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[18] Measurement of protein-ligand interactions by gel chromatography
346
MACROMOLECULES" LIGANDS AND LINKAGES
[18]
parison to the total ligand concentration. This source of error can be minimized by employing conditions that yield a relatively small excluded volume, namely a high ratio of resin volume to total volume. In the experiment illustrated in Fig. IA, the total volume was 1.0 ml and the excluded volume was 0.45 ml. With these conditions, values of AlL] ranged from 153% of [L] E° at the lowest concentration of AMP to 115% of ILl E° at the highest concentration. At higher concentrations of ligand the values of A[L] became unreliable. Sephadex G-75 would provide a lower proportion of excluded volume, but would require at least 24 hr for complete swelling and equilibration. The value for KD (0.7 x 10 -6 M) is smaller than the values (-105-10 -5 M) reported by earlier workers. H However, the earlier experiments were carried out with a proteolytically modified form of the enzyme, which Traniello et al. 13have reported to be considerably less sensitive to inhibition by AMP. More recent studies in which the equilibrium gel penetration method was employed with preparations of native, neutral fructose1,6-bisphosphatase ~5have confirmed the value of Ko reported here. 15A. Vita, H. Kido, S. Pontremoli,and B. L. Horecker,Arch. Biochem. Biophys. 209, 598 (1981).
[ 18] M e a s u r e m e n t
of Protein-Ligand Interactions by Gel Chromatography
By JosI~ MANUEL ANDREU
The measurement of ligand binding to protein by application of the gel chromatography technique of Hummel and Dreyer I will be discussed in this chapter. Particular attention will be given to the criteria of application and the meas',rement of weak binding effects. Principle of Measurement and Possible Sources of Error The method consists in the chromatography of protein through a size exclusion gel column equilibrated with a known ligand concentration. The perturbations in ligand concentration in the effluent due to binding are observed. This technique 1-3 is based on three requirements: (1) the chroi j. p. H u m m e l and W. J. Dreyer, Biochim. Biophys. Acta 63, 530 (1962). 2 G. F. Fairclough and J. S. Fruton, Biochemistry 5, 673 (1966). 3 G. K. Ackers, this series, Vol. 27, p. 441.
METHODS IN ENZYMOLOGY,VOL. 117
Copyright © 1985by Academic Press, Inc. All rights of reproduction in any form reserved.
[18]
PROTEIN--LIGAND INTERACTIONS
347
matographic gel efficiently excludes the protein while including the ligand, (2) attainment of binding equilibrium is faster than the separation process, and (3) there are convenient methods to measure accurately both protein and ligand in the effluent. Under the first and second conditions the procedure can be regarded as an equilibrium thermodynamic technique, even though it involves a transport separation process. In this sense the gel technique is formally analogous to equilibrium dialysis, where the inside of the dialysis bag is now the excluded volume of the column (outer volume Vo, accessible to both the macromolecule P and the ligand A) and the outer dialysis compartment is equivalent to the included volume (inner volume Vi, accessible only to ligand A). Binding equilibrium is established in the vicinity of the macromolecule as it passes through the column. The total macromolecule concentration, [P] + [PA], and the total ligand concentration, [A] + [PA], of the outer volume can be measured in the effluent. The unbound ligand concentration [A] is measured in the vicinity of the macromolecule fractions and is essentially the same as the ligand concentration with which the column has been equilibrated. Therefore, we can adjust the free ligand concentration, and the bound ligand concentration in the protein fractions is calculated by its difference to the total ligand concentration measured. The sample applied to the column usually includes a total ligand concentration exactly equal to the ligand concentration with which the column is equilibrated and therefore a trough is observed in the effluent at Vo + V~due to the depletion of ligand in the sample by binding to the protein. The decrease of ligand in the trough is equal to the increase of ligand in the peak. A related approach is employed in binding measurements by batch gel partition procedures, 4,5 with the important difference that the free ligand concentration is not adjusted, but calculated from the difference of bound to total ligand concentration. These methods are valuable for small samples and estimate binding from the ligand concentration of the outer phase of gel suspensions with and without macromolecule, with the appropriate volumetric corrections. However, they involve more measurements, calculations, and corrections than the Hummel and Dreyer I technique and will not be discussed here. The gel chromatography equilibrium technique is of great value when the binding assay required has to be simple and considerably faster than equilibrium dialysis, provided enough ligand is available to run the columns. This technique can be automated 2 and also performed with highperformance liquid chromatography systems. 6 Provided equilibrium is at4 p.
Fasella, G. G. Harnmes, and P. R. Schimmel, Biochim. Biophys. Acta 103, 708 (1965). M. Hirose and Y. Kano, Biochim. Biophys. Acta 251, 376 (1971). 6 B, Sebille, N. Thuaud, and J. P. Tillement, J. Chromatogr. 167, 159 (1978).
348
MACROMOLECULES: LIGANDS AND LINKAGES
[18]
tained the technique is still subject to several potential pitfalls, most of which can be adequately circumvented. (1) The technique is not applicable to ligands that adsorb strongly to the gel; however, this can be avoided by changing, for example, from dextran to polyacrylamide. A small adsorption of ligand to the columns (that is, a retardation of its elution beyond the position of small molecules) should not cause problems once the column is well equilibrated with ligand. (2) When the ligand under study is charged, the Donnan effect must be taken into account, as in the case of equilibrium dialysis. Provided the charge of the macromolecule is known under the conditions of the experiment, the uneven distribution of ions in both compartments can be calculated 7 and corrected for. This effect can be practically abolished by working with dilute macromolecule solutions in moderately high concentrations of neutral salt. 7 (3) The volume occupied by the macromolecule can be a substantial portion of the outer gel compartment and therefore the small solute concentration can be reduced. This effect may cause an artifactual decrease in ligand con":entration associated to protein elution, so that in cases of no interaction an apparent negative binding would be measured. This effect can be corrected if the partial specific volume of the macromolecule is known, or made very small by the use of low macromolecule concentrations (<1%). (4) Large binding effects can lead to substantial depletion of ligand from the inner gel compartment so that the actual free ligand concentration of the protein solution is smaller than the concentration used to equilibrate the gel. This may be noticed by a relatively large peak surrounded by ligand concentrations smaller than in the rest of the effluent. This effect is easily avoided using a larger column or, as above, low protein concentrations.
Experimental Procedures The procedures and examples discussed below are those with which the author is most familiar and proceed from work with the protein tubulin. However, they are easily applied to other systems.
Sample Preparation and Chromatography Samples containing 1 to 4 mg of Protein and a chosen total ligand concentration in the desired buffer (final volume < I ml) were prepared by careful dilution of concentrated protein and ligand solutions (this can be made gravimetrically if great precision is required in order to measure binding from the trough) were taken to temperature and applied to 0.9 × 25-cm Sephadex G-25 columns previously equilibrated with the same 7 C. Tanford, "Physical Chemistry of Macromolecules," p. 221. Wiley, New York, 1961.
[18]
PROTEIN--LIGAND INTERACTIONS
349
buffer of identical ligand concentration. Column temperature was controlled to +--0.2° by means of water jackets and a circulating bath. The column flow was not interrupted during sample application and was kept constant during the whole experiment by means of adjustable LKB peristaltic pumps. The binding time (taken as the mean chromatographic elution time of the protein) could be varied among different experiments, in order to ensure attainment of equilibrium, between 5 and 100 min, with an accuracy of +-5% by simply changing the pump setting. Fractions of 1.05 --- 0.05 ml were collected and the protein was determined spectrophotometrically, subtracting any small contributions of bound ligand when necessary. Ligand concentrations in the effluent were measured as described below. Experiments were performed at different free ligand concentrations in order to obtain a binding isotherm, which was then analyzed according to the pertinent binding scheme. This was done at different temperatures and the apparent thermodynamic parameters of the binding reaction measured by van't Hoff analysis of the d a t a . 8,9
Measurement of Labeled Ligand Concentration The radioactive ligand concentration was measured throughout the column effluent by means of carefully taken aliquots (using a 0.5-ml delivery pipet that afforded a reproducibility of -0.25%, as determined by weighing buffer and protein solutions) that were added to 10 ml of aqueous counting scintillant and counted twice to a statistical counting error smaller than 0.3% (95% confidence) in a liquid scintillation spectrometer. Duplicate aliquots were taken in the peak region. The baseline counts per minute were determined from the regions outside the peak and trough typically to a standard deviation <0.5% of the absolute value. The radioactive ligand concentrations in the effluent had to be very close to that applied to the column. Experiments without stable baseline in the vicinity of the peak or not giving a good separation of peak and trough were discarded. The amount of bound ligand was calculated from the measured increment in eluate radioactivity coupled to protein elution; this had to be coincident, within experimental error, with the absolute value of the decrement in the trough. The standard deviation of the baseline was taken as an estimate of the standard deviation of measurements of bound ligand. As an example of the sensitivity of this procedure, Fig. 1 shows an equilibrium binding gel chromatography experiment in which the weak binding of [3H]tropolone methyl ether to tubulin was measured [under these conditions a binding equilibrium constant of (7.5 +- 2) x 102 M -~ was s j. M. Andreu and S. N. Timasheff, Biochemistry 21, 534 (1982). 9 j. M. Andreu, M. J. Gorbunoff, J. C. Lee, and S. N. Timasheff, Biochemistry 23, 1742 (1984).
350
MACROMOLECULES" LIGANDS AND LINKAGES
[18]
KD 0
I
r
i
2f7 o
i
[3.],ME
.-~'" ?
• o
•
~-e
.]rME [
~1
+COL °
-2
02 E 0
..j \
.....
I
0
,
.
I
.
.
.
.
.
i
I0
I
20
Fraction number
Flo. 1. Interaction of tropolone methyl ether (TME) with tubulin. The results of a typical equilibrium binding Sephadex G-25 chromatography experiment with 1.05 x 10-4 M [3H]TME (1.4 x 105 cpm m l - 0 in 10 mM sodium phosphate buffer 0.1 mM GTP, pH 7.0 at 18" are shown by the open circles. The changes in ligand concentration of the upper profile were produced by chromatography of 3.2 mg of tubulin, while the middle profile was obtained under identical conditions after the addition of 10-4 M colchicine (COL) to column and sample; the vertical bars show the standard deviation of the measurements. The filled circles are the average of three (upper profile) and two (middle profile) runs, which reduce noise and show unequivocally the position of the peak and trough. Reproduced from Ref. 8.
obtainedS]. These binding measurements involved ligand concentration increments in the order of only 1% of the absolute value and the specificity of these effects was verified by their inhibition by colchicine (see Fig. 1), a high affinity ligand that binds to the same site. 8 The purity of the ligands used was carefully checked since it is well known that the presence of radiochemical impurities in the labeled ligand preparation employed may lead to large errors that have been discussed by others.~° to S. E. Builder and I. H. Segel, Anal. Biochem. 85, 413 (1978).
[18]
PROTEIlq-LIGAND INTERACTIONS
35 1
Measurement of Ligand Concentration by Light Absorption The ligand concentration in the column effluent is often measured spectrophotometrically. This procedure is subject to the same general considerations as radioactive counting. In particular, it relies frequently on the measurement of small absorbance increments due to bound ligand and is therefore subjected to several potential sources of error: interference of light absorption by the protein, absorption increments due to protein effects on bound ligand, interfering light scattering by the protein and instrumental noise. In our case, the ligand concentration measurements were made at a wavelength where the protein does not absorb light. The second effect was not large and could be easily overcome if needed by measuring the ligand absorbance at an isosbestic point of the proteinligand interaction difference spectrum. 9 The third source of error is a systematic one and is important in proteins which, like tubulin, have a tendency to aggregate. The second and third effects were easily avoided by careful addition of a small volume of concentrated sodium dodecyl sulfate to each fraction, giving a final concentration of 0.4% detergent; this displaced the protein-ligand interaction and solubilized any aggregated protein (giving typically A350 ~ 0.001 in the absence of ligand). Since the detergent may also interact with the ligand, the extinction coefficients of the protein and ligand in detergent solution were determined independently. It was also ascertained that the detergent was in excess in the protein-containing fractions of the effluent (that is, above its effective critical micelle concentration in the presence of protein) since otherwise artifactual absorption increments could be generated. Finally, noise was reduced by carefully repeated measurements through the whole column effluent, leading to a good statistical estimation of the baseline concentration. The absorption increments due to bound ligand were estimated by subtraction of the baseline values as in the case of radioactive counting. When small absorbance increments had to be measured this was done from recordings of the difference spectra of the peak fractions versus those from the baseline. 9 Figure 2 shows the elution profile generated by the interaction of 1.8 mg of tubulin chromatographed in a column equilibrated with 1.63 × 10-5 M 2-methoxy-5-(2,3,4-trimethoxyphenyl)tropone at 25 ° and its inhibition by 2 × 10-4 M podophyllotoxin, a ligand that does not absorb light at the wavelength employed and binds to a partially overlapping site. 8,9 Typically, these measurements were useful for ligands binding to protein with equilibrium constants - 103 M -l. Any other procedure to measure ligand concentration that is found to give good accuracy can, in principle, be employed.
352
[18]
MACROMOLECULES: LIGANDS AND LINKAGES i
r
O
r
I
HI oo
E
cO
O
--o-~-teo
° o-o,o.o 8 o °
o
o o o o o'o'°'°'o 0
0 o
O0 o
.cI
b
I O
~
I IO Froction
*
I 20
i
I 30
number
FIG. 2. Gel chromatography of tubulin in Sephadex G-25 columns equilibrated with 2methoxy-5-(2',3',4'-trimethoxyphenyl)tropone. (a) Tubulin (1.8 rag) was chromatographed
in a column equilibrated with 1.63 x 10-5 M ligand in 10 mM sodium phosphate buffer 0.1 mM GTP pH 7.0 at 25°. (b) An identical experiment in which the elution buffer contained 1.63 x 10-~ M ligand and 2 x 10-4 M podophyllotoxin. The flow rate was 40 ml/hr and fractions were approximately 1.5 ml. Taken from Ref. 9.
Experimental Criteria of Applicability; Abnormal Profiles; Protein Self-Association There are several easily verifiable characteristics to ensure attainment o f equilibrium in the H u m m e l and D r e y e r technique. 1-3 First, and most important, the ligand concentration immediately before and after the protein peak must return to baseline, so that the peak and the trough are • separated by a region o f nil concentration increment. Any other type o f profile may lead to incorrect measurements. For example, a tailing peak ~hat does not separate from the trough indicates imperfect equilibrium. When the ligand concentration used in the sample is increased or decreased, the rest o f the experiment remaining the same, this will be reflected by a corresponding change in the trough, but the ligand increment o f the peak must remain constant. Second, the number of moles of ligand bound per mole protein must be independent o f small changes of the chromatographic flow, otherwise the separation time is too close to the time needed to attain equilibrium. Third, the binding measured must be independent o f protein concentration. This is easily checked by loading
[18]
353
PROTEIN--LIGAND INTERACTIONS I
I
lel
,,' ~, 107 E
d°~
I
I
•
\,
•
el"~" ~lll-
./ ttll..., ]
•
E oo 10E
I
0o#X
•
•
I
5
I
l0 Fraction
I
15
t
20
2
number
FIG. 3. Interaction of octylglucoside with tubulin. Tubulin (1.5 mg) was chromatographed in a 0.9 x 26-cm Sephacryl S-300 column equilibrated with 2.1 × 10-2 M [14C]octylglucoside in 10 mM sodium phosphate 0.1 mM GTP pH 7.0 at 25°. Three identical experiments were averaged to obtain the upper radioactivity elution profile. In the lower part (o) is the protein elution profile, ( - - - ) is the protein elution profile without detergent, and (o) the protein elution profile with 4.0 x 10 -2 M octylglucoside. From Ref. 13.
different protein amounts on the columns and also, within the same run, by determining the binding separately in each protein containing fraction and looking for any trend with protein concentration. A dependence of ligand binding on protein concentration indicates protein self-association linked to ligand binding tl and therefore the measurements no longer reflect intrinsic ligand binding to monomeric protein, but a combination of the bindings to all macromolecular species. Ignoring such linkage effects can lead to large errors, as has been shown, for example, with the tubulin-vinblastine system.12 On the other hand, ligand-induced protein selfassociation can also give abnormal Hummel and Dreyer profiles. Besides performing the pertinent self-association studies, gross self-association effects induced by ligand may be detected during the binding measurements. For this purpose a chromatographic support that partially includes the protein is used. Figure 3 shows the ligand concentration profiles obtained by interaction of the detergent [14C]octylglucoside with tubulin in Sephacryl S-300 at 25o.13 The amphiphile concentration employed was 2. l × l0 -2 M, just below its critical miceUe concentration and the protein elution profile (filled circles) was not significantly different from one obi1 j. Wyman, Adv. Protein Chem. 19, 223 (1964). 12 G. C. Na and S. N. Timasheff, Biochemistry 19, 1355 (1980). is j. M. Andreu, EMBO. J. 1, 1105 (1982).
354
MACROMOLECULES." LIGANDS AND LINKAGES
[19]
tained without detergent. Increasing the detergent concentration led to an abnormal ligand elution profile in which the peak and trough were not well separated (not shown) together with the bimodal protein profile shown by the open circles in Fig. 3, where most of the protein was no longer included but eluted in the void volume of the column, indicating the formation of large aggregates. Acknowledgment I wish to thank Dr. A. Hargreavesfor his critical readingof the manuscript.
[19] U l t r a f i l t r a t i o n in L i g a n d - B i n d i n g S t u d i e s
By ALKIS J. SOPHIANOPOULOSand JUDITH A. SOPHIANOPOULOS Studies of the reversible interaction of small molecules with macromolecules have played a major role in the unraveling of biological mechanisms. Equilibrium dialysis has been virtually the only method for measuring free ligand directly. The introduction of permselective membranes and related apparatus has facilitated various operations with biological materials such as desalting and concentrating of macromolecular solutions; their use in binding studies however has been limited. There are two major approaches to binding studies using semipermeable membranes. In ultrafiltration, a portion of the contents of the macromolecular solution is filtered through a membrane impermeable to the macromolecule. No components are added to the macromolecular solution in this process. In diafiltration, simultaneous to the filtering process, components are added to the macromolecular solution to maintain its volume nearly constant. It was thought that ultrafiltration would give only approximate values of free ligand in binding studies and thus was not used in highly precise binding studies. It was shown recently that ultrafiltration is theoretically equivalent to equilibrium dialysis.l The conclusion t was that during ultrafiltration, the concentration of free ligand remains constant, although the concentration of the other components increases. During ultrafiltration of a system at equilibrium, although the concentration of the macromoleJ J. A. Sophianopoulos, S. J. Durham, A. J, Sophianopoulos, H. L. Ragsdale, and W. P. Cropper, Arch. Biochem. Biophys. 187, 132 (1978),
METHODS IN ENZYMOLOGY,VOL. 117
Copyright © 1985by Academic Press, Inc. All rights of reproduction in any form reserved.
MACROMOLECULES" LIGANDS AND LINKAGES
[18]
parison to the total ligand concentration. This source of error can be minimized by employing conditions that yield a relatively small excluded volume, namely a high ratio of resin volume to total volume. In the experiment illustrated in Fig. IA, the total volume was 1.0 ml and the excluded volume was 0.45 ml. With these conditions, values of AlL] ranged from 153% of [L] E° at the lowest concentration of AMP to 115% of ILl E° at the highest concentration. At higher concentrations of ligand the values of A[L] became unreliable. Sephadex G-75 would provide a lower proportion of excluded volume, but would require at least 24 hr for complete swelling and equilibration. The value for KD (0.7 x 10 -6 M) is smaller than the values (-105-10 -5 M) reported by earlier workers. H However, the earlier experiments were carried out with a proteolytically modified form of the enzyme, which Traniello et al. 13have reported to be considerably less sensitive to inhibition by AMP. More recent studies in which the equilibrium gel penetration method was employed with preparations of native, neutral fructose1,6-bisphosphatase ~5have confirmed the value of Ko reported here. 15A. Vita, H. Kido, S. Pontremoli,and B. L. Horecker,Arch. Biochem. Biophys. 209, 598 (1981).
[ 18] M e a s u r e m e n t
of Protein-Ligand Interactions by Gel Chromatography
By JosI~ MANUEL ANDREU
The measurement of ligand binding to protein by application of the gel chromatography technique of Hummel and Dreyer I will be discussed in this chapter. Particular attention will be given to the criteria of application and the meas',rement of weak binding effects. Principle of Measurement and Possible Sources of Error The method consists in the chromatography of protein through a size exclusion gel column equilibrated with a known ligand concentration. The perturbations in ligand concentration in the effluent due to binding are observed. This technique 1-3 is based on three requirements: (1) the chroi j. p. H u m m e l and W. J. Dreyer, Biochim. Biophys. Acta 63, 530 (1962). 2 G. F. Fairclough and J. S. Fruton, Biochemistry 5, 673 (1966). 3 G. K. Ackers, this series, Vol. 27, p. 441.
METHODS IN ENZYMOLOGY,VOL. 117
Copyright © 1985by Academic Press, Inc. All rights of reproduction in any form reserved.
[18]
PROTEIN--LIGAND INTERACTIONS
347
matographic gel efficiently excludes the protein while including the ligand, (2) attainment of binding equilibrium is faster than the separation process, and (3) there are convenient methods to measure accurately both protein and ligand in the effluent. Under the first and second conditions the procedure can be regarded as an equilibrium thermodynamic technique, even though it involves a transport separation process. In this sense the gel technique is formally analogous to equilibrium dialysis, where the inside of the dialysis bag is now the excluded volume of the column (outer volume Vo, accessible to both the macromolecule P and the ligand A) and the outer dialysis compartment is equivalent to the included volume (inner volume Vi, accessible only to ligand A). Binding equilibrium is established in the vicinity of the macromolecule as it passes through the column. The total macromolecule concentration, [P] + [PA], and the total ligand concentration, [A] + [PA], of the outer volume can be measured in the effluent. The unbound ligand concentration [A] is measured in the vicinity of the macromolecule fractions and is essentially the same as the ligand concentration with which the column has been equilibrated. Therefore, we can adjust the free ligand concentration, and the bound ligand concentration in the protein fractions is calculated by its difference to the total ligand concentration measured. The sample applied to the column usually includes a total ligand concentration exactly equal to the ligand concentration with which the column is equilibrated and therefore a trough is observed in the effluent at Vo + V~due to the depletion of ligand in the sample by binding to the protein. The decrease of ligand in the trough is equal to the increase of ligand in the peak. A related approach is employed in binding measurements by batch gel partition procedures, 4,5 with the important difference that the free ligand concentration is not adjusted, but calculated from the difference of bound to total ligand concentration. These methods are valuable for small samples and estimate binding from the ligand concentration of the outer phase of gel suspensions with and without macromolecule, with the appropriate volumetric corrections. However, they involve more measurements, calculations, and corrections than the Hummel and Dreyer I technique and will not be discussed here. The gel chromatography equilibrium technique is of great value when the binding assay required has to be simple and considerably faster than equilibrium dialysis, provided enough ligand is available to run the columns. This technique can be automated 2 and also performed with highperformance liquid chromatography systems. 6 Provided equilibrium is at4 p.
Fasella, G. G. Harnmes, and P. R. Schimmel, Biochim. Biophys. Acta 103, 708 (1965). M. Hirose and Y. Kano, Biochim. Biophys. Acta 251, 376 (1971). 6 B, Sebille, N. Thuaud, and J. P. Tillement, J. Chromatogr. 167, 159 (1978).
348
MACROMOLECULES: LIGANDS AND LINKAGES
[18]
tained the technique is still subject to several potential pitfalls, most of which can be adequately circumvented. (1) The technique is not applicable to ligands that adsorb strongly to the gel; however, this can be avoided by changing, for example, from dextran to polyacrylamide. A small adsorption of ligand to the columns (that is, a retardation of its elution beyond the position of small molecules) should not cause problems once the column is well equilibrated with ligand. (2) When the ligand under study is charged, the Donnan effect must be taken into account, as in the case of equilibrium dialysis. Provided the charge of the macromolecule is known under the conditions of the experiment, the uneven distribution of ions in both compartments can be calculated 7 and corrected for. This effect can be practically abolished by working with dilute macromolecule solutions in moderately high concentrations of neutral salt. 7 (3) The volume occupied by the macromolecule can be a substantial portion of the outer gel compartment and therefore the small solute concentration can be reduced. This effect may cause an artifactual decrease in ligand con":entration associated to protein elution, so that in cases of no interaction an apparent negative binding would be measured. This effect can be corrected if the partial specific volume of the macromolecule is known, or made very small by the use of low macromolecule concentrations (<1%). (4) Large binding effects can lead to substantial depletion of ligand from the inner gel compartment so that the actual free ligand concentration of the protein solution is smaller than the concentration used to equilibrate the gel. This may be noticed by a relatively large peak surrounded by ligand concentrations smaller than in the rest of the effluent. This effect is easily avoided using a larger column or, as above, low protein concentrations.
Experimental Procedures The procedures and examples discussed below are those with which the author is most familiar and proceed from work with the protein tubulin. However, they are easily applied to other systems.
Sample Preparation and Chromatography Samples containing 1 to 4 mg of Protein and a chosen total ligand concentration in the desired buffer (final volume < I ml) were prepared by careful dilution of concentrated protein and ligand solutions (this can be made gravimetrically if great precision is required in order to measure binding from the trough) were taken to temperature and applied to 0.9 × 25-cm Sephadex G-25 columns previously equilibrated with the same 7 C. Tanford, "Physical Chemistry of Macromolecules," p. 221. Wiley, New York, 1961.
[18]
PROTEIN--LIGAND INTERACTIONS
349
buffer of identical ligand concentration. Column temperature was controlled to +--0.2° by means of water jackets and a circulating bath. The column flow was not interrupted during sample application and was kept constant during the whole experiment by means of adjustable LKB peristaltic pumps. The binding time (taken as the mean chromatographic elution time of the protein) could be varied among different experiments, in order to ensure attainment of equilibrium, between 5 and 100 min, with an accuracy of +-5% by simply changing the pump setting. Fractions of 1.05 --- 0.05 ml were collected and the protein was determined spectrophotometrically, subtracting any small contributions of bound ligand when necessary. Ligand concentrations in the effluent were measured as described below. Experiments were performed at different free ligand concentrations in order to obtain a binding isotherm, which was then analyzed according to the pertinent binding scheme. This was done at different temperatures and the apparent thermodynamic parameters of the binding reaction measured by van't Hoff analysis of the d a t a . 8,9
Measurement of Labeled Ligand Concentration The radioactive ligand concentration was measured throughout the column effluent by means of carefully taken aliquots (using a 0.5-ml delivery pipet that afforded a reproducibility of -0.25%, as determined by weighing buffer and protein solutions) that were added to 10 ml of aqueous counting scintillant and counted twice to a statistical counting error smaller than 0.3% (95% confidence) in a liquid scintillation spectrometer. Duplicate aliquots were taken in the peak region. The baseline counts per minute were determined from the regions outside the peak and trough typically to a standard deviation <0.5% of the absolute value. The radioactive ligand concentrations in the effluent had to be very close to that applied to the column. Experiments without stable baseline in the vicinity of the peak or not giving a good separation of peak and trough were discarded. The amount of bound ligand was calculated from the measured increment in eluate radioactivity coupled to protein elution; this had to be coincident, within experimental error, with the absolute value of the decrement in the trough. The standard deviation of the baseline was taken as an estimate of the standard deviation of measurements of bound ligand. As an example of the sensitivity of this procedure, Fig. 1 shows an equilibrium binding gel chromatography experiment in which the weak binding of [3H]tropolone methyl ether to tubulin was measured [under these conditions a binding equilibrium constant of (7.5 +- 2) x 102 M -~ was s j. M. Andreu and S. N. Timasheff, Biochemistry 21, 534 (1982). 9 j. M. Andreu, M. J. Gorbunoff, J. C. Lee, and S. N. Timasheff, Biochemistry 23, 1742 (1984).
350
MACROMOLECULES" LIGANDS AND LINKAGES
[18]
KD 0
I
r
i
2f7 o
i
[3.],ME
.-~'" ?
• o
•
~-e
.]rME [
~1
+COL °
-2
02 E 0
..j \
.....
I
0
,
.
I
.
.
.
.
.
i
I0
I
20
Fraction number
Flo. 1. Interaction of tropolone methyl ether (TME) with tubulin. The results of a typical equilibrium binding Sephadex G-25 chromatography experiment with 1.05 x 10-4 M [3H]TME (1.4 x 105 cpm m l - 0 in 10 mM sodium phosphate buffer 0.1 mM GTP, pH 7.0 at 18" are shown by the open circles. The changes in ligand concentration of the upper profile were produced by chromatography of 3.2 mg of tubulin, while the middle profile was obtained under identical conditions after the addition of 10-4 M colchicine (COL) to column and sample; the vertical bars show the standard deviation of the measurements. The filled circles are the average of three (upper profile) and two (middle profile) runs, which reduce noise and show unequivocally the position of the peak and trough. Reproduced from Ref. 8.
obtainedS]. These binding measurements involved ligand concentration increments in the order of only 1% of the absolute value and the specificity of these effects was verified by their inhibition by colchicine (see Fig. 1), a high affinity ligand that binds to the same site. 8 The purity of the ligands used was carefully checked since it is well known that the presence of radiochemical impurities in the labeled ligand preparation employed may lead to large errors that have been discussed by others.~° to S. E. Builder and I. H. Segel, Anal. Biochem. 85, 413 (1978).
[18]
PROTEIlq-LIGAND INTERACTIONS
35 1
Measurement of Ligand Concentration by Light Absorption The ligand concentration in the column effluent is often measured spectrophotometrically. This procedure is subject to the same general considerations as radioactive counting. In particular, it relies frequently on the measurement of small absorbance increments due to bound ligand and is therefore subjected to several potential sources of error: interference of light absorption by the protein, absorption increments due to protein effects on bound ligand, interfering light scattering by the protein and instrumental noise. In our case, the ligand concentration measurements were made at a wavelength where the protein does not absorb light. The second effect was not large and could be easily overcome if needed by measuring the ligand absorbance at an isosbestic point of the proteinligand interaction difference spectrum. 9 The third source of error is a systematic one and is important in proteins which, like tubulin, have a tendency to aggregate. The second and third effects were easily avoided by careful addition of a small volume of concentrated sodium dodecyl sulfate to each fraction, giving a final concentration of 0.4% detergent; this displaced the protein-ligand interaction and solubilized any aggregated protein (giving typically A350 ~ 0.001 in the absence of ligand). Since the detergent may also interact with the ligand, the extinction coefficients of the protein and ligand in detergent solution were determined independently. It was also ascertained that the detergent was in excess in the protein-containing fractions of the effluent (that is, above its effective critical micelle concentration in the presence of protein) since otherwise artifactual absorption increments could be generated. Finally, noise was reduced by carefully repeated measurements through the whole column effluent, leading to a good statistical estimation of the baseline concentration. The absorption increments due to bound ligand were estimated by subtraction of the baseline values as in the case of radioactive counting. When small absorbance increments had to be measured this was done from recordings of the difference spectra of the peak fractions versus those from the baseline. 9 Figure 2 shows the elution profile generated by the interaction of 1.8 mg of tubulin chromatographed in a column equilibrated with 1.63 × 10-5 M 2-methoxy-5-(2,3,4-trimethoxyphenyl)tropone at 25 ° and its inhibition by 2 × 10-4 M podophyllotoxin, a ligand that does not absorb light at the wavelength employed and binds to a partially overlapping site. 8,9 Typically, these measurements were useful for ligands binding to protein with equilibrium constants - 103 M -l. Any other procedure to measure ligand concentration that is found to give good accuracy can, in principle, be employed.
352
[18]
MACROMOLECULES: LIGANDS AND LINKAGES i
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FIG. 2. Gel chromatography of tubulin in Sephadex G-25 columns equilibrated with 2methoxy-5-(2',3',4'-trimethoxyphenyl)tropone. (a) Tubulin (1.8 rag) was chromatographed
in a column equilibrated with 1.63 x 10-5 M ligand in 10 mM sodium phosphate buffer 0.1 mM GTP pH 7.0 at 25°. (b) An identical experiment in which the elution buffer contained 1.63 x 10-~ M ligand and 2 x 10-4 M podophyllotoxin. The flow rate was 40 ml/hr and fractions were approximately 1.5 ml. Taken from Ref. 9.
Experimental Criteria of Applicability; Abnormal Profiles; Protein Self-Association There are several easily verifiable characteristics to ensure attainment o f equilibrium in the H u m m e l and D r e y e r technique. 1-3 First, and most important, the ligand concentration immediately before and after the protein peak must return to baseline, so that the peak and the trough are • separated by a region o f nil concentration increment. Any other type o f profile may lead to incorrect measurements. For example, a tailing peak ~hat does not separate from the trough indicates imperfect equilibrium. When the ligand concentration used in the sample is increased or decreased, the rest o f the experiment remaining the same, this will be reflected by a corresponding change in the trough, but the ligand increment o f the peak must remain constant. Second, the number of moles of ligand bound per mole protein must be independent o f small changes of the chromatographic flow, otherwise the separation time is too close to the time needed to attain equilibrium. Third, the binding measured must be independent o f protein concentration. This is easily checked by loading
[18]
353
PROTEIN--LIGAND INTERACTIONS I
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FIG. 3. Interaction of octylglucoside with tubulin. Tubulin (1.5 mg) was chromatographed in a 0.9 x 26-cm Sephacryl S-300 column equilibrated with 2.1 × 10-2 M [14C]octylglucoside in 10 mM sodium phosphate 0.1 mM GTP pH 7.0 at 25°. Three identical experiments were averaged to obtain the upper radioactivity elution profile. In the lower part (o) is the protein elution profile, ( - - - ) is the protein elution profile without detergent, and (o) the protein elution profile with 4.0 x 10 -2 M octylglucoside. From Ref. 13.
different protein amounts on the columns and also, within the same run, by determining the binding separately in each protein containing fraction and looking for any trend with protein concentration. A dependence of ligand binding on protein concentration indicates protein self-association linked to ligand binding tl and therefore the measurements no longer reflect intrinsic ligand binding to monomeric protein, but a combination of the bindings to all macromolecular species. Ignoring such linkage effects can lead to large errors, as has been shown, for example, with the tubulin-vinblastine system.12 On the other hand, ligand-induced protein selfassociation can also give abnormal Hummel and Dreyer profiles. Besides performing the pertinent self-association studies, gross self-association effects induced by ligand may be detected during the binding measurements. For this purpose a chromatographic support that partially includes the protein is used. Figure 3 shows the ligand concentration profiles obtained by interaction of the detergent [14C]octylglucoside with tubulin in Sephacryl S-300 at 25o.13 The amphiphile concentration employed was 2. l × l0 -2 M, just below its critical miceUe concentration and the protein elution profile (filled circles) was not significantly different from one obi1 j. Wyman, Adv. Protein Chem. 19, 223 (1964). 12 G. C. Na and S. N. Timasheff, Biochemistry 19, 1355 (1980). is j. M. Andreu, EMBO. J. 1, 1105 (1982).
354
MACROMOLECULES." LIGANDS AND LINKAGES
[19]
tained without detergent. Increasing the detergent concentration led to an abnormal ligand elution profile in which the peak and trough were not well separated (not shown) together with the bimodal protein profile shown by the open circles in Fig. 3, where most of the protein was no longer included but eluted in the void volume of the column, indicating the formation of large aggregates. Acknowledgment I wish to thank Dr. A. Hargreavesfor his critical readingof the manuscript.
[19] U l t r a f i l t r a t i o n in L i g a n d - B i n d i n g S t u d i e s
By ALKIS J. SOPHIANOPOULOSand JUDITH A. SOPHIANOPOULOS Studies of the reversible interaction of small molecules with macromolecules have played a major role in the unraveling of biological mechanisms. Equilibrium dialysis has been virtually the only method for measuring free ligand directly. The introduction of permselective membranes and related apparatus has facilitated various operations with biological materials such as desalting and concentrating of macromolecular solutions; their use in binding studies however has been limited. There are two major approaches to binding studies using semipermeable membranes. In ultrafiltration, a portion of the contents of the macromolecular solution is filtered through a membrane impermeable to the macromolecule. No components are added to the macromolecular solution in this process. In diafiltration, simultaneous to the filtering process, components are added to the macromolecular solution to maintain its volume nearly constant. It was thought that ultrafiltration would give only approximate values of free ligand in binding studies and thus was not used in highly precise binding studies. It was shown recently that ultrafiltration is theoretically equivalent to equilibrium dialysis.l The conclusion t was that during ultrafiltration, the concentration of free ligand remains constant, although the concentration of the other components increases. During ultrafiltration of a system at equilibrium, although the concentration of the macromoleJ J. A. Sophianopoulos, S. J. Durham, A. J, Sophianopoulos, H. L. Ragsdale, and W. P. Cropper, Arch. Biochem. Biophys. 187, 132 (1978),
METHODS IN ENZYMOLOGY,VOL. 117
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