), another mode of sievorptive chromatography that rapidly separates similar molecules under conditions of maximal resolution. The strength, flexibility, and gentleness of the new procedures promise to make sievorptive chromatography an important method for enzyme purification. Because of the inherent advantages of sievorptive chromatography, enzymes may be purified in a small fraction of the time previously required. Proc. Nat. Acad. Sci. USA 70 (1973) We thank Professors Nelson J. Leonard and John M. Clark, Jr. for supporting this work by providing laboratory facilities. C.C.A. is a NIH Postdoctoral Research Fellow (GM-51047). This work was supported by Research Grants (USPHS-GM awarded to Nelson J. Leonard and USPHS-GM awarded to John M. Clark, Jr.) from the National Institutes of Health, U.S. Public Health Service. 1. Ackers, G. K. (1970) Advan. Protein Chem. 24, Kirkegaard, L. H., Johnson, T. J. A. & Bock, R. M. (1972) Anal. Biochem. 50, Traub, P., Mizushima S., Lowry, C. (1971) Methods Enzymol. XX, V. & Nomura, M. 4. Kurland, C. G., Hardy, S. J. S. & Mora, G. (1971) Methods Enzymol. XX, Fogel, S. & Sypherd, P. S. (1968) J. Bacteriol. 96, Tanford, C. (1968) Advan. Protein Chem. 23, Tanford, C. (1970) Advan. Protein Chem. 24, Morris, C. J. 0. R. & Morris, P. (1963) in Separation Methods in Biochemistry (Interscience Publishers, New York), pp Bethune, J L. & Kegeles, G. (1961) J. Phys. Chem. 65, 10. Cann, J. R. (1970) in Interacting Macromolecules (Academic Press, New York), pp Clark, J. M., Jr., Chang, A. Y., Spiegelman, S. & Reichmann, M. E. (1965) Proc. Nat. Acad. Sci. USA 54, Salas, M., Smith, M. A., Stanley, Jr., W. M., Wahba, A. J. & Ochoa, S. (1965) J. Biol. Chem. 240,