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Affinity electrophoresis
ANALYTICAL
BIOCHEMISTRY
112, l-8
(1981)
REVIEW Affinity
Electrophoresis
VACLAV Institute
of Molecular
HOi%EJSf
Genetics, Czechoslovak Academy 142 20 Prague 4, Czechoslovakia Received
November
of Sciences,
VideAskri
1083,
20, 1980
the interaction, and sometimes precipitation occurs. Some modifications of this method are analogous to rocket immunoelectrophoresis, fused-rocket electrophoresis, classical Grabar immunoelectrophoresis, and crossed immunoelectrophoresis, as reviewed by Bog-Hansen (5,7). The chief advantage of the use of lectins, as compared with antibodies, for the identification and quantitation of glycoproteins by immunoelectrophoresis-like affinity electrophoresis (7) is that the interaction, is not affected by denaturation of the glycoproteins. Of course, this approach is not limited only to the study of lectin-glycoprotein interactions; the binding of staphylococcal protein A to immunoglobulins (8) and interactions of a glial protein with small group-specific ligands (9) were also studied in this way. The ancestor of the present agarose-gel affinity electrophoresis was the interesting technique of “crossing diagrams” devised by Nakamura and his colleagues (lo- 12). This technique, reviewed in (12), was used to demonstrate, e.g., the interaction of serum glycoproteins with crude Con A (11) or trypsin with trypsin inhibitors (12). The second approach to affinity electrophoresis is based on electrophoresis in polyacrylamide affinity gels containing immobilized ligands (2,14- 16). This approach is more closely related to the principles of affinity chromatography, and also similar problems are encountered, i.e., methodology of ligand immobilization, specificity
The term “affinity electrophoresis” which was suggested in analogy to affinity chromatography (l-3) should, in principle, denote all techniques in which charged molecules (A) move in an electric field within a medium containing molecules B capable of interaction with A resulting in AB complex formation. Such a definition would also include some immunoelectrophoretic techniques (i.e., those in which antibody is incorporated in the gel) in which the interaction between A (antigens) and B (antibodies) leads to insoluble precipitate formation. Despite this principal similarity between immunoelectrophoresis and “true” affinity electrophoresis, the former techniques will not be discussed in this article. Short reviews on affinity electrophoresis have been published recently (4-6). TWO APPROACHES-THE USE OF AGAROSE AND POLYACRYLAMIDE GELS
At present, there are basically two approaches to affinity electrophoresis. One approach has been developed mainly by T. C. Bog-Hansen and his colleagues and is closely related to immunoelectrophoretic techniques. These authors use certain ligandbinding proteins (mostly lectins) as analogs of antibodies incorporated (either free or covalently bound) into agarose gels. When glycoproteins capable of interaction with these lectins are electrophoresed in such an “affinity gel,” they are retarded due to 1
0003-2697/81/050001-08$02.00/O Copyright 0 1981 by Academic Press. Inc. All rights of reproduction in any form reserved.
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VkLAV
of interaction, the use of suitable spacers, etc. However, there is in fact no sharp distinction between these two approaches: the agarose gel methods using covalently immobilized ligands are also analogous to affinity chromatography. Both these variants of affinity electrophoresis possess certain features which are advantageous for studying and solving different problems: the immunoelectrophoresis-like modification using agarose gels is especially useful in investigating the macromoleculemacromolecule interactions, whereas the polyacrylamide gel modification can be used to study various aspects of protein-small ligand interactions. The agarose gel variant is usually combined with crossed immunoelectrophoresis; this requires preparation of good antisera containing enough precipitating antibodies. On the other hand, in the polyacrylamide gel modification the higher resolving power allows the use of direct detection of separated components. PREPARATION
OF AFFINITY
GELS
So far four methods of affinity gel preparation have been used: (i) Incorporation of soluble lectins (or their ligand-binding proteins) (5 -8,17-24) into agarose gels. As stated above, this
method closely resembles immunoelectrophoresis. The pH of the buffer used and electroendoosmosis of agarose must be chosen so that the lectin incorporated into the agarose gel is immobile or nearly immobile. The characteristic feature of this method is that the soluble lectin-glycoprotein complexes possess a nonzero mobility-the mobility is always lower than that of free glycoprotein, however. (ii) Incorporation of agarose beads substituted with a protein (or other ligand) into agarose gel. In the first step a protein, such
as lectin (1,18,25,26), antibody (29-31), or hydrocarbon residue, Cibacron Blue F3GA
glycoprotein (27,28), a small ligand (e.g., chelating ligand, or (9,32), is coupled to
HOl?EJSf
CNBr-activated agarose beads. Such a modified agarose gel (which does not melt any more due to crosslinking during CNBr activation) is then added to melted agarose and the affinity gel, i.e., the suspension of “affinity beads,” is poured. Such affinity gels are usually used as first-dimension gels or intermediate gel strips in crossed immunoaffinoelectrophoresis. (iii) Incorporation of a macromolecular derivative of a ligand into polyacrylamide
gel. In this method, a macromolecular substance of sufficiently high molecular weight and carrying a ligand is simply dissolved in the polymerization mixture used normally for the preparation of polyacrylamide gels; after completion of polymerization reaction, the macromolecules are physically entrapped within the polyacrylamide gel network and the ligand is thus effectively immobilized. At present this is practically the only method used for preparation of polyacrylamide affinity gels. It has been originally introduced by Siepmann and Stegemann (14); in this case and in a number of other studies unmodified natural polysaccharides were used as the macromolecules to be entrapped within the gel network (14,15,33-4051). High-molecularweight dextrans can be substituted with a ligand after suitable “activation” reaction (e.g., periodate oxidation) to yield macromolecular-soluble derivatives of the ligand. This approach has been adopted in preparation of affinity gels containing immobilized sugars (51), p-aminobenzamidine (an inhibitor of trypsin) (52), or the dye Cibacron Blue F3G specifically binding to some proteins (53). An alternative way in immobilization of ligands via macromolecular carrier molecules represents the use of synthetic copolymers of acrylamide and unsaturated derivatives of the ligands. Copolymerization of these two components yields linear, soluble copolymers in which molecular weight and ligand content can be controlled to a great extent under the conditions used
AFFINITY
(41). Affinity gels prepared in this way contained immobilized glycosidically bound carbohydrates (16,42-48) or AMP (49,SO). Similarly, synthetic copolymers can be prepared, carrying reactive groups which can be subsequently reacted with a suitable ligand. Thus, hydroxypropyl methacrylamide copolymers containing 4-nitrophenyl ester groups were used as carriers of paminobenzamidine or AMP (52,54). All these methods of preparation of macromolecular-soluble derivatives of ligands seem to be generally applicablepractically any ligand can be covalently attached to dextran or some other macromolecular carrier, or an unsaturated derivative of a ligand can be synthesized and copolymerized with acrylamide. However, these methods have also some drawbacks: (a) Macromolecules of very high molecular weight should be used (depending also on porosity of the polyacrylamide gel); especially in the case of synthetic polymers, immobilization may not be complete. (b) Synthesis of an unsaturated derivative may be laborious, whereas derivatization of a dextran may lead to insoluble products (e.g., during CNBr activation or when hydrophobic ligands are attached). In any case, preparation of a soluble macromolecular derivative is usually more difficult than derivatization of an agarose gel. (c) Microdistribution of the ligand in affinity gels prepared in this way is nonhomogeneous (islet like), which may seriously affect the value of the effective concentration of immobilized ligand. (iv) Direct preparation amide gels with covalently
3
ELECTROPHORESIS
of polyaqylbound ligands.
This type of affinity gels was prepared by addition of ally1 glycosides to the polymerization mixture normally used for polyacrylamide gel electrophoresis. After polymerization reaction, the gel rods must be removed from glass tubes, washed free from the unreacted ally1 glycoside, equilibrated with the gel buffer, and inserted back into glass tubes (2,3). This method allows com-
plete and reliable immobilization of the ligand. Besides, very high concentrations of immobilized ligand can be achieved and its distribution throughout the gel is absolutely homogeneous. However, from the experimental point of view, the processing of affinity gels (washing, equilibration, insertion back to the tubes) may be too laborious and sometimes cumbersome. For these reasons, the method was abandoned when the above-mentioned methods (i.e., immobilization via macromolecular-soluble carriers) became available. Nevertheless, this method deserves attention; after some technical improvements are made, it might still find application for some special purposes. In exceptional cases, a specific interaction between the gel matrix itself and a protein was observed. Thus, some Dglucose-specific lectins have an anomalously low mobility on starch gel, which increases upon addition of free D-glucose and related sugars (56). QUALITATIVE
APPLICATIONS
The simplest application of affinity electrophoresis is to identify the component(s) in a complex mixture capable of binding the ligand immobilized in the affinity gel. Such a ligand-binding component is readily identified by comparison of the pattern observed on control (noninteracting) and affinity gels. The ligand-binding component is more or less retarded on affinity gel as a result of interaction; in the case of strong interaction or at high concentration of immobilized ligand, it is captured at the very starting point. The specificity of interaction can be checked by using proper control gels or by adding a free ligand to the affinity gel; free ligand in sufficiently high concentration should abolish the retardation. When the ligand-binding component is heterogeneous with respect to interaction with the immobilized ligand, the heterogeneity is manifested by the presence on affinity gels of several bands of different retarda-
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tion (7,17,18,24,42,44,45). Thus, affinity electrophoresis was used to identify lectinbinding glycoproteins (1,6,7,17-24,26) in agarose gel, lectins (2,3,17,25,28) and phosphorylases, amylases, and pectinases interacting with natural polysaccharides (14,15, 33-36,40) in polyacrylamide gel, and to demonstrate the heterogeneity of glycoproteins (5,17,18,24) and lectins (42,44,45). Moreover, affinity electrophoresis can be used for monitoring the course and results of modification reactions of proteins. If, for example, a modification reaction results in a 90% loss of the activity of a protein, it may be difficult to decide whether 90% of the molecules are completely inactivated and the remaining 10% are fully active, whether all molecules are modified in such a way that they retain only 10% of activity, or whether a complex mixture of reaction products is formed. Affinity electrophoresis enables to decide readily between these alternatives and, in addition, it provides quantitative information on the binding activities of the components of the reaction mixture (4). So far, the results of demetalization, sulfenylation, and maleylation of lectins (42) and of various modifications of the Cu-binding site of galactose oxidase (48) have been monitored by affinity electrophoresis. The method was also used to demonstrate the lack of sugar-binding activity in isolated lectin subunits (42) and in native insulin (57). Furthermore, affinity electrophoresis can be used to predict the results of affinity chromatography and to optimize the conditions of affinity interaction (1,24,29,30). As affinity electrophoresis can be performed rapidly with small amounts of gels and using simultaneously many samples, it can spare considerable time and material in pretesting the affinity chromatography systems. However, a recent study (9) (in which binding of the glial fibrillary acidic protein to affinity gels prepared of alkyl-agaroses, iminodiacetic acid-agarose, and Cibacron Blue-agarose was tested) has indicated
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that there may not be a simple correlation between the results of affinity electrophoresis and affinity chromatography. In recent years a technique of electrophoretic elution of proteins adsorbed on affinity adsorbents (in chromatographic arrangement) has been developed (58-60) and some authors have considered it a modification of affinity electrophoresis. QUANTITATIVE
APPLICATIONS
It is well known that affinity chromatography is a valuable tool not only for preparative purposes but also for quantitive evaluation of the strength of interaction between the protein and the ligand (for review on quantitative applications of affinity chromatography see (61)). Affinity electrophoresis can also be used for similar purposes. The mobility of the protein decreases with increasing concentration of the immobilized ligand and this concentration dependence can be used for a simple evaluation of apparent dissociation constant (Ki) of the complex protein-immobilized ligand. Such a quantitative application of affinity electrophoresis (although the authors did not call their method so) was described nearly simultaneously by Gerbrandy and Doorgeest (36) and Takeo and Nakamura (15). Later on, it was demonstrated that incorporation of increasing concentrations of free ligand (c) into the affinity gels containing constant concentration of immobilized ligand ( ci) results in increased mobility of the protein (due to competition between free and immobilized ligands for the ligand binding sites); at a sufficiently high c the mobility approaches that on the control (noninteracting) gels. This dependence of mobility on c can be again used for a simple evaluation of dissociation constants (K) of the protein-free ligand complexes; Ki is obtained as a “by-product” (16). These principles were used for quantitative evaluation of the strength of interaction of enzymes with their macromolecular sub-
AFFINITY
ELECTROPHORESIS
strates (15,36,62), glycoproteins with lectins (6), protein A with immunoglobulins and their fragments (8), lectins with immobilized and free sugars (16,42-47,51), enzymes and other proteins with Blue Dextran (i.e., immobilized Cibacron Blue) (53), or with immobilized AMP (49,50,54), trypsin with immobilized p-aminobenzamidine (52), galactose oxidase with free and immobilized sugars (48), and carbohydrate-specific myeloma immunoglobulins with immobilized polysaccharides and free sugars (37-39). These parameters ( Ki and K ) were followed also as functions of the pH (46), ionic strength (47), and spacer length (51,52,54,55). The use of affinity electrophoresis for estimation of dissociation constants of the protein-ligand complexes possesses a number of advantages: the method is economic, rapid, and simple to perform; the sample can be a crude mixture; dissociation constants can be determined simultaneously for all electrophoretically different forms of the ligand binding protein (isoenzymes, isolectins) and for estimation of K the value of Ci need not be known; the method is especially suitable for measuring very weak interactions (dissociation constants up to 10-l to 10” M). However, there are also some limitations: the protein under study must have good electrophoretic properties in the particular system (i.e., relatively high mobility and sharp band); the K values are difficult to measure for charged free ligands; the number of the ligand-binding sites in the protein molecule cannot be determined; the interpretation of Kj value is difficult (see the following paragraph). THEORY
OF THE METHOD
While for qualitative applications of affinity electrophoresis a purely empirical basis is quite sufficient, the quantitative uses of the method are crucially dependent on the theoretical background. The simple equations used originally for evaluation of Ki (15,36) and K (16) are valid exactly
5
only under several assumptions: (a) The protein molecule contains a single ligandbinding site, (b) immobilization of the ligand is complete, (c) all molecules of immobilized ligand are accessible to interaction with the protein (i.e., effective ci is identical with total ci), (d) mobility of the protein-free ligand complex is identical with that of free protein, (e) concentration of the protein in the migrating zone is much lower than concentration of free and immobilized ligand, (f) complex formation is a very rapid reaction, (g) microdistribution of immobilized ligand is homogeneous. Most of these assumptions do not hold true under the actual experimental conditions, and this may more or less seriously affect the Ki and K values obtained. The effects of violation of the above assumptions on the Kj and K values have been theoretically dealt with so far only in two studies (63,64) in which several equations were derived, describing the effects of various factors on the results of affinity electrophoresis. The results from these studies can be summarized as follows: (a) Dissociation constants Ki and K can be estimated even if immobilization of the ligand is not complete and if the mobility of the protein-free ligand complex differs from that of free protein. Similar conclusions were also reached by Bog-Hansen and Takeo (6). (b) The adverse effects of the high protein concentration on Ki estimation can be prevented using a proper plot. (c) The kinetics of protein-ligand complex formation has in most cases negligible effects on the results of affinity electrophoresis; the method might be principally used to study the kinetics of very slow complexformation reactions. (d) The value of K can be determined also for proteins with multiple ligand-binding sites (n) in the molecule; the Kiln value is obtained instead of Ki in such cases. (e) The value of the effective concentration of immobilized ligand (ci) can be determined from the dependence of mobility on protein concentration at constant Ci.
6
VACLAV HOItEJSf
It should be noted that some of the limitations of the method (especially problems connected with estimation of effective ci, the effects of multiple binding sites) are also important in quantitative affinity chromatography (65,66). CONCLUSIONS AND FUTURE PROSPECTS
Although affinity electrophoresis has been used in a relatively large number of studies, the method is not yet “mature,” as some important aspects are still to be explored. (a) It is necessary to test thoroughly some of the theoretical conclusions (63,64), first of all, to determine the effective concentration of immobilized ligand in affinity gels prepared by different methods and thus to estimate “true” Ki. Preliminary results indicate that, at least in the case of affinity gels prepared by incorporation of macromolecular-soluble carriers (e.g., O-glycosyl polyacrylamide copolymers), the effective ci is much lower than total ci (67) and thus, the true Ki is much lower than the apparent constants which have been estimated earlier (16). Similarly, a thorough study of the effect of multivalency of the protein and microdistribution of immobilized ligand would be useful. (b) The present techniques of preparation of affinity polyacrylamide gels are not quite satisfactory. It would be very helpful to work out simple and general methods comparable to the preparation of affinity carriers in aflinity chromatography. Several ways can be suggested: either a ligand could be bound to a beaded gel matrix (agarose, dextran, or other gels) by well-established simple methods (CNBr or periodate activation, etc.) and such particles could be incorporated directly into polyacrylamide gels. Such nonhomogeneous affinity gels (analogous to those used in agarose-gel affinity electrophoresis (1,9,18,29)) would be clearly applicable rather for qualitative
purposes. Alternatively, agarose or some other gel could be substituted by a ligand in such a way that the gel could be subsequently solubilized (either chemically or melted) and incorporated into polyacrylamide gel network. This way would overcome difficulties in preparation of soluble macromolecular derivatives and it would retain the simplicity of affinity gels preparation. Both these methods have appeared very promising in our preliminary experiments (68). (c) A serious drawback of affinity electrophoresis is its limitation in estimating the K for charged free ligands. A technique which would solve this problem would be extremely valuable. (d) The principle of affinity electrophoresis might be extended also to other electromigration methods, such as isoelectric focusing and isotachophoresis. Indeed, affinity isoelectric focusing was introduced recently as a method for the detection of ligand-binding proteins and for similar qualitative purposes (69). On the other hand, electrophoretic equivalents of covalent-affinity chromatography (70) and possibly also of ion-exchange chromatography might be useful. Electrophoretic methods analogous to hydrophobic chromatography have already been introduced (9,32,55). For some special purposes also preparative modifications of affinity electrophoresis might be considered; an easy separation of electrophoretic forms (isoenzymes) differing slightly in affinity toward immobilized ligand would be particularly advantageous. (e) So far, affinity electrophoresis has only been used to study the interactions between proteins and small or macromolecular ligands or between two proteins. It might be useful to develop procedures allowing its application also in studies of the interactions between nucleic acids and proteins, between two kinds of nucleic acid molecules and in other cases of interacting molecules. In conclusion, affinity electrophoresis has
AFFINITY
made considerable progress in recent years but much remains to be done before all potential applications of this method are exploited. ACKNOWLEDGMENT I am indebted to Dr. Marie Ticha for careful critical reading of the manuscript and many valuable suggestions.
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66. Kasai, K., and Ishii, S. (1978)5. Biochem. (Tokyo) 84, 1051-1060. 67. HoiejSi, V., and Ticha, M., in preparation. 68. HoiejSi, V., and Ticha, M., in preparation. 69. HorejU, V., and Ticha, M. And. Biochem. submitted for publication. 70. Voss, H. F., Ashani, Y., and Wilson, I. B. (1974) in Methods in Enzymology (Jakoby, W. B., ed.), Vol. 34, pp. 581-590, Academic Press, New York.
BIOCHEMISTRY
112, l-8
(1981)
REVIEW Affinity
Electrophoresis
VACLAV Institute
of Molecular
HOi%EJSf
Genetics, Czechoslovak Academy 142 20 Prague 4, Czechoslovakia Received
November
of Sciences,
VideAskri
1083,
20, 1980
the interaction, and sometimes precipitation occurs. Some modifications of this method are analogous to rocket immunoelectrophoresis, fused-rocket electrophoresis, classical Grabar immunoelectrophoresis, and crossed immunoelectrophoresis, as reviewed by Bog-Hansen (5,7). The chief advantage of the use of lectins, as compared with antibodies, for the identification and quantitation of glycoproteins by immunoelectrophoresis-like affinity electrophoresis (7) is that the interaction, is not affected by denaturation of the glycoproteins. Of course, this approach is not limited only to the study of lectin-glycoprotein interactions; the binding of staphylococcal protein A to immunoglobulins (8) and interactions of a glial protein with small group-specific ligands (9) were also studied in this way. The ancestor of the present agarose-gel affinity electrophoresis was the interesting technique of “crossing diagrams” devised by Nakamura and his colleagues (lo- 12). This technique, reviewed in (12), was used to demonstrate, e.g., the interaction of serum glycoproteins with crude Con A (11) or trypsin with trypsin inhibitors (12). The second approach to affinity electrophoresis is based on electrophoresis in polyacrylamide affinity gels containing immobilized ligands (2,14- 16). This approach is more closely related to the principles of affinity chromatography, and also similar problems are encountered, i.e., methodology of ligand immobilization, specificity
The term “affinity electrophoresis” which was suggested in analogy to affinity chromatography (l-3) should, in principle, denote all techniques in which charged molecules (A) move in an electric field within a medium containing molecules B capable of interaction with A resulting in AB complex formation. Such a definition would also include some immunoelectrophoretic techniques (i.e., those in which antibody is incorporated in the gel) in which the interaction between A (antigens) and B (antibodies) leads to insoluble precipitate formation. Despite this principal similarity between immunoelectrophoresis and “true” affinity electrophoresis, the former techniques will not be discussed in this article. Short reviews on affinity electrophoresis have been published recently (4-6). TWO APPROACHES-THE USE OF AGAROSE AND POLYACRYLAMIDE GELS
At present, there are basically two approaches to affinity electrophoresis. One approach has been developed mainly by T. C. Bog-Hansen and his colleagues and is closely related to immunoelectrophoretic techniques. These authors use certain ligandbinding proteins (mostly lectins) as analogs of antibodies incorporated (either free or covalently bound) into agarose gels. When glycoproteins capable of interaction with these lectins are electrophoresed in such an “affinity gel,” they are retarded due to 1
0003-2697/81/050001-08$02.00/O Copyright 0 1981 by Academic Press. Inc. All rights of reproduction in any form reserved.
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of interaction, the use of suitable spacers, etc. However, there is in fact no sharp distinction between these two approaches: the agarose gel methods using covalently immobilized ligands are also analogous to affinity chromatography. Both these variants of affinity electrophoresis possess certain features which are advantageous for studying and solving different problems: the immunoelectrophoresis-like modification using agarose gels is especially useful in investigating the macromoleculemacromolecule interactions, whereas the polyacrylamide gel modification can be used to study various aspects of protein-small ligand interactions. The agarose gel variant is usually combined with crossed immunoelectrophoresis; this requires preparation of good antisera containing enough precipitating antibodies. On the other hand, in the polyacrylamide gel modification the higher resolving power allows the use of direct detection of separated components. PREPARATION
OF AFFINITY
GELS
So far four methods of affinity gel preparation have been used: (i) Incorporation of soluble lectins (or their ligand-binding proteins) (5 -8,17-24) into agarose gels. As stated above, this
method closely resembles immunoelectrophoresis. The pH of the buffer used and electroendoosmosis of agarose must be chosen so that the lectin incorporated into the agarose gel is immobile or nearly immobile. The characteristic feature of this method is that the soluble lectin-glycoprotein complexes possess a nonzero mobility-the mobility is always lower than that of free glycoprotein, however. (ii) Incorporation of agarose beads substituted with a protein (or other ligand) into agarose gel. In the first step a protein, such
as lectin (1,18,25,26), antibody (29-31), or hydrocarbon residue, Cibacron Blue F3GA
glycoprotein (27,28), a small ligand (e.g., chelating ligand, or (9,32), is coupled to
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CNBr-activated agarose beads. Such a modified agarose gel (which does not melt any more due to crosslinking during CNBr activation) is then added to melted agarose and the affinity gel, i.e., the suspension of “affinity beads,” is poured. Such affinity gels are usually used as first-dimension gels or intermediate gel strips in crossed immunoaffinoelectrophoresis. (iii) Incorporation of a macromolecular derivative of a ligand into polyacrylamide
gel. In this method, a macromolecular substance of sufficiently high molecular weight and carrying a ligand is simply dissolved in the polymerization mixture used normally for the preparation of polyacrylamide gels; after completion of polymerization reaction, the macromolecules are physically entrapped within the polyacrylamide gel network and the ligand is thus effectively immobilized. At present this is practically the only method used for preparation of polyacrylamide affinity gels. It has been originally introduced by Siepmann and Stegemann (14); in this case and in a number of other studies unmodified natural polysaccharides were used as the macromolecules to be entrapped within the gel network (14,15,33-4051). High-molecularweight dextrans can be substituted with a ligand after suitable “activation” reaction (e.g., periodate oxidation) to yield macromolecular-soluble derivatives of the ligand. This approach has been adopted in preparation of affinity gels containing immobilized sugars (51), p-aminobenzamidine (an inhibitor of trypsin) (52), or the dye Cibacron Blue F3G specifically binding to some proteins (53). An alternative way in immobilization of ligands via macromolecular carrier molecules represents the use of synthetic copolymers of acrylamide and unsaturated derivatives of the ligands. Copolymerization of these two components yields linear, soluble copolymers in which molecular weight and ligand content can be controlled to a great extent under the conditions used
AFFINITY
(41). Affinity gels prepared in this way contained immobilized glycosidically bound carbohydrates (16,42-48) or AMP (49,SO). Similarly, synthetic copolymers can be prepared, carrying reactive groups which can be subsequently reacted with a suitable ligand. Thus, hydroxypropyl methacrylamide copolymers containing 4-nitrophenyl ester groups were used as carriers of paminobenzamidine or AMP (52,54). All these methods of preparation of macromolecular-soluble derivatives of ligands seem to be generally applicablepractically any ligand can be covalently attached to dextran or some other macromolecular carrier, or an unsaturated derivative of a ligand can be synthesized and copolymerized with acrylamide. However, these methods have also some drawbacks: (a) Macromolecules of very high molecular weight should be used (depending also on porosity of the polyacrylamide gel); especially in the case of synthetic polymers, immobilization may not be complete. (b) Synthesis of an unsaturated derivative may be laborious, whereas derivatization of a dextran may lead to insoluble products (e.g., during CNBr activation or when hydrophobic ligands are attached). In any case, preparation of a soluble macromolecular derivative is usually more difficult than derivatization of an agarose gel. (c) Microdistribution of the ligand in affinity gels prepared in this way is nonhomogeneous (islet like), which may seriously affect the value of the effective concentration of immobilized ligand. (iv) Direct preparation amide gels with covalently
3
ELECTROPHORESIS
of polyaqylbound ligands.
This type of affinity gels was prepared by addition of ally1 glycosides to the polymerization mixture normally used for polyacrylamide gel electrophoresis. After polymerization reaction, the gel rods must be removed from glass tubes, washed free from the unreacted ally1 glycoside, equilibrated with the gel buffer, and inserted back into glass tubes (2,3). This method allows com-
plete and reliable immobilization of the ligand. Besides, very high concentrations of immobilized ligand can be achieved and its distribution throughout the gel is absolutely homogeneous. However, from the experimental point of view, the processing of affinity gels (washing, equilibration, insertion back to the tubes) may be too laborious and sometimes cumbersome. For these reasons, the method was abandoned when the above-mentioned methods (i.e., immobilization via macromolecular-soluble carriers) became available. Nevertheless, this method deserves attention; after some technical improvements are made, it might still find application for some special purposes. In exceptional cases, a specific interaction between the gel matrix itself and a protein was observed. Thus, some Dglucose-specific lectins have an anomalously low mobility on starch gel, which increases upon addition of free D-glucose and related sugars (56). QUALITATIVE
APPLICATIONS
The simplest application of affinity electrophoresis is to identify the component(s) in a complex mixture capable of binding the ligand immobilized in the affinity gel. Such a ligand-binding component is readily identified by comparison of the pattern observed on control (noninteracting) and affinity gels. The ligand-binding component is more or less retarded on affinity gel as a result of interaction; in the case of strong interaction or at high concentration of immobilized ligand, it is captured at the very starting point. The specificity of interaction can be checked by using proper control gels or by adding a free ligand to the affinity gel; free ligand in sufficiently high concentration should abolish the retardation. When the ligand-binding component is heterogeneous with respect to interaction with the immobilized ligand, the heterogeneity is manifested by the presence on affinity gels of several bands of different retarda-
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tion (7,17,18,24,42,44,45). Thus, affinity electrophoresis was used to identify lectinbinding glycoproteins (1,6,7,17-24,26) in agarose gel, lectins (2,3,17,25,28) and phosphorylases, amylases, and pectinases interacting with natural polysaccharides (14,15, 33-36,40) in polyacrylamide gel, and to demonstrate the heterogeneity of glycoproteins (5,17,18,24) and lectins (42,44,45). Moreover, affinity electrophoresis can be used for monitoring the course and results of modification reactions of proteins. If, for example, a modification reaction results in a 90% loss of the activity of a protein, it may be difficult to decide whether 90% of the molecules are completely inactivated and the remaining 10% are fully active, whether all molecules are modified in such a way that they retain only 10% of activity, or whether a complex mixture of reaction products is formed. Affinity electrophoresis enables to decide readily between these alternatives and, in addition, it provides quantitative information on the binding activities of the components of the reaction mixture (4). So far, the results of demetalization, sulfenylation, and maleylation of lectins (42) and of various modifications of the Cu-binding site of galactose oxidase (48) have been monitored by affinity electrophoresis. The method was also used to demonstrate the lack of sugar-binding activity in isolated lectin subunits (42) and in native insulin (57). Furthermore, affinity electrophoresis can be used to predict the results of affinity chromatography and to optimize the conditions of affinity interaction (1,24,29,30). As affinity electrophoresis can be performed rapidly with small amounts of gels and using simultaneously many samples, it can spare considerable time and material in pretesting the affinity chromatography systems. However, a recent study (9) (in which binding of the glial fibrillary acidic protein to affinity gels prepared of alkyl-agaroses, iminodiacetic acid-agarose, and Cibacron Blue-agarose was tested) has indicated
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that there may not be a simple correlation between the results of affinity electrophoresis and affinity chromatography. In recent years a technique of electrophoretic elution of proteins adsorbed on affinity adsorbents (in chromatographic arrangement) has been developed (58-60) and some authors have considered it a modification of affinity electrophoresis. QUANTITATIVE
APPLICATIONS
It is well known that affinity chromatography is a valuable tool not only for preparative purposes but also for quantitive evaluation of the strength of interaction between the protein and the ligand (for review on quantitative applications of affinity chromatography see (61)). Affinity electrophoresis can also be used for similar purposes. The mobility of the protein decreases with increasing concentration of the immobilized ligand and this concentration dependence can be used for a simple evaluation of apparent dissociation constant (Ki) of the complex protein-immobilized ligand. Such a quantitative application of affinity electrophoresis (although the authors did not call their method so) was described nearly simultaneously by Gerbrandy and Doorgeest (36) and Takeo and Nakamura (15). Later on, it was demonstrated that incorporation of increasing concentrations of free ligand (c) into the affinity gels containing constant concentration of immobilized ligand ( ci) results in increased mobility of the protein (due to competition between free and immobilized ligands for the ligand binding sites); at a sufficiently high c the mobility approaches that on the control (noninteracting) gels. This dependence of mobility on c can be again used for a simple evaluation of dissociation constants (K) of the protein-free ligand complexes; Ki is obtained as a “by-product” (16). These principles were used for quantitative evaluation of the strength of interaction of enzymes with their macromolecular sub-
AFFINITY
ELECTROPHORESIS
strates (15,36,62), glycoproteins with lectins (6), protein A with immunoglobulins and their fragments (8), lectins with immobilized and free sugars (16,42-47,51), enzymes and other proteins with Blue Dextran (i.e., immobilized Cibacron Blue) (53), or with immobilized AMP (49,50,54), trypsin with immobilized p-aminobenzamidine (52), galactose oxidase with free and immobilized sugars (48), and carbohydrate-specific myeloma immunoglobulins with immobilized polysaccharides and free sugars (37-39). These parameters ( Ki and K ) were followed also as functions of the pH (46), ionic strength (47), and spacer length (51,52,54,55). The use of affinity electrophoresis for estimation of dissociation constants of the protein-ligand complexes possesses a number of advantages: the method is economic, rapid, and simple to perform; the sample can be a crude mixture; dissociation constants can be determined simultaneously for all electrophoretically different forms of the ligand binding protein (isoenzymes, isolectins) and for estimation of K the value of Ci need not be known; the method is especially suitable for measuring very weak interactions (dissociation constants up to 10-l to 10” M). However, there are also some limitations: the protein under study must have good electrophoretic properties in the particular system (i.e., relatively high mobility and sharp band); the K values are difficult to measure for charged free ligands; the number of the ligand-binding sites in the protein molecule cannot be determined; the interpretation of Kj value is difficult (see the following paragraph). THEORY
OF THE METHOD
While for qualitative applications of affinity electrophoresis a purely empirical basis is quite sufficient, the quantitative uses of the method are crucially dependent on the theoretical background. The simple equations used originally for evaluation of Ki (15,36) and K (16) are valid exactly
5
only under several assumptions: (a) The protein molecule contains a single ligandbinding site, (b) immobilization of the ligand is complete, (c) all molecules of immobilized ligand are accessible to interaction with the protein (i.e., effective ci is identical with total ci), (d) mobility of the protein-free ligand complex is identical with that of free protein, (e) concentration of the protein in the migrating zone is much lower than concentration of free and immobilized ligand, (f) complex formation is a very rapid reaction, (g) microdistribution of immobilized ligand is homogeneous. Most of these assumptions do not hold true under the actual experimental conditions, and this may more or less seriously affect the Ki and K values obtained. The effects of violation of the above assumptions on the Kj and K values have been theoretically dealt with so far only in two studies (63,64) in which several equations were derived, describing the effects of various factors on the results of affinity electrophoresis. The results from these studies can be summarized as follows: (a) Dissociation constants Ki and K can be estimated even if immobilization of the ligand is not complete and if the mobility of the protein-free ligand complex differs from that of free protein. Similar conclusions were also reached by Bog-Hansen and Takeo (6). (b) The adverse effects of the high protein concentration on Ki estimation can be prevented using a proper plot. (c) The kinetics of protein-ligand complex formation has in most cases negligible effects on the results of affinity electrophoresis; the method might be principally used to study the kinetics of very slow complexformation reactions. (d) The value of K can be determined also for proteins with multiple ligand-binding sites (n) in the molecule; the Kiln value is obtained instead of Ki in such cases. (e) The value of the effective concentration of immobilized ligand (ci) can be determined from the dependence of mobility on protein concentration at constant Ci.
6
VACLAV HOItEJSf
It should be noted that some of the limitations of the method (especially problems connected with estimation of effective ci, the effects of multiple binding sites) are also important in quantitative affinity chromatography (65,66). CONCLUSIONS AND FUTURE PROSPECTS
Although affinity electrophoresis has been used in a relatively large number of studies, the method is not yet “mature,” as some important aspects are still to be explored. (a) It is necessary to test thoroughly some of the theoretical conclusions (63,64), first of all, to determine the effective concentration of immobilized ligand in affinity gels prepared by different methods and thus to estimate “true” Ki. Preliminary results indicate that, at least in the case of affinity gels prepared by incorporation of macromolecular-soluble carriers (e.g., O-glycosyl polyacrylamide copolymers), the effective ci is much lower than total ci (67) and thus, the true Ki is much lower than the apparent constants which have been estimated earlier (16). Similarly, a thorough study of the effect of multivalency of the protein and microdistribution of immobilized ligand would be useful. (b) The present techniques of preparation of affinity polyacrylamide gels are not quite satisfactory. It would be very helpful to work out simple and general methods comparable to the preparation of affinity carriers in aflinity chromatography. Several ways can be suggested: either a ligand could be bound to a beaded gel matrix (agarose, dextran, or other gels) by well-established simple methods (CNBr or periodate activation, etc.) and such particles could be incorporated directly into polyacrylamide gels. Such nonhomogeneous affinity gels (analogous to those used in agarose-gel affinity electrophoresis (1,9,18,29)) would be clearly applicable rather for qualitative
purposes. Alternatively, agarose or some other gel could be substituted by a ligand in such a way that the gel could be subsequently solubilized (either chemically or melted) and incorporated into polyacrylamide gel network. This way would overcome difficulties in preparation of soluble macromolecular derivatives and it would retain the simplicity of affinity gels preparation. Both these methods have appeared very promising in our preliminary experiments (68). (c) A serious drawback of affinity electrophoresis is its limitation in estimating the K for charged free ligands. A technique which would solve this problem would be extremely valuable. (d) The principle of affinity electrophoresis might be extended also to other electromigration methods, such as isoelectric focusing and isotachophoresis. Indeed, affinity isoelectric focusing was introduced recently as a method for the detection of ligand-binding proteins and for similar qualitative purposes (69). On the other hand, electrophoretic equivalents of covalent-affinity chromatography (70) and possibly also of ion-exchange chromatography might be useful. Electrophoretic methods analogous to hydrophobic chromatography have already been introduced (9,32,55). For some special purposes also preparative modifications of affinity electrophoresis might be considered; an easy separation of electrophoretic forms (isoenzymes) differing slightly in affinity toward immobilized ligand would be particularly advantageous. (e) So far, affinity electrophoresis has only been used to study the interactions between proteins and small or macromolecular ligands or between two proteins. It might be useful to develop procedures allowing its application also in studies of the interactions between nucleic acids and proteins, between two kinds of nucleic acid molecules and in other cases of interacting molecules. In conclusion, affinity electrophoresis has
AFFINITY
made considerable progress in recent years but much remains to be done before all potential applications of this method are exploited. ACKNOWLEDGMENT I am indebted to Dr. Marie Ticha for careful critical reading of the manuscript and many valuable suggestions.
T. C. (1973)
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