Affinity isoelectric focusing in polyacrylamide gel—A method to detect ligand-binding proteins

ANALYTICAL

Affinity

116,

BIOCHEMISTRY

Isoelectric

22-26

( 198 I)

Focusing in Polyacrylamide Gel-A Ligand-Binding Proteins V. Ho~EJ~~*

AND MARIE

Method

to Detect

TICHAT

*Institute of Molecular Genetics. Czechoslovak Academy of Sciences. Videriskd 1083, Praha Czechoslovakia; and TDepartment of Biochemistry, Charles University, Albertov 2030, Praha 2. Czechoslovakia Received Isoelectric

focusing

of the position of the containing immobilized with lectins can be used homogeneity

December

in polyacrylamide bands of proteins sugars or Blue

or lactate dehydrogenase, for qualitative detection of purified

protein

gel containing

l/130022-05$02.00/O

Copyright ib 1981 by Academic Press. Inc. All rights of reproduction I” any form reserved.

1980 immobilized

capable Dextran

of interaction 2000 were

respectively. of ligand-binding

This

ligands

results

in a shift

with these ligands. Affinity gels used to demonstrate the interaction

technique, affinity isoelectric focusing, proteins and for checking the functional

preparations

phoresis, developed mainly by T. C. BQgHansen and his co-workers ( 1) employs agarose gel as supporting medium, whereas other authors have used a polyacrylamide gel-based variant of the method. Both these modifications possess their own advantages and limitations (2); the former one closely resembles some immunoelectrophoretic techniques, whereas the latter may be considered as an electrophoretic analog of affinity chromatography. When using affinity electrophoresis in polyacrylamide gel, it may be difficult to find a buffer system optimal for resolution of a particular protein mixture. Isoelectric focusing in polyacrylamide gel is a method with excellent resolving power and it is nearly universally applicable for complex proteinaceous samples of various origin. Hence, in analogy with affinity electrophoresis in the present communication we have combined the principles of separation by means of isoelectric focusing and biospecific interactions. The resulting new technique, affinity isoelectric focusing (AIEF),’ com-

Affinity electrophoresis combines the principles of electrophoretic separation of complex protein mixtures and biospecific interaction of a component(s) of the mixture with a ligand incorporated into the gel (for review see Refs. (1,2)). In brief, the sample is electrophoresed in the gel containing ligand molecules immobilized either by covalent attachment to the gel matrix or by physical entrapment of a macromolecular derivative of the ligand within the gel network. As a result of the interaction with the immobilized ligand, the mobility of the protein possessing the ligand-binding site(s) is decreased as compared to that observed in a control (noninteracting) gel; thus, the band of such a specific ligand-binding protein is usually located near the site of sample application and can be readily identified by comparison of the electrophoretic patterns obtained on affinity and control gels, respectively. It can be shown that the degree of retardation is in simple relation to the strength of protein-ligand interaction. Thus, the method enables both qualitative identification of ligand-binding proteins and quantitative estimation of apparent dissociation constants of the protein-ligand complexes (3-6). One modification of affinity electro0003-2697/B

10,

4.

’ Abbreviations cusing; PsA,

ithrina isoelectric 22

indica

pea

lectin; focusing;

used: lectin; LDH, LcA.

AIEF, SBA,

affinity soybean

isoelectric lectin; EiA,

lactate dehydrogenase; lentil lectin.

foErIEF.

AFFINITY

ISOELECTRIC

FOCUSING

plements the set of existing variants of affinity electrophoretic methods and it might be useful especially as a widely applicable sensitive tool for qualitative identification of various ligand-binding proteins. MATERIALS

AND METHODS

Crude protein fractions. Crude fractions of seed proteins containing lectins were prepared by ammonium sulfate precipitation of water-buffer extracts of the seeds. The fractions of proteins precipitating at 55% saturation (350 g (NH4)S04 per liter) (pea and lentil seeds) or at 80% saturation (560 g (NH,),SO, per liter) (soybean seeds) were used. Purified lectins and enzymes. Pea lectin (PsA) was prepared by affinity chromatography on Sephadex G- 100 (7) soybean lectin (SBA) and Erythrina indica lectin ( EiA) by affinity chromatography on a-t>-galactosyl polyacryiamide gels (8,9). Crystalline lactate dehydrogenase (LDH) (Boehringer, Mannheim, F.R.G.) was kindly provided by Dr. A. Bass. Other materials. Blue Dextran 2000 was purchased from Pharmacia Fine Chemicals, Uppsala, Sweden, carrier ampholytes from LKB, Bromma, Sweden, acrylamide and N,N’-methylenebisacrylamide from Serva, Heidelberg, West Germany. Soluble glycosyl polyacrylamide copolymers were prepared by copolymerization of ally1 glycosides and acrylamide as described earlier ( 10); the copolymers used throughout this work contained 14.6% mannose (a-D-mannosy] polyacrylamide copolymer) or 6.8% galactose (a-D-galactosyl polyacrylamide copolymer). Isoelectric focusing. Isoelectric focusing (IEF) in polyacrylamide gel was performed according to Righetti and Drysdale (11) using 5% polyacrylamide gel rods (65 X 2.5 mm) containing 2% Ampholine pH range 3.5-10. Samples containing 35 pg of crude protein mixture or 5 pg purified proteins in 20 ~1 sample solution (20% glycerol, 2% Ampholine pH 3.5- 10, trace of bromphenol blue) were loaded on the top of gel rods

IN POLYACRYLAMIDE

GEL

23

(without prefocusing), covered by 10 ~1 of overlay solution (10% glycerol, 1% Ampholine pH 3.5-10). Focusing was performed usually from cathode to anode (in some cases in the opposite direction) at 100 V (1 h) and 200 V (additional 1.5 h), if not stated otherwise. The gels were then fixed in 10% trichloroacetic acid and stained by Coomassie Blue R-250. Affinity gels. Affinity gels were prepared by addition of soluble glycosyl polyacrylamide copolymers (0. I- 1%) into the solution normally used for preparation of IEF gels. Immobilization of glycosyf residues in the gel was achieved by physical entrapment of these macromolecular glycosylated derivatives within the polyacrylamide gel network as described earlier (4). Affinity gels used for ALEF of LDH were prepared analogously by addition of Blue Dextran (O.OOl0.1%) into the gel mixture as described in affinity electrophoresis ( 12); 0.1% Blue Dextran solution corresponds to 40 pM Cibacron Blue F3GA (12). Control gels contained usually corresponding amounts of soluble (non-crosslinked) polyacrylamide. RESULTS

AND DISCUSSION

The principle of the method is obvious from Fig. 1. The bands of lectins capable of interaction with sugar residues immobilized in the gel are shifted on affinity gels as compared with the control gels. The overall difference between the band patterns on control and affinity gels is quite similar to that observed after affinity disc electrophoresis in polyacrylamide gel. Normal band pattern can be restored by addition of free ligand (sugar) into the affinity gels (Fig. 1, gels a3, b-3, c-3), confirming specific nature of the retardation. Thus, comparison of band patterns observed on the control and affinity gels represents an easy way of identification of ligand-binding proteins in a complex protein mixture. For this purpose, it may be advantageous to use such conditions, that all (or most) of the proteins are focused in the lower part of the gel and the only band pres-

24 1

HOl?EJ!?i 2

3

1

2

3

12

+

+

c

b

0

3

FIG. 1. Affinity isoelectric focusing of crude protein fractions containing lectins. (a) Crude protein fraction from soybean seeds; (b) crude protein fraction from lentil seeds; (c) crude protein fraction from pea seeds. Gels: 1, Control gels; 2, affinity gels containing 1% a-D-galactosyl polyacrylamide copolymer (a) or 1% a-D-mannosy1 polyacrylamide copolymer (b and c); 3, the same as 2 but containing in addition 2% corresponding free sugars.

ent in the region near the upper end of affinity gel is that corresponding to the ligandbinding protein (Fig. lc). The region of the gel where most of the proteins are focused could be regulated to great extent by the type of carrier ampholytes used. The method can also be used for checking the ligand-binding homogeneity of purified protein preparations (Fig. 2). Thus, all the major electrophoretically distinct components present in the affinity-purified PsA and EiA preparations (isolectins) do interact with immobilized sugar residues and essentially no contaminating inactive components 1

2

1

AND

TICHA

are present. Similarly, all the protein bands (isoenzymes) present in the LDH preparation were able to interact with immobilized Cibacron Blue (i.e., Blue Dextran) (Fig. 3b), demonstrating again high degree of purity of this preparation. It should be noted that in the case of LDH free ligand could not be incorporated into the gel in order to demonstrate the specificity of the interaction because the dye would have been subjected to focusing itself. However, the specific nature of retardation of LDH on Blue Dextran-containing gels could be inferred from the fact that the IEF-banding pattern of several complex protein mixtures was unchanged on the gels containing up to 0.1% Blue Dextran (figure not shown). This observation may be of importance because it demonstrates that AIEF can be performed even in the cases when immobilized ligand is charged (Cibacron Blue is an amphoteric ion). However, detailed studies on the effects of various immobilized charged ligands on IEF are still to be performed. Certainly, even relatively low concentrations of immobilized highly dissociated anions and cations would be incompatible with IEF, due to appreciable electroendoosmosis. Similarly as in the case of affinity electrophoresis, the degree of retardation of the ligand-binding protein on the affinity gel 1

12345

2

3

4 . -

2

+ b

FIG. 2. Affinity isoelectric focusing of purified lectins. (a) PsA; (b) EiA. Gels: 1, control gels; 2, affinity gels containing 1% a-D-IWinnOSyl polyacrylamide copolymer (a) or 1% cu-D-galactosyt polyacrylamide copolymer (b).

FIG. 3. The effect of concentration of immobilized ligand on the position of the ligand-binding protein in AIEF. (a) Crude protein fraction from lentil seeds; (b) purified LDH. Gels 1. control gels. Gels 2-5 in a contained 0.05, 0.1, 0.25, and 0.5% a-D-mannosyl polyacrylamide copolymer, respectively. Gels 2-4 in b contained 0.001,0.01, and 0.1% Blue Dextran, respectively.

AFFINITY

ISOELECTRIC

FOCUSING

depends on the concentration of immobilized ligand in the affinity gels (Fig. 3). The affinity effect is most obvious at high ligand concentrations (gels a-4, b-4). In affinity electrophoresis the concentration dependence of mobility can be used for quantitative evaluation of the strength of proteinligand interaction. Similar quantitative application is not feasible in AIEF because of a number of complicating factors which are not encountered in affinity electrophoresis (variability of pH along the gel, pH dependence of protein-ligand interaction, nonsteady state of the interacting protein band, etc.). It should be noted that application of relatively low voltage is necessary to obtain good results in AIEF (see Materials and Methods). When higher voltage is used, the retardation on affinity gels is apparently lower and interacting bands are usually blurred (although noninteracting bands are sharper at higher voltage) (results not shown). This phenomenon is direct implication of the very principle of the method: all the proteins are forced by external voltage to move to their p1 regions. This effect of voltage is selectively counteracted by the interaction of the ligand-binding protein with the immobilized ligand and quite obviously (although detailed theory of AIEF is completely lacking at present) the degree of retardation of the interacting protein must be dependent on the ratio of both these opposite forces, i.e., voltage and affinity interaction; the lower is the former, the more efficient is apparently the latter. Interesting, also, is the question of time dependence of the position of interacting bands in the affinity gel. Ideally, one would assume that after a sufficiently long period of time the interacting protein should reach its p1 position even on the affinity gel; the difference between the position on control and affinity gels should be maximal after a relatively short time of IEF, when equilibrium or near-equilibrium is already reached on control gel, whereas the interacting band

IN POLYACRYLAMIDE

GEL

25

is still far from equilibrium position on affinity gel. The results obtained experimentally are shown in Fig. 4. In principle, the behavior of LcA corresponds to that expected from the above considerations. The interacting LcA bands move initially slowly toward their equilibrium positions, whereas the positions of noninteracting proteins are changed only slightly during this time, demonstrating the existence of near-equilibrium state in the control gels. However, the time necessary for reaching the equilibrium p1 position of the interacting band on affinity gel may be too long, so that meanwhile the cathodic drift of pH gradient may cause the migration of the band into the cathodic electrode space before it could reach the equilibrium position (gel 4 and partially gel 3 in Fig. 4). The ends of IEF affinity gels where the AIEF effects are usually observed may be of extremely acidic or basic pH, which may inhibit the protein-ligand interactions; e.g., lectins usually interact only weakly with sugars at pH ~3-4 (I 3). This pH dependence of lectin-sugar interactions is probably also the reason for the results shown in Fig. 5. When crude protein fraction from lentil seeds was subjected to AIEF from anode to cathode, the affinity effect was less obvious than in the case of AIEF from cathode to anode (Fig. lb). Although the bands of lec-

FIG. 4. The dependence of AIEF pattern on time. Crude protein fraction from lentil seeds. Gels 1-4 were focused for 2, 5, 9. and 24 h, respectively. All gels contained 1% ol-D-mannosyl polyacrylamide copolymer. The bands in Gel 4 correspond to those at the bottom of Gels 1-3.

26

HOi?EJSi

AND

TICHA

timation of pH range where protein-ligand interaction can occur, and for similar qualitative purposes. From a practical point of view, relatively low voltage, focusing times as short as possible, high ligand concentration, and suitable ampholyte pH range should be used to obtain best results with this technique. REFERENCES FG. 5. AIEF details identical polarity.

of crude lentil seed protein fraction. Al1 with those in Fig. 1b; note the reversed

tins disappeared on affinity gel from their positions observed on control gels, they did not emerge as a sharp band at the upper (acidic) end of the gel, but were localized as a broad blurred zone rather distant from the top of the gel (indicated by an arrow in Fig. 5). Obviously, the low pH at the top region of the gel was unsuitable for lectinsugar interaction and the interaction could occur just in the lower part with less extremely acidic pH. However, it is interesting that LDH interacted with immobilized Blue Dextran quite strongly even at this acidic end of the AIEF gel (Fig. 3b). It seems likely that AIEF can be useful for qualitative detection and identification of ligand-binding proteins in complex mixtures, for checking the functional homogeneity of purified preparations, for rough es-

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