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Analysis of Lectin–Carbohydrate Interactions by Capillary Affinophoresis
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Analysis of Lectin–Carbohydrate Interactions by Capillary Affinophoresis By Kiyohito Shimura and Ken-ichi Kasai
Introduction
Electrophoresis has long been applied to the analysis of interactions between proteins and ligands. Among these, lectin–carbohydrate interactions represent a typical area where affinity constants have been determined by observing the migration of lectins in gel matrices to which carbohydrates are immobilized.1–3 The migration of lectins is diminished by interaction with the immobilized carbohydrates in these applications. We describe a different approach, in which carbohydrate ligands are attached to soluble ionic polymers. In this case, the electrophoretic migration of lectins is enhanced by the interactions. We refer to the ligand–ionic polymer conjugate as an ‘‘affinophore’’ and electrophoresis using the affinophore as ‘‘affinophoresis.’’4,5 Typically, polyliganded affinophores are prepared by coupling p-aminophenyl glycosides to an anionic polymer, succinylpolylysine, at a glycoside:succinyllysine ratio of about 10%.6 Capillary electrophoresis has many characteristics that recommend it in the analysis of molecular interactions, that is, the ability to analyze interactions in free solutions, a short analysis time, precise temperature control, and a small sample size.7–10 Although polyliganded affinophores are also effective in detecting lectin–carbohydrate interactions in a capillary, they are not suitable for the determination of affinity constants because of the multivalency of most lectins.11 Monoliganded affinophores were developed to solve this problem, and affinity constants between divalent lectins and 1
K. Takeo, in ‘‘Advances in Electrophoresis’’ (A. Chrambach and M. J. Dunn, eds.), Vol. 1, p. 229. VCH, New York, 1987. 2 T. C. Bøg-Hansen, in ‘‘Affinity Chromatography and Molecular Interactions: INSERM Symposia Series’’ (J. M. Egly, ed.), Vol. 86, p. 399. INSERM, Paris, 1979. 3 V. Horˇejsˇi and M. Ticha´, J. Chromatogr. 376, 49 (1986). 4 K. Shimura, J. Chromatogr. 510, 251 (1990). 5 K. Shimura and K. Kasai, Methods Enzymol. 271, 203 (1996). 6 K. Shimura and K. Kasai, J. Chromatogr. 400, 353 (1987). 7 S. Honda, A. Taga, K. Suzuki, S. Suzuki, and K. Kakehi, J. Chromatogr. 597, 377 (1992). 8 Y.-H. Chu, L. Z. Avila, J. Gao, and G. M. Whitesides, Acc. Chem. Res. 28, 461 (1995). 9 K. Shimura and K. Kasai, Anal. Biochem. 251, 1 (1997). 10 N. H. H. Heegaard and R. T. Kennedy, Electrophoresis 20, 3122 (1999). 11 K. Shimura and K. Kasai, Electrophoresis 19, 397 (1998).
METHODS IN ENZYMOLOGY, VOL. 362
Copyright 2003, Elsevier (USA). All rights reserved. 0076-6879/03 $35.00
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carbohydrates were determined by capillary affinophoresis performed in a competitive manner.12,13 Outline of Experimental Procedures
Interactions with an anionic affinophore increase the mobility of a lectin toward the positive electrode and result in a change in detection time for the lectin. The mobility change can be calculated from the change in the detection time. Because mobility change is proportional to the degree of saturation of the binding site of the lectin with the affinophore, the dissociation constants (Kd) between the lectin and the affinophore, and the maximum mobility change (max) of the lectin, can be determined by affinophoresis, using different concentrations of affinophore. Once the Kd and max are determined, the affinity constants for other sugars can be determined by competition experiments.12,13 A neutral sugar competes with the affinophore for binding to the lectin and diminishes the effect of the affinophore. As in enzyme kinetics, the competition apparently increases the Kd value between the lectin and the affinophore. The affinity constant of the lectin and the neutral sugar can be calculated as a Ki value in manner identical to that used for the competitive inhibition of enzyme kinetics. Preparation of Monoliganded Affinophore
Glutathione is used as an affinophore matrix for monoliganded affinophores. A carbohydrate ligand is attached by utilizing the unique reactivity of the thiol group of glutathione with iodoacetylated p-aminophenylglycosides.12,13 Succinylation of the amino group of the glutathione moiety confers three negative charges to the affinophore that can produce a sufficient change in the mobility of the lectins, on binding to it (Fig. 1). p-Aminophenylglycoside (AP-glycoside, 58 mol; Sigma, St. Louis, MO) is dissolved in 1 ml of 2-(N-morpholino)ethanesulfonic acid (MES)–NaOH buffer (pH 6.0), and N-iodoacetoxysuccinimide14 (25 mg, 88 mol) in 80 l of N,N-dimethylformamide is then added to the solution. After a 1-h reaction at room temperature in the dark, a mixed-bed ion-exchange resin (45 mg, AG-X8 resin; Bio-Rad, Hercules, CA) is added to the mixture and it is vortexed mainly to remove free iodoacetic 12
K. Shimura and K. Kasai, J. Biochem. 120, 1146 (1996). K. Shimura, Y. Arata, N. Uchiyama, J. Hirabayashi, and K. Kasai, J. Chromatogr. B 768, 199 (2002). 14 A. Hampton, L. A. Slotin, and R. R. Chawla, J. Med. Chem. 19, 1279 (1976). 13
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Fig. 1. Structure of a monoliganded affinophore bearing a lactoside moiety (sGS-AP-Lac). Reproduced from Ref. 13.
acid formed by the hydrolysis of N-iodoacetoxysuccinimide. The supernatant is recovered and 300 l of 0.5 M sodium phosphate buffer (pH 7.5) containing 25 mM EDTA is added. Glutathione (GSH, reduced form, 23 mg, 75 mol) is added to the mixture, followed by a 2-h reaction at room temperature in the dark. The mixture is acidified to pH 2–3 by the addition of 1 M hydrochloric acid. The coupling product (GS-APglycoside) is purified by high-performance liquid chromatography (HPLC) on a reversed-phase chromatographic column (TSK-Gel ODS-80TS, 4.6 mm i.d. 25 cm) with a cartridge guard column (TSK guard gel ODS-80TS, 3.2 mm i.d. 1.5 cm) (Tosoh, Tokyo, Japan). About one-fifth of each of the reaction mixtures is applied to the column, which had been equilibrated with 0.1% trifluoroacetic acid, and eluted with a gradient of 0 to 25% acetonitrile in 0.1% trifluoroacetic acid over a period of 25 min at a flow rate of 1 ml=min at room temperature, with detection by measurement of the absorption at 280 nm. GS-AP-glycoside, a new peak, is collected, combined, and dried by evaporation. The GS-AP-glycoside preparation is dissolved in 700 l of 0.1 M sodium phosphate buffer (pH 7.5) containing 25 mM EDTA and succinic anhydride, 20 mg, is added to the solution. The mixture is allowed to react for 10 min, while maintaining the pH at 7–8 by the addition of 5 M NaOH. Aliquots, 100 l, of the reaction mixture are acidified to pH 2–3 by the addition of hydrochloric acid, and applied to the chromatographic column, followed by elution under the same conditions as described above. The affinophore, succinylated GS-AP-glycoside (sGS-AP-glycoside), eluting as a new peak, is collected, combined, and dried by evaporation. It is then dissolved in 1 ml of water and stored in a freezer. The concentration of the affinophore is determined by means of the phenol–sulfuric acid method with corresponding sugar solution as a standard. The molecular extinction coefficient of the affinophores in water at 248 nm has been determined to be 10,400 M1 cm1. The overall yield of the affinophore from the p-aminophenylglycoside is 40–50%.
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Electroendosmosis and Capillary Affinophoresis
A distinctive feature of free solution capillary electrophoresis, in comparison with the electrophoresis in gels, is the apparent contribution of electroendosmosis to the results of the electrophoresis. The inner surface of the capillaries used in the affinophoresis is negatively charged, and the application of an electric field generates an asymmetric migration of the counter ions of the immobilized negative charges on the surface. The migrating counter ions push water molecules and produce a plug flow, in which the flow speed is identical irrespective of the distance from the wall. The speed of the flow can be sufficiently high to carry all the molecular species participating in the interactions toward the negative electrode. This enables the detection of the entire sample components injected at the positive end of the capillary in a relatively short time with a fixed detector on the capillary. Electrically neutral species are transported only by electroendosmosis to the detection point (Fig. 2). On the other hand, negatively charged species migrate toward the positive electrode against the overwhelming electroendosmosis, and are detected after the neutral species. The faster the migration toward the positive electrode, the later they are detected. When an anionic affinophore is involved in the electrophoresis, the interaction should increase the detection time for the lectin. Anionic affinophores migrate toward the positive electrode much faster than lectins but not fast enough to move backward against the electroendosmotic flow. The affinophore solution needs to be loaded only in the capillary, and this allows the lectin, injected at the positive end, to interact with the affinophore from the beginning of the electrophoresis to its detection. In competition experiments, a competing sugar needs to be added only in the positive electrode buffer. The neutral sugar is transported along the capillary only by the electroendosmosis. The lectin and the affinophore electrophoretically migrate into the zone of the neutral sugar, and competition can then be observed (Fig. 2). Calculation of Mobility Change
To determine the affinity constant, the mobility changes of a lectin must be calculated from the detection time obtained under different conditions of affinophoresis. In free solution capillary electrophoresis, the detection time is a function of both the electrophoretic mobility of a sample and the electroendosmotic mobility of a capillary. Coelectrophoresis with a reference molecule, that is, an electrophoresis marker that does not interact
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Fig. 2. Capillary affinophoresis and electroendosmosis. (A1) A sample solution is injected at the positive end of the capillary. (A2) Application of an electric field generates electroendosmosis due to the negatively charged surface of the capillary. If the lectin is negatively charged and migrates toward the positive side, a neutral marker is detected before the lectin. (B1) In the case of affinophoresis, an affinophore is included only in the capillary. (B2) The affinophore migrates toward the positive end much faster than the lectin, and the lectin migrates in the affinophore solution until it is detected. Interaction between the lectin
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with the affinophore, allows the calculation of the mobility change from a change in the detection time as shown below.15,16 The detection time (t) of a lectin is a function of its electrophoretic mobility () as well as of the electroendosmotic mobility (eo) of a capillary: t¼
L 1 E þ eo
ð1Þ
where L is the distance (cm) from the injection end to a detection point, and E is the field strength (V=cm). The same relation should be applied for the detection time of an electrophoresis marker (tr): tr ¼
L 1 E r þ eo
ð2Þ
where r represents the electrophoretic mobility of the marker. When the lectin and the marker are subjected to simultaneous electrophoresis, eo, E, and L are the same in Eqs. (1) and (2). As a result, the difference between the reciprocals of each detection time is proportional to the difference between the mobility of the lectin and the marker, that is, r. 1 1 E E ¼ ½ð þ eo Þ ðr þ eo Þ ¼ ð r Þ t tr L L
ð3Þ
Thus the mobility difference between the lectin and the marker in the same run can be expressed as follows: L 1 1 r ¼ ð4Þ E t tr The mobility difference in the presence of the affinophore can also be calculated using Eq. (4). When the mobility of the lectin changes from 0 to by the affinophoresis, the mobility change ð ¼ 0 ) induced by the affinophoresis can be extracted from the two mobility differences from the marker in the presence and the absence of the affinophore, that is, and the affinophore increases the mobility of the lectin and retards its detection. (C1) To observe competition by a neutral sugar, it is included only in the positive electrode solution. (C2) The neutral sugar is rapidly transported toward the negative side by electroendosmosis, and the lectin migrates in a mixed zone of the affinophore and the neutral sugar. Effective competition suppresses the acceleration of the electrophoresis of the lectin by the affinophore. 15 16
K. Shimura and K. Kasai, Anal. Biochem. 227, 186 (1995). F. A. Gomez, L. Z. Avila, Y.-H. Chu, and G. M. Whitesides, Anal. Chem. 66, 1785 (1994).
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¼ 0 ¼ ð r Þ ð0 r Þ L 1 1 1 1 ¼ E t tr t0 tr0
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ð5Þ
where t0 is the detection time of lectin in the absence of the affinophore. The values of tr and t0 r might be different because of variations in the electroendosmosis between the two runs. The calculation of by Eq. (5) permits the variation in electroendosmosis between the runs to be determined. Instrument and Capillary
A capillary electrophoresis instrument with an ultraviolet absorption detector is required. The instrument should have a temperature-controlling system for the capillary, because the interactions occur during the electrophoresis run. Fused silica capillaries (25–50 m i.d., 375 m o.d.) with a polyimide covering (Polymicro Technologies, Phoenix, AZ) should be suitable for most applications. A fused silica capillary has a tendency to adsorb proteins mainly because of the anionic characteristics of its surface caused by the dissociation of silanol groups. The adsorption largely depends on the nature of the particular protein under consideration. If the protein does not pose any problems related to adsorption, that is, the electropherograms are reproducible without obvious tailing and broadening of peaks, the use of a fused silica capillary is the most straightforward. Coating of the inner surface of the capillary may alleviate the problem, should it occur. Capillary Coating
We typically use capillaries that are coated with an anionic polymer, succinylpolylysine. An epoxy group is introduced onto the inner surface of the capillary by reaction with an epoxysilane and polylysine is then reacted to cover the surface. Finally, the amino function of polylysine is succinylated.15 An aqueous solution of 3-glycidoxypropyltrimethoxysilane [5% (v=v)] is adjusted to pH 5.5–5.8 with 5 mM KOH and clarified by centrifugation. The fused silica capillary (60 cm long) is filled with the solution and the ends are closed by inserting them into rubber septa used for gas chromatography. The capillaries are heated in a boiling water bath for 30 min and rinsed by passing 0.1 ml each of water and acetone through them, followed by drying by passing air by suction. The capillaries are filled with
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a poly(l-lysine) solution (Sigma; average degree of polymerization, 270; 10 mg=ml in 1 M NaCl; adjusted to pH 10 with NaOH) and reacted overnight at room temperature. They are then washed with 0.1 ml of water and filled with a mixture [1:1 (v=v)] of succinic anhydride in dimethylformamide (25 mg=ml) and N-ethylmorpholine. They are allowed to react for 30 min at room temperature and, finally, rinsed with 0.1 ml of water. The coating procedure can be facilitated by the use of a syringe (50– 100 l; a pressure-lock or gas-tight syringe with a 26-gauge needle with a tip cut at 90 ), with a segment of Teflon tubing (2 cm long, 0.3 mm i.d. 1.5 mm o.d.; Alltech Associates, Deerfield, IL) attached to the tip of the needle. The tube is fitted to both the needle of the syringe and the capillary (375 m o.d.) by thrusting the tips of them into each end of the tube after softening by mild heating with an electric heater. After cooling, the capillary can be detached and reconnected without heating, and the connection permits the rapid injection of solutions into capillaries with the syringe. Equations for Affinophoresis
Most lectins are divalent and must be treated accordingly in the analysis. We start with the equations for affinophoresis of monovalent proteins and then return to divalent systems. For a monovalent lectin, L, and an affinophore, A, we assume the following binding equilibrium: L þ A Ð LA
ð6Þ
The original electrophoretic mobility of the lectin is 0 and that of the complex with the affinophore is c. Because the lectin and the affinophore are in dynamic binding equilibrium, the macroscopic mobility of the lectin, , in equilibrium should be the weighted average of 0 and c: ¼ ð1 Þ0 þ c
ð7Þ
The weight, , is the degree of saturation of the lectin with the affinophore and is a function of the dissociation constant Kd (Kd ¼ [L][A]= [LA]) of the complex and the concentration of the affinophore, [A]: ¼
½A Kd þ ½A
ð8Þ
From Eq. (7) and (8), the mobility change ( ¼ 0) of the lectin by affinophoresis can be expressed as follows:
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¼ max
½A Kd þ ½A
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ð9Þ
where max (max ¼ c 0) represents the maximum mobility change of the lectin. It should be noted that the equation has the same form as the Michaelis–Menten equation for enzyme kinetics and, thus, the Kd and max can be determined in a manner similar to that used for determining Km and Vmax in enzyme kinetics. A plot of versus =[A] according to the following linear equation yields a straight line that intercepts the ordinate at max with a slope of Kd: ¼ max Kd
½A
ð10Þ
We call this a ‘‘Woolf–Hofstee plot’’ after the investigators who contributed to the development of the corresponding plot in enzyme kinetics.17 The values of and =[A] for each run can be easily calculated with spreadsheet software by providing t, tr, and [A] as input data. Divalency
The lectin is assumed to be monovalent in the above-described treatments. In the case of divalent lectins, two monoliganded affinophores can simultaneously bind to the lectin. An identical analysis is applicable to such dimeric lectins under the following conditions: (1) the two binding sites are equivalent; (2) the two binding sites are independent; and (3) the mobility change induced by the binding of the second affinophore is identical to that induced by the first one.12 In the case of a homodimeric lectin, the two binding sites are apparently equivalent. The independency of the binding sites cannot be known in advance. It will, however, be revealed by a linear relation of the plot according to Eq. (10). The mobility change caused by the successive binding of each affinophore can be considered to be approximately identical, because the electrophoretic mobility is proportional to the number of charges on a particle with an identical electrophoretic drag, that is, a negligible change in the molecular size of the lectin on binding with the affinophores. The relatively small molecular size of the monoliganded affinophore in comparison with a protein should warrant this approximation. In the analysis of divalent lectins, it should be noted that the Kd that appears in the equations is that for a single binding site and not for the entire protein.
17
M. Dixon and E. C. Webb, in ‘‘Enzymes,’’ 3rd ed., p. 62. Longman, London, 1979.
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Affinophoresis
The buffer for the electrophoresis should not have a large electric conductivity. When 0.1 M Tris–acetic acid buffer (pH 7.9) is used as in the example described below, the current is about 25 A at 300 V=cm with a 50 m i.d. capillary. As a criterion, electric power at less than 1 W=m of capillary length can be considered safe. At 350 V=cm, 30 A corresponds to about 1 W=m. An affinophore solution is included only in the capillary (Fig. 2). The volume of the capillary is less than 1 l and this is the volume of affinophore solution actually consumed in a single run of affinophoresis. Experimentally, however, it would be better to prepare affinophore solutions in a greater volume (about 50 l) in order to ensure precision in the concentration of the affinophore against the effect of evaporation, as well as possible errors in the use of micropipettes. Cooling of the vial rack is effective in minimizing the evaporation of water. Evaporation depends on the humidity of the surrounding air and overcooling might result in the condensation of water under conditions of high humidity. An overlay of a small quantity of mineral oil has been shown not to interfere with the injection processes and to be effective in suppressing evaporation.18,19 A lectin zone formed by injecting a sample does not contain affinophore and there should be a presteady state period before achieving a binding equilibrium between a lectin sample and the affinophore. During this period, the macroscopic mobility of the lectin gradually changes from 0 to as expressed by Eq. (7). The length of the period is a function of the relative concentration of the lectin to that of affinophore. When this period is not negligibly small, the mobility change of the lectin becomes smaller than would be predicted from Eq. (9), especially at low concentration ranges of affinophore. This results in a downward curvature of the plot according to Eq. (10) in the vicinity of the abscissa. The concentration of the lectin sample should be nearly identical to or lower than that of the affinophore on a molar basis. In the following experiments, lectin solutions of 0.2–1.0 g=l are used with absorption detection at 214 nm. This phenomenon might be problematic, when the affinity for an affinophore is high, where considerable binding occurs, even at low affinophore concentration. As an example, the affinophoresis of pea lectin (Pisum sativum) using a mannoside affinophore is shown in Fig. 3. Pea lectin is a homodimeric lectin
18 19
N. H. H. Heegaard, J. W. Sen, and M. H. Nissen, J. Chromatogr. A 894, 319 (2000). K. Shimura, N. Uchiyama, and K. Kasai, Electrophoresis 22, 3471 (2001).
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Fig. 3. Affinophoresis of pea lectin with a mannoside. Left: A sample (0.7 nl) containing pea lectin (**, 0.2 g=l) and cytidine (*, 0.2 mM) was injected by means of pressure at the positive end of a capillary (25 m i.d., 375 m o.d., 57 cm total length, 50 cm separation distance, and inner-coated with succinylpolylysine) filled with a solution of the affinophore (sGS-AP-Man). Electrophoresis was carried out at a field strength of 350 V=cm (7 A) at 25 , with detection at A214. Tris–acetate buffer (0.1 M, pH 7.9) was used throughout the system. The concentration of the affinophore was (a) 0 mM; (b) 0.05 mM; (c) 0.1 mM; (d) 0.2 mM; (e) 0.3 mM; (f) 0.4 mM; (g) 0.5 mM. Right: A plot according to Eq. (10) was prepared, using the data shown on the left, in order to determine the Kd and max values. Reproduced from Ref. 12 .
with a molecular mass of 49,000 Da and has two carbohydrate-binding sites.20 Its structure is closely related to concanavalin A and the affinity constants for mannose and some other carbohydrates have been reported.20,21 20 21
I. S. Trowbridge, J. Biol. Chem. 249, 6004 (1974). F. P. Schwarz, K. D. Puri, R. G. Bhat, and A. Sorolia, J. Biol. Chem. 268, 7668 (1993).
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The main component of pea lectin has been purified by anion-exchange chromatography and this preparation is used in the experiments. Electrophoresis is carried out with an automated capillary electrophoresis instrument (P=ACE 2210 with a UV detector; Beckman, Fullerton, CA). A fused silica capillary (25 m i.d., 375 m o.d.) coated with succinylpolylysine is installed in a capillary cartridge with a separation distance of 50 cm, from the positive end to the detection point. The capillary is filled with an electrophoresis buffer (0.1 M Tris–acetic acid buffer, pH 7.9, containing 0.02% NaN3 as a preservative) with or without the mannoside affinophore (sGSPA-Man), containing p-aminophenyl--d-mannoside as an affinity ligand, using a pressure of 20 lb=in2 for 3 min. The sample, which contains pea lectin (0.2 g=l) and 0.2 mM cytidine as a neutral marker in the electrophoresis buffer, is injected at the positive end for 10 s under a pressure of 0.5 lb=in2 (injection volume of 0.67 nl, which corresponds to 0.13 ng of pea lectin). Electrophoresis is carried out at a field strength of 350 V=cm with an electric current of 6.7 A. The cartridge temperature is set at 25 and pea lectin and cytidine are detected by absorption measurement at 214 nm. The electrophoresis buffer is also used as an electrode solution in each electrode vessel. The solution of pea lectin (20 l) and that of the affinophore (50 l) at different concentrations are placed in polypropylene microvials and the vials are set in a cooled (typically 10 ) rotating tray to minimize evaporation. After electrophoresis, the capillary is rinsed with 0.1 M sodium carbonate buffer (pH 10) containing 1 M NaCl and 10 mM EDTA in the high-pressure rinse mode for 3 min and then with water for 3 min. In the absence of affinophore, the mobility of the lectin is low as indicated by the close separation of the peaks from that of the neutral marker, cytidine. The detection time of the lectin gradually increases as the concentration of the affinophore becomes higher, indicating binding of the affinophore. The mobility change, , at each concentration of the affinophore is calculated using Eq. (5), and is plotted against =[A] according to Eq. (10) (Fig. 3, right). A linear regression provides Kd as the slope of the line and max at the intercept with the ordinate, that is, Kd ¼ 0.202 mM and max ¼ 5.25 105 cm2 V1 s1. Five identical runs yield an average value for Kd of 0.199 mM (coefficient of variation, 6.0%) and max of 5.35 105 cm2 V1 s1 (coefficient of variation, 1.6%). Competitive Capillary Affinophoresis
The addition of a neutral sugar to the affinophoresis system results in suppression of the mobility change induced by the affinophore. As described in the previous section, a neutral sugar can be added only in the buffer in the positive electrode reservoir. The electroendosmosis in
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the capillary transports the sugar solution toward the negative end, and a lectin and an affinophore migrate in the neutral sugar zone (Fig. 2). The volume of the positive electrode buffer containing the neutral sugar can be as small as 100 l. The concentration of an affinophore should be set so as to be between the Kd and 2 Kd. Other conditions should be the same as those used for the determination of Kd and max of the affinophore. The detailed experimental conditions for a competitive capillary affinophoresis experiment using a lactoside affinophore are described for the determination of affinity constants of recombinant human galectins for neutral sugars. Recombinant human galectin-1 (rhGal-1) is a dimeric protein composed of two identical 14-kDa polypeptide chains with 134 amino acid residues.22 The apparent difference between rhGal-1 and the native galectin-1 found in human tissues is the absence of an acetyl group at the N terminus of rhGal-1. The C2S mutant of rhGal-1 has one amino acid substitution at the second residue from the N terminus, from cysteine to serine. This mutation at one of the six cysteine residues found in galectin-1 has been reported to substantially increase the stability of sugar-binding activity of the lectin in the absence of reducing agents.22 A fused silica capillary (50 m i.d., 375 m o.d.) coated with succinylpolylysine is used, with a separation distance of 20 cm. The electrophoresis instrument and the detection system are the same as those used in the experiments described for pea lectin, and the cartridge temperature is set at 20 . Solutions, 20 l each of a lactoside affinophore (sGS-AP-Lac) bearing p-aminophenyl--lactoside as an affinity ligand, a lectin at 1 g=l, and references of 1 mM acrylamide and 0.3 mM acetyltryptophan in the electrophoresis buffer, are placed in polypropylene minivials (200 l) and overlaid with 20 l each of mineral oil (light white oil, density ¼ 0.84 g=ml; g=ml; Sigma) to prevent the evaporation of water.18,19 The capillary is filled with the affinophore solution at a pressure of 20 lb=in2 for 30 s. A solution of electrophoresis markers and that of the lectin are consecutively injected at the positive end for 1 s each under a pressure of 0.5 lb=in2 (injection volume of 2.2 nl of each solution). Electrophoresis is carried out at a field strength of 300 V=cm with an electric current of about 25 A, using a neutral sugar solution of 200 l in the electrophoresis buffer as the positive electrode solution. After electrophoresis, the capillary is rinsed with water at a pressure of 20 lb=in2 for 3 min. The electropherograms shown in Fig. 4 were obtained by the affinophoresis of the C2S variant of rhGal-1 with the lactoside affinophore. The Kd value of the lectin for the affinophore was determined to be
22
J. Hirabayashi and K. Kasai, J. Biol. Chem. 266, 23648 (1991).
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Fig. 4. Competition of affinophoresis of the C2S variant of rhGal-1 by N-acetyllactosamine. Affinophoresis of C2S (1 g=l) was carried out with the lactoside affinophore (sGSAP-Lac) at a concentration of 0.51 mM in a succinylpolylysine-coated capillary (50 m i.d., 375 m o.d., 23 cm total length, 20 cm separation distance) with a field strength of 300 V=cm at 20 . N-Acetyllactosamine (LacNAc) was added to the electrophoresis buffer at the anode at the concentrations indicated above each electropherogram. Acrylamide and acetyltryptophan (Ac-Trp) were injected with the lectin sample as electrophoresis markers. Detection was carried out by absorption measurement at 214 nm. Reproduced from Ref. 13 .
0.40 mM, with a max value of 4.69 105 cm2 V1 s1. The affinophore at a concentration of 0.51 mM lengthens the detection time for the lectin by about 1 min (Fig. 4, second electropherogram from top). The addition of N-acetyllactosamine to the affinophoresis system cancels the effect of the affinophore, and the detection time is reduced as a function of the concentration of N-acetyllactosamine (Fig. 4, third and fourth electropherograms from top).
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Calculation of Affinity Constants for Neutral Sugars
The mobility change of the lectin in the presence of the affinophore and the neutral sugar, i, can be written as follows: i ¼ max
½A Kd app þ ½A
ð11Þ
where the Kd app is the apparent dissociation constant for the affinophore in the presence of the neutral sugar, and has a relation with the dissociation constant, Ki (Ki ¼ [L][I]=[LI]), for a neutral sugar, I: ½I Kd app ¼ Kd 1 þ ð12Þ Ki Because max has already been determined, Kd app can be calculated by measurement of i in the presence of a neutral sugar according to Eq. (11). Kd is also already known by the plot of Eq. (10) and Ki can be calculated from Eq. (12). Alternatively, Eq. (12) can be rearranged as Kd app ½I ¼ þ1 Ki Kd
ð13Þ
A plot of Kd app=Kd against different [I] should result in a straight line with a slope of 1=Ki. It is preferable to use the competing sugar at concentrations around its Ki value. The results of competition experiments with some neutral sugars in the affinophoresis of pea lectin with the mannoside affinophore are plotted according to Eq. (13) (Fig. 5). The reciprocal of the slope of the lines corresponds to the dissociation constant Ki for the lectin–neutral sugar complexes. The values are compared with those determined by calorimetry21 (Table I). The affinophoresis is carried out at 25 but some of the calorimetry values are determined at different temperatures. A close agreement is found for methyl--d-mannoside and d-mannose, which are determined at identical temperatures. Other calorimetry values are determined at 16 or 14 and are considerably smaller than those obtained by affinophoresis. This discrepancy can be largely attributed to the difference in temperature used in the different determinations and the positive H values for the dissociation of the pea lectin–sugar complexes, that is, 10 and 13.6 kJ=mol kJ=mol for d-glucose and methyl--d-glucoside, respectively.21 The dissociation constants of recombinant human galectin-1 (rhGal-1), its C2S variant,22 and recombinant human galectin-3 (rhGal-3)23,24 for 23 24
Y. Oda, H. Leffler, Y. Sakakura, K. Kasai, and S. H. Barondes, Gene 99, 279 (1991). J. Hirabayashi, Y. Sakakura, and K. Kasai, in ‘‘Lectins and Glycobiology’’ (H.-J. Gabius and S. Gabius, eds.), p. 474. Springer-Verlag, New York, 1993.
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capillary affinophoresis
Fig. 5. Determination of the dissociation constants of pea lectin for neutral sugars (I) through competition experiments of affinophoresis using the mannoside affinophore. The results are plotted according to Eq. (13), (d) p-Aminophenyl--d-mannoside; ( ) methyl-d-mannoside; (m) d-mannose; (4) maltose; (&) methyl--d-glucoside; (h) d-glucose. Reproduced from Ref. 12 .
various neutral sugars, as determined by capillary affinophoresis, are summarized in Fig. 6. The striking preference of rhGal-1 and its C2S variant for N-acetyllactosamine [Gal(1–4)GlcNAc] and lactose [Gal(1–4)Glc] is clear, as has been repeatedly reported.25,26 The affinity of the two lectins for N-acetyllactosamine, lactose, and galactose is hardly distinguishable, indicating that the effect of the substitution of the second cysteine residue of rhGal-1 to a serine residue on affinity is negligible, as has been previously noted.22 The most relevant work that can be compared with the present results for rhGal-1 is that reported by Lee et al.26 They have reported the concentrations, I50, for various sugars that cause a 50% reduction in the binding of 125I-labeled galectin-1 to lactose-immobilized agarose beads. The values of I50, with relative values of 1=I50 to lactose in parentheses, are as follows: N-acetyllactosamine, 0.13 mM (350%); lactose, 0.46 mM (100%); methyl--galactoside, 26 mM (1.8%); melibiose [Gal(1–6)Glc], 25 26
C. P. Sparrow, H. Leffler, and S. H. Barondes, J. Biol. Chem. 262, 7383 (1987). R. T. Lee, Y. Ichikawa, H. J. Allen, and Y. C. Lee, J. Biol. Chem. 265, 7864 (1990).
414
[28]
general techniques TABLE 1 Affinity Constants of Pea Lectin for Neutral Sugars
Sugar
Affinophoresisa (mM)
Calorimetryb
p-Aminophenyl--d-mannoside Methyl--d-mannoside d-Mannose Maltose Methyl--d-glucoside d-Glucose
0.25 0.65 1.6 2.1 2.4 3.6
Not determined 0.61 mMc 1.3 mMd Not determined 1.3 mMe 2.8 mMf
a
0.1 M Tris–acetic acid buffer (pH 7.9) containing 0.02% NaN3, 25 (Ref. 12). 0.02 M sodium phosphate buffer (pH 7.4) containing 0.15 M NaCl (Ref. 21). c 25 . d 24.3 . e 16 . f 14.2 . b
30 mM (1.5%); galactose, 63 mM (0.7%) and methyl--galactoside, 85 mM (0.5%). The Ki values of C2S and its relative affinities determined by affinophoresis are as follows, in the same order as above: N-acetyllactosamine, 0.067 mM (352%); lactose, 0.23 mM (100%); methyl--galactoside, 16 mM (1.5%); melibiose, 21 mM (1.1%); galactose, 30 mM (0.78%); methyl--galactoside, 39 mM (0.6%). Although the I50 values are about twice as large as the Ki values determined by capillary affinophoresis, the consistency of the relative magnitude of the affinity is remarkable. When lactose-immobilized beads are used at a concentration equivalent to the Kd value for the lectin and the modified beads, as was the case in their experiments,26 the I50 value for each sugar should be twice as large as the corresponding Ki value. This theory may simply explain the difference, although there are some additional factors to be considered, that is, the divalency of the lectin in the interaction with the lactose-immobilized beads and the difference in temperature between their experiments, 25 , and ours, 20 . rhGal-3 also has an exclusively high affinity for N-acetyllactosamine and lactose. rhGal-3 was shown to have a 2 to 10 times higher affinity than rhGal-1 for sugars containing galactose at their nonreducing ends, whereas the affinities for sucrose [Glc(1-2)Fru] are almost the same. This result indicates a higher preference of galectin-3 for galactosides than galectin-1. The preference for methyl--galactoside over methyl--galactoside is not observed for rhGal-3. Sparrow et al. report I50 values for a variety of sugars
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415
Fig. 6. Dissociation constants of recombinant human galectins for neutral sugars determined by capillary affinophoresis. The Ki values (M) are the dissociation constants for a single binding site of lectins. rhGal-1, Recombinant human galectin-1; C2S, a C2S variant of rhGal-1; rhGal-3, recombinant human galectin-3; Gal, d-galactose; Man, d-mannose; Glc, d-glucose; Fru, d-fructose; GalNAc, N-acetyl-d-galactosamine; GlcNAc, N-acetyl-d-glucosamine; IPTG, isopropyl--d-thiogalactoside; me-, methyl. Original data were taken from Ref. 13 .
and oligosaccharides in the binding of human galectin-3 (called HL-29 at that time) to asialofetuin–agarose beads.25 A portion of the data that are relevant to our results is as follows (with a relative value of 1=I50 to lactose in parentheses): lactose (100%), N-acetyllactosamine (1130%), galactose (1.8%), methyl--galactoside (2.6%), and methyl--galactoside (1.6%). Overall agreement in the relative affinity data based on the I50 values and Ki values of our determinations by capillary affinophoresis
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is apparent, although the affinity for N-acetyllactosamine has been emphasized in their data. Divalency Revisited
The monoliganded affinophore was developed to circumvent difficulty related to the divalent nature of lectins. After the determination of affinity constants for neutral sugars had been established by capillary affinophoresis, we attempted a new approach for estimating the degree of contribution of divalent interactions between lectins and polyliganded matrices. The interactions between a divalent pea lectin and polyliganded affinophores, succinylpolylysine bearing a p-aminophenyl--d-mannoside at different ligand densities, were investigated.11 The overall dissociation constant between the lectin and the affinophore was determined by capillary affinophoresis to show a higher affinity for the affinophore with higher ligand densities. The overall interactions should comprise both monovalent and divalent interactions. When a neutral sugar, methyl -d-mannoside, interferes with these interactions, the contribution of divalent interactions should decrease more rapidly than monovalent interactions. The reason for this is that one binding site is already occupied with the neutral sugar, and the lectin should behave as a monovalent molecule. The ratio of the temporary monovalent lectins to the divalent lectins with two free binding sites increases with increasing degree of saturation by the neutral sugar. When affinophoresis using a polyliganded affinophore was interfered with by a neutral sugar, the plot according to Eq. (13) became nonlinear with a gradual increase in the slope with increasing concentration of neutral sugar, indicating the decreasing contribution of divalency11: the more curvature, the higher the contribution of divalent interactions. Because the Ki value of the neutral sugar had already been determined, nonlinear regression allows the determination of two elemental equilibrium constants, one for the monovalent binding (K1 ¼ [L][A]=[LA], where LA denotes the monovalent complexes) and the other for the divalent interactions (K2 ¼ [LA]=[L ¼ A], where L ¼ A denotes the divalent complexes). The divalency value, representing the mole fraction of the divalent species in the complex, can be calculated from K2 values. Polyliganded affinophores bearing a mannoside ligand at different ligand densities from 4.2 to 17.5% of mannoside over a succinyllysine residue were found to interact with pea lectin with divalency values of 24 to 62%.10 This principle, based on competition with a monovalent ligand, should be applicable to the analysis of interactions with polyliganded cell surface or extracellular matrices in conjunction with a wide variety of techniques for analyzing molecular interactions.
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417
Conclusion
Capillary affinophoresis, performed with an automated electrophoresis instrument, is a useful technique for determining the affinity constants of divalent lectins for neutral sugars. The affinity constants obtained are strictly for a single binding site and are free from the components related to multivalent interactions. These values should be useful in comparing affinities of various lectins and sugars. Once a suitable affinophore is established for a target lectin, the method can be carried out with a relatively small amount of lectin sample and time for a variety of sugars with good precision and reproducibility.
[29] Describing Topology of Bound Ligand by Transferred Nuclear Overhauser Effect Spectroscopy and Molecular Modeling By Hans-Christian Siebert, Jesu´ s Jime´ nez-Barbero, Sabine Andre´ , Herbert Kaltner, and Hans-Joachim Gabius Introduction
The perspective needed to translate progress in unraveling the role of protein–carbohydrate interactions in clinically relevant processes (e.g., initiation of infections by pathogen attachment or lectin-dependent endocytosis) into therapeutic concepts is one reason for the structural analysis of lectin–ligand complexes.1–4 Despite the exquisite sensitivity of powerful X-ray diffraction techniques, concerns about whether the crystal structures fully reflect structural details in solution cannot be ignored.5,6 To address this issue, nuclear magnetic resonance (NMR) spectroscopy can provide salient input on ligand topology even without meeting the requirement for a complete signal assignment. This is especially helpful when the size 1
Y. C. Lee and R. T. Lee, J. Biomed. Sci. 3, 221 (1996). K.-A. Karlsson, Mol. Microbiol. 29, 1 (1998). 3 H. Ru¨ dı´ger, H.-C. Siebert, D. Solı´s, J. Jime´ nez-Barbero, A. Romero, C.-W. von der Lieth, T. Dı´az-Maurin˜ o, and H.-J. Gabius, Curr. Med. Chem. 7, 389 (2000). 4 N. Yamazaki, S. Kojima, N. V. Bovin, S. Andre´ , S. Gabius, and H.-J. Gabius, Adv. Drug Deliv. Rev. 43, 225 (2000). 5 G. Wagner, S. G. Hyberts, and T. F. Havel, Annu. Rev. Biophys. Biomol. Struct. 21, 167 (1992). 6 M. W. MacArthur, P. C. Driscoll, and J. M. Thornton, Trends Biotechnol. 12, 149 (1994). 2
METHODS IN ENZYMOLOGY, VOL. 362
Copyright 2003, Elsevier (USA). All rights reserved. 0076-6879/03 $35.00
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general techniques
Analysis of Lectin–Carbohydrate Interactions by Capillary Affinophoresis By Kiyohito Shimura and Ken-ichi Kasai
Introduction
Electrophoresis has long been applied to the analysis of interactions between proteins and ligands. Among these, lectin–carbohydrate interactions represent a typical area where affinity constants have been determined by observing the migration of lectins in gel matrices to which carbohydrates are immobilized.1–3 The migration of lectins is diminished by interaction with the immobilized carbohydrates in these applications. We describe a different approach, in which carbohydrate ligands are attached to soluble ionic polymers. In this case, the electrophoretic migration of lectins is enhanced by the interactions. We refer to the ligand–ionic polymer conjugate as an ‘‘affinophore’’ and electrophoresis using the affinophore as ‘‘affinophoresis.’’4,5 Typically, polyliganded affinophores are prepared by coupling p-aminophenyl glycosides to an anionic polymer, succinylpolylysine, at a glycoside:succinyllysine ratio of about 10%.6 Capillary electrophoresis has many characteristics that recommend it in the analysis of molecular interactions, that is, the ability to analyze interactions in free solutions, a short analysis time, precise temperature control, and a small sample size.7–10 Although polyliganded affinophores are also effective in detecting lectin–carbohydrate interactions in a capillary, they are not suitable for the determination of affinity constants because of the multivalency of most lectins.11 Monoliganded affinophores were developed to solve this problem, and affinity constants between divalent lectins and 1
K. Takeo, in ‘‘Advances in Electrophoresis’’ (A. Chrambach and M. J. Dunn, eds.), Vol. 1, p. 229. VCH, New York, 1987. 2 T. C. Bøg-Hansen, in ‘‘Affinity Chromatography and Molecular Interactions: INSERM Symposia Series’’ (J. M. Egly, ed.), Vol. 86, p. 399. INSERM, Paris, 1979. 3 V. Horˇejsˇi and M. Ticha´, J. Chromatogr. 376, 49 (1986). 4 K. Shimura, J. Chromatogr. 510, 251 (1990). 5 K. Shimura and K. Kasai, Methods Enzymol. 271, 203 (1996). 6 K. Shimura and K. Kasai, J. Chromatogr. 400, 353 (1987). 7 S. Honda, A. Taga, K. Suzuki, S. Suzuki, and K. Kakehi, J. Chromatogr. 597, 377 (1992). 8 Y.-H. Chu, L. Z. Avila, J. Gao, and G. M. Whitesides, Acc. Chem. Res. 28, 461 (1995). 9 K. Shimura and K. Kasai, Anal. Biochem. 251, 1 (1997). 10 N. H. H. Heegaard and R. T. Kennedy, Electrophoresis 20, 3122 (1999). 11 K. Shimura and K. Kasai, Electrophoresis 19, 397 (1998).
METHODS IN ENZYMOLOGY, VOL. 362
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carbohydrates were determined by capillary affinophoresis performed in a competitive manner.12,13 Outline of Experimental Procedures
Interactions with an anionic affinophore increase the mobility of a lectin toward the positive electrode and result in a change in detection time for the lectin. The mobility change can be calculated from the change in the detection time. Because mobility change is proportional to the degree of saturation of the binding site of the lectin with the affinophore, the dissociation constants (Kd) between the lectin and the affinophore, and the maximum mobility change (max) of the lectin, can be determined by affinophoresis, using different concentrations of affinophore. Once the Kd and max are determined, the affinity constants for other sugars can be determined by competition experiments.12,13 A neutral sugar competes with the affinophore for binding to the lectin and diminishes the effect of the affinophore. As in enzyme kinetics, the competition apparently increases the Kd value between the lectin and the affinophore. The affinity constant of the lectin and the neutral sugar can be calculated as a Ki value in manner identical to that used for the competitive inhibition of enzyme kinetics. Preparation of Monoliganded Affinophore
Glutathione is used as an affinophore matrix for monoliganded affinophores. A carbohydrate ligand is attached by utilizing the unique reactivity of the thiol group of glutathione with iodoacetylated p-aminophenylglycosides.12,13 Succinylation of the amino group of the glutathione moiety confers three negative charges to the affinophore that can produce a sufficient change in the mobility of the lectins, on binding to it (Fig. 1). p-Aminophenylglycoside (AP-glycoside, 58 mol; Sigma, St. Louis, MO) is dissolved in 1 ml of 2-(N-morpholino)ethanesulfonic acid (MES)–NaOH buffer (pH 6.0), and N-iodoacetoxysuccinimide14 (25 mg, 88 mol) in 80 l of N,N-dimethylformamide is then added to the solution. After a 1-h reaction at room temperature in the dark, a mixed-bed ion-exchange resin (45 mg, AG-X8 resin; Bio-Rad, Hercules, CA) is added to the mixture and it is vortexed mainly to remove free iodoacetic 12
K. Shimura and K. Kasai, J. Biochem. 120, 1146 (1996). K. Shimura, Y. Arata, N. Uchiyama, J. Hirabayashi, and K. Kasai, J. Chromatogr. B 768, 199 (2002). 14 A. Hampton, L. A. Slotin, and R. R. Chawla, J. Med. Chem. 19, 1279 (1976). 13
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Fig. 1. Structure of a monoliganded affinophore bearing a lactoside moiety (sGS-AP-Lac). Reproduced from Ref. 13.
acid formed by the hydrolysis of N-iodoacetoxysuccinimide. The supernatant is recovered and 300 l of 0.5 M sodium phosphate buffer (pH 7.5) containing 25 mM EDTA is added. Glutathione (GSH, reduced form, 23 mg, 75 mol) is added to the mixture, followed by a 2-h reaction at room temperature in the dark. The mixture is acidified to pH 2–3 by the addition of 1 M hydrochloric acid. The coupling product (GS-APglycoside) is purified by high-performance liquid chromatography (HPLC) on a reversed-phase chromatographic column (TSK-Gel ODS-80TS, 4.6 mm i.d. 25 cm) with a cartridge guard column (TSK guard gel ODS-80TS, 3.2 mm i.d. 1.5 cm) (Tosoh, Tokyo, Japan). About one-fifth of each of the reaction mixtures is applied to the column, which had been equilibrated with 0.1% trifluoroacetic acid, and eluted with a gradient of 0 to 25% acetonitrile in 0.1% trifluoroacetic acid over a period of 25 min at a flow rate of 1 ml=min at room temperature, with detection by measurement of the absorption at 280 nm. GS-AP-glycoside, a new peak, is collected, combined, and dried by evaporation. The GS-AP-glycoside preparation is dissolved in 700 l of 0.1 M sodium phosphate buffer (pH 7.5) containing 25 mM EDTA and succinic anhydride, 20 mg, is added to the solution. The mixture is allowed to react for 10 min, while maintaining the pH at 7–8 by the addition of 5 M NaOH. Aliquots, 100 l, of the reaction mixture are acidified to pH 2–3 by the addition of hydrochloric acid, and applied to the chromatographic column, followed by elution under the same conditions as described above. The affinophore, succinylated GS-AP-glycoside (sGS-AP-glycoside), eluting as a new peak, is collected, combined, and dried by evaporation. It is then dissolved in 1 ml of water and stored in a freezer. The concentration of the affinophore is determined by means of the phenol–sulfuric acid method with corresponding sugar solution as a standard. The molecular extinction coefficient of the affinophores in water at 248 nm has been determined to be 10,400 M1 cm1. The overall yield of the affinophore from the p-aminophenylglycoside is 40–50%.
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401
Electroendosmosis and Capillary Affinophoresis
A distinctive feature of free solution capillary electrophoresis, in comparison with the electrophoresis in gels, is the apparent contribution of electroendosmosis to the results of the electrophoresis. The inner surface of the capillaries used in the affinophoresis is negatively charged, and the application of an electric field generates an asymmetric migration of the counter ions of the immobilized negative charges on the surface. The migrating counter ions push water molecules and produce a plug flow, in which the flow speed is identical irrespective of the distance from the wall. The speed of the flow can be sufficiently high to carry all the molecular species participating in the interactions toward the negative electrode. This enables the detection of the entire sample components injected at the positive end of the capillary in a relatively short time with a fixed detector on the capillary. Electrically neutral species are transported only by electroendosmosis to the detection point (Fig. 2). On the other hand, negatively charged species migrate toward the positive electrode against the overwhelming electroendosmosis, and are detected after the neutral species. The faster the migration toward the positive electrode, the later they are detected. When an anionic affinophore is involved in the electrophoresis, the interaction should increase the detection time for the lectin. Anionic affinophores migrate toward the positive electrode much faster than lectins but not fast enough to move backward against the electroendosmotic flow. The affinophore solution needs to be loaded only in the capillary, and this allows the lectin, injected at the positive end, to interact with the affinophore from the beginning of the electrophoresis to its detection. In competition experiments, a competing sugar needs to be added only in the positive electrode buffer. The neutral sugar is transported along the capillary only by the electroendosmosis. The lectin and the affinophore electrophoretically migrate into the zone of the neutral sugar, and competition can then be observed (Fig. 2). Calculation of Mobility Change
To determine the affinity constant, the mobility changes of a lectin must be calculated from the detection time obtained under different conditions of affinophoresis. In free solution capillary electrophoresis, the detection time is a function of both the electrophoretic mobility of a sample and the electroendosmotic mobility of a capillary. Coelectrophoresis with a reference molecule, that is, an electrophoresis marker that does not interact
402
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Fig. 2. Capillary affinophoresis and electroendosmosis. (A1) A sample solution is injected at the positive end of the capillary. (A2) Application of an electric field generates electroendosmosis due to the negatively charged surface of the capillary. If the lectin is negatively charged and migrates toward the positive side, a neutral marker is detected before the lectin. (B1) In the case of affinophoresis, an affinophore is included only in the capillary. (B2) The affinophore migrates toward the positive end much faster than the lectin, and the lectin migrates in the affinophore solution until it is detected. Interaction between the lectin
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403
with the affinophore, allows the calculation of the mobility change from a change in the detection time as shown below.15,16 The detection time (t) of a lectin is a function of its electrophoretic mobility () as well as of the electroendosmotic mobility (eo) of a capillary: t¼
L 1 E þ eo
ð1Þ
where L is the distance (cm) from the injection end to a detection point, and E is the field strength (V=cm). The same relation should be applied for the detection time of an electrophoresis marker (tr): tr ¼
L 1 E r þ eo
ð2Þ
where r represents the electrophoretic mobility of the marker. When the lectin and the marker are subjected to simultaneous electrophoresis, eo, E, and L are the same in Eqs. (1) and (2). As a result, the difference between the reciprocals of each detection time is proportional to the difference between the mobility of the lectin and the marker, that is, r. 1 1 E E ¼ ½ð þ eo Þ ðr þ eo Þ ¼ ð r Þ t tr L L
ð3Þ
Thus the mobility difference between the lectin and the marker in the same run can be expressed as follows: L 1 1 r ¼ ð4Þ E t tr The mobility difference in the presence of the affinophore can also be calculated using Eq. (4). When the mobility of the lectin changes from 0 to by the affinophoresis, the mobility change ð ¼ 0 ) induced by the affinophoresis can be extracted from the two mobility differences from the marker in the presence and the absence of the affinophore, that is, and the affinophore increases the mobility of the lectin and retards its detection. (C1) To observe competition by a neutral sugar, it is included only in the positive electrode solution. (C2) The neutral sugar is rapidly transported toward the negative side by electroendosmosis, and the lectin migrates in a mixed zone of the affinophore and the neutral sugar. Effective competition suppresses the acceleration of the electrophoresis of the lectin by the affinophore. 15 16
K. Shimura and K. Kasai, Anal. Biochem. 227, 186 (1995). F. A. Gomez, L. Z. Avila, Y.-H. Chu, and G. M. Whitesides, Anal. Chem. 66, 1785 (1994).
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¼ 0 ¼ ð r Þ ð0 r Þ L 1 1 1 1 ¼ E t tr t0 tr0
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ð5Þ
where t0 is the detection time of lectin in the absence of the affinophore. The values of tr and t0 r might be different because of variations in the electroendosmosis between the two runs. The calculation of by Eq. (5) permits the variation in electroendosmosis between the runs to be determined. Instrument and Capillary
A capillary electrophoresis instrument with an ultraviolet absorption detector is required. The instrument should have a temperature-controlling system for the capillary, because the interactions occur during the electrophoresis run. Fused silica capillaries (25–50 m i.d., 375 m o.d.) with a polyimide covering (Polymicro Technologies, Phoenix, AZ) should be suitable for most applications. A fused silica capillary has a tendency to adsorb proteins mainly because of the anionic characteristics of its surface caused by the dissociation of silanol groups. The adsorption largely depends on the nature of the particular protein under consideration. If the protein does not pose any problems related to adsorption, that is, the electropherograms are reproducible without obvious tailing and broadening of peaks, the use of a fused silica capillary is the most straightforward. Coating of the inner surface of the capillary may alleviate the problem, should it occur. Capillary Coating
We typically use capillaries that are coated with an anionic polymer, succinylpolylysine. An epoxy group is introduced onto the inner surface of the capillary by reaction with an epoxysilane and polylysine is then reacted to cover the surface. Finally, the amino function of polylysine is succinylated.15 An aqueous solution of 3-glycidoxypropyltrimethoxysilane [5% (v=v)] is adjusted to pH 5.5–5.8 with 5 mM KOH and clarified by centrifugation. The fused silica capillary (60 cm long) is filled with the solution and the ends are closed by inserting them into rubber septa used for gas chromatography. The capillaries are heated in a boiling water bath for 30 min and rinsed by passing 0.1 ml each of water and acetone through them, followed by drying by passing air by suction. The capillaries are filled with
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405
a poly(l-lysine) solution (Sigma; average degree of polymerization, 270; 10 mg=ml in 1 M NaCl; adjusted to pH 10 with NaOH) and reacted overnight at room temperature. They are then washed with 0.1 ml of water and filled with a mixture [1:1 (v=v)] of succinic anhydride in dimethylformamide (25 mg=ml) and N-ethylmorpholine. They are allowed to react for 30 min at room temperature and, finally, rinsed with 0.1 ml of water. The coating procedure can be facilitated by the use of a syringe (50– 100 l; a pressure-lock or gas-tight syringe with a 26-gauge needle with a tip cut at 90 ), with a segment of Teflon tubing (2 cm long, 0.3 mm i.d. 1.5 mm o.d.; Alltech Associates, Deerfield, IL) attached to the tip of the needle. The tube is fitted to both the needle of the syringe and the capillary (375 m o.d.) by thrusting the tips of them into each end of the tube after softening by mild heating with an electric heater. After cooling, the capillary can be detached and reconnected without heating, and the connection permits the rapid injection of solutions into capillaries with the syringe. Equations for Affinophoresis
Most lectins are divalent and must be treated accordingly in the analysis. We start with the equations for affinophoresis of monovalent proteins and then return to divalent systems. For a monovalent lectin, L, and an affinophore, A, we assume the following binding equilibrium: L þ A Ð LA
ð6Þ
The original electrophoretic mobility of the lectin is 0 and that of the complex with the affinophore is c. Because the lectin and the affinophore are in dynamic binding equilibrium, the macroscopic mobility of the lectin, , in equilibrium should be the weighted average of 0 and c: ¼ ð1 Þ0 þ c
ð7Þ
The weight, , is the degree of saturation of the lectin with the affinophore and is a function of the dissociation constant Kd (Kd ¼ [L][A]= [LA]) of the complex and the concentration of the affinophore, [A]: ¼
½A Kd þ ½A
ð8Þ
From Eq. (7) and (8), the mobility change ( ¼ 0) of the lectin by affinophoresis can be expressed as follows:
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¼ max
½A Kd þ ½A
[28]
ð9Þ
where max (max ¼ c 0) represents the maximum mobility change of the lectin. It should be noted that the equation has the same form as the Michaelis–Menten equation for enzyme kinetics and, thus, the Kd and max can be determined in a manner similar to that used for determining Km and Vmax in enzyme kinetics. A plot of versus =[A] according to the following linear equation yields a straight line that intercepts the ordinate at max with a slope of Kd: ¼ max Kd
½A
ð10Þ
We call this a ‘‘Woolf–Hofstee plot’’ after the investigators who contributed to the development of the corresponding plot in enzyme kinetics.17 The values of and =[A] for each run can be easily calculated with spreadsheet software by providing t, tr, and [A] as input data. Divalency
The lectin is assumed to be monovalent in the above-described treatments. In the case of divalent lectins, two monoliganded affinophores can simultaneously bind to the lectin. An identical analysis is applicable to such dimeric lectins under the following conditions: (1) the two binding sites are equivalent; (2) the two binding sites are independent; and (3) the mobility change induced by the binding of the second affinophore is identical to that induced by the first one.12 In the case of a homodimeric lectin, the two binding sites are apparently equivalent. The independency of the binding sites cannot be known in advance. It will, however, be revealed by a linear relation of the plot according to Eq. (10). The mobility change caused by the successive binding of each affinophore can be considered to be approximately identical, because the electrophoretic mobility is proportional to the number of charges on a particle with an identical electrophoretic drag, that is, a negligible change in the molecular size of the lectin on binding with the affinophores. The relatively small molecular size of the monoliganded affinophore in comparison with a protein should warrant this approximation. In the analysis of divalent lectins, it should be noted that the Kd that appears in the equations is that for a single binding site and not for the entire protein.
17
M. Dixon and E. C. Webb, in ‘‘Enzymes,’’ 3rd ed., p. 62. Longman, London, 1979.
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Affinophoresis
The buffer for the electrophoresis should not have a large electric conductivity. When 0.1 M Tris–acetic acid buffer (pH 7.9) is used as in the example described below, the current is about 25 A at 300 V=cm with a 50 m i.d. capillary. As a criterion, electric power at less than 1 W=m of capillary length can be considered safe. At 350 V=cm, 30 A corresponds to about 1 W=m. An affinophore solution is included only in the capillary (Fig. 2). The volume of the capillary is less than 1 l and this is the volume of affinophore solution actually consumed in a single run of affinophoresis. Experimentally, however, it would be better to prepare affinophore solutions in a greater volume (about 50 l) in order to ensure precision in the concentration of the affinophore against the effect of evaporation, as well as possible errors in the use of micropipettes. Cooling of the vial rack is effective in minimizing the evaporation of water. Evaporation depends on the humidity of the surrounding air and overcooling might result in the condensation of water under conditions of high humidity. An overlay of a small quantity of mineral oil has been shown not to interfere with the injection processes and to be effective in suppressing evaporation.18,19 A lectin zone formed by injecting a sample does not contain affinophore and there should be a presteady state period before achieving a binding equilibrium between a lectin sample and the affinophore. During this period, the macroscopic mobility of the lectin gradually changes from 0 to as expressed by Eq. (7). The length of the period is a function of the relative concentration of the lectin to that of affinophore. When this period is not negligibly small, the mobility change of the lectin becomes smaller than would be predicted from Eq. (9), especially at low concentration ranges of affinophore. This results in a downward curvature of the plot according to Eq. (10) in the vicinity of the abscissa. The concentration of the lectin sample should be nearly identical to or lower than that of the affinophore on a molar basis. In the following experiments, lectin solutions of 0.2–1.0 g=l are used with absorption detection at 214 nm. This phenomenon might be problematic, when the affinity for an affinophore is high, where considerable binding occurs, even at low affinophore concentration. As an example, the affinophoresis of pea lectin (Pisum sativum) using a mannoside affinophore is shown in Fig. 3. Pea lectin is a homodimeric lectin
18 19
N. H. H. Heegaard, J. W. Sen, and M. H. Nissen, J. Chromatogr. A 894, 319 (2000). K. Shimura, N. Uchiyama, and K. Kasai, Electrophoresis 22, 3471 (2001).
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Fig. 3. Affinophoresis of pea lectin with a mannoside. Left: A sample (0.7 nl) containing pea lectin (**, 0.2 g=l) and cytidine (*, 0.2 mM) was injected by means of pressure at the positive end of a capillary (25 m i.d., 375 m o.d., 57 cm total length, 50 cm separation distance, and inner-coated with succinylpolylysine) filled with a solution of the affinophore (sGS-AP-Man). Electrophoresis was carried out at a field strength of 350 V=cm (7 A) at 25 , with detection at A214. Tris–acetate buffer (0.1 M, pH 7.9) was used throughout the system. The concentration of the affinophore was (a) 0 mM; (b) 0.05 mM; (c) 0.1 mM; (d) 0.2 mM; (e) 0.3 mM; (f) 0.4 mM; (g) 0.5 mM. Right: A plot according to Eq. (10) was prepared, using the data shown on the left, in order to determine the Kd and max values. Reproduced from Ref. 12 .
with a molecular mass of 49,000 Da and has two carbohydrate-binding sites.20 Its structure is closely related to concanavalin A and the affinity constants for mannose and some other carbohydrates have been reported.20,21 20 21
I. S. Trowbridge, J. Biol. Chem. 249, 6004 (1974). F. P. Schwarz, K. D. Puri, R. G. Bhat, and A. Sorolia, J. Biol. Chem. 268, 7668 (1993).
[28]
capillary affinophoresis
409
The main component of pea lectin has been purified by anion-exchange chromatography and this preparation is used in the experiments. Electrophoresis is carried out with an automated capillary electrophoresis instrument (P=ACE 2210 with a UV detector; Beckman, Fullerton, CA). A fused silica capillary (25 m i.d., 375 m o.d.) coated with succinylpolylysine is installed in a capillary cartridge with a separation distance of 50 cm, from the positive end to the detection point. The capillary is filled with an electrophoresis buffer (0.1 M Tris–acetic acid buffer, pH 7.9, containing 0.02% NaN3 as a preservative) with or without the mannoside affinophore (sGSPA-Man), containing p-aminophenyl--d-mannoside as an affinity ligand, using a pressure of 20 lb=in2 for 3 min. The sample, which contains pea lectin (0.2 g=l) and 0.2 mM cytidine as a neutral marker in the electrophoresis buffer, is injected at the positive end for 10 s under a pressure of 0.5 lb=in2 (injection volume of 0.67 nl, which corresponds to 0.13 ng of pea lectin). Electrophoresis is carried out at a field strength of 350 V=cm with an electric current of 6.7 A. The cartridge temperature is set at 25 and pea lectin and cytidine are detected by absorption measurement at 214 nm. The electrophoresis buffer is also used as an electrode solution in each electrode vessel. The solution of pea lectin (20 l) and that of the affinophore (50 l) at different concentrations are placed in polypropylene microvials and the vials are set in a cooled (typically 10 ) rotating tray to minimize evaporation. After electrophoresis, the capillary is rinsed with 0.1 M sodium carbonate buffer (pH 10) containing 1 M NaCl and 10 mM EDTA in the high-pressure rinse mode for 3 min and then with water for 3 min. In the absence of affinophore, the mobility of the lectin is low as indicated by the close separation of the peaks from that of the neutral marker, cytidine. The detection time of the lectin gradually increases as the concentration of the affinophore becomes higher, indicating binding of the affinophore. The mobility change, , at each concentration of the affinophore is calculated using Eq. (5), and is plotted against =[A] according to Eq. (10) (Fig. 3, right). A linear regression provides Kd as the slope of the line and max at the intercept with the ordinate, that is, Kd ¼ 0.202 mM and max ¼ 5.25 105 cm2 V1 s1. Five identical runs yield an average value for Kd of 0.199 mM (coefficient of variation, 6.0%) and max of 5.35 105 cm2 V1 s1 (coefficient of variation, 1.6%). Competitive Capillary Affinophoresis
The addition of a neutral sugar to the affinophoresis system results in suppression of the mobility change induced by the affinophore. As described in the previous section, a neutral sugar can be added only in the buffer in the positive electrode reservoir. The electroendosmosis in
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[28]
the capillary transports the sugar solution toward the negative end, and a lectin and an affinophore migrate in the neutral sugar zone (Fig. 2). The volume of the positive electrode buffer containing the neutral sugar can be as small as 100 l. The concentration of an affinophore should be set so as to be between the Kd and 2 Kd. Other conditions should be the same as those used for the determination of Kd and max of the affinophore. The detailed experimental conditions for a competitive capillary affinophoresis experiment using a lactoside affinophore are described for the determination of affinity constants of recombinant human galectins for neutral sugars. Recombinant human galectin-1 (rhGal-1) is a dimeric protein composed of two identical 14-kDa polypeptide chains with 134 amino acid residues.22 The apparent difference between rhGal-1 and the native galectin-1 found in human tissues is the absence of an acetyl group at the N terminus of rhGal-1. The C2S mutant of rhGal-1 has one amino acid substitution at the second residue from the N terminus, from cysteine to serine. This mutation at one of the six cysteine residues found in galectin-1 has been reported to substantially increase the stability of sugar-binding activity of the lectin in the absence of reducing agents.22 A fused silica capillary (50 m i.d., 375 m o.d.) coated with succinylpolylysine is used, with a separation distance of 20 cm. The electrophoresis instrument and the detection system are the same as those used in the experiments described for pea lectin, and the cartridge temperature is set at 20 . Solutions, 20 l each of a lactoside affinophore (sGS-AP-Lac) bearing p-aminophenyl--lactoside as an affinity ligand, a lectin at 1 g=l, and references of 1 mM acrylamide and 0.3 mM acetyltryptophan in the electrophoresis buffer, are placed in polypropylene minivials (200 l) and overlaid with 20 l each of mineral oil (light white oil, density ¼ 0.84 g=ml; g=ml; Sigma) to prevent the evaporation of water.18,19 The capillary is filled with the affinophore solution at a pressure of 20 lb=in2 for 30 s. A solution of electrophoresis markers and that of the lectin are consecutively injected at the positive end for 1 s each under a pressure of 0.5 lb=in2 (injection volume of 2.2 nl of each solution). Electrophoresis is carried out at a field strength of 300 V=cm with an electric current of about 25 A, using a neutral sugar solution of 200 l in the electrophoresis buffer as the positive electrode solution. After electrophoresis, the capillary is rinsed with water at a pressure of 20 lb=in2 for 3 min. The electropherograms shown in Fig. 4 were obtained by the affinophoresis of the C2S variant of rhGal-1 with the lactoside affinophore. The Kd value of the lectin for the affinophore was determined to be
22
J. Hirabayashi and K. Kasai, J. Biol. Chem. 266, 23648 (1991).
[28]
capillary affinophoresis
411
Fig. 4. Competition of affinophoresis of the C2S variant of rhGal-1 by N-acetyllactosamine. Affinophoresis of C2S (1 g=l) was carried out with the lactoside affinophore (sGSAP-Lac) at a concentration of 0.51 mM in a succinylpolylysine-coated capillary (50 m i.d., 375 m o.d., 23 cm total length, 20 cm separation distance) with a field strength of 300 V=cm at 20 . N-Acetyllactosamine (LacNAc) was added to the electrophoresis buffer at the anode at the concentrations indicated above each electropherogram. Acrylamide and acetyltryptophan (Ac-Trp) were injected with the lectin sample as electrophoresis markers. Detection was carried out by absorption measurement at 214 nm. Reproduced from Ref. 13 .
0.40 mM, with a max value of 4.69 105 cm2 V1 s1. The affinophore at a concentration of 0.51 mM lengthens the detection time for the lectin by about 1 min (Fig. 4, second electropherogram from top). The addition of N-acetyllactosamine to the affinophoresis system cancels the effect of the affinophore, and the detection time is reduced as a function of the concentration of N-acetyllactosamine (Fig. 4, third and fourth electropherograms from top).
412
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[28]
Calculation of Affinity Constants for Neutral Sugars
The mobility change of the lectin in the presence of the affinophore and the neutral sugar, i, can be written as follows: i ¼ max
½A Kd app þ ½A
ð11Þ
where the Kd app is the apparent dissociation constant for the affinophore in the presence of the neutral sugar, and has a relation with the dissociation constant, Ki (Ki ¼ [L][I]=[LI]), for a neutral sugar, I: ½I Kd app ¼ Kd 1 þ ð12Þ Ki Because max has already been determined, Kd app can be calculated by measurement of i in the presence of a neutral sugar according to Eq. (11). Kd is also already known by the plot of Eq. (10) and Ki can be calculated from Eq. (12). Alternatively, Eq. (12) can be rearranged as Kd app ½I ¼ þ1 Ki Kd
ð13Þ
A plot of Kd app=Kd against different [I] should result in a straight line with a slope of 1=Ki. It is preferable to use the competing sugar at concentrations around its Ki value. The results of competition experiments with some neutral sugars in the affinophoresis of pea lectin with the mannoside affinophore are plotted according to Eq. (13) (Fig. 5). The reciprocal of the slope of the lines corresponds to the dissociation constant Ki for the lectin–neutral sugar complexes. The values are compared with those determined by calorimetry21 (Table I). The affinophoresis is carried out at 25 but some of the calorimetry values are determined at different temperatures. A close agreement is found for methyl--d-mannoside and d-mannose, which are determined at identical temperatures. Other calorimetry values are determined at 16 or 14 and are considerably smaller than those obtained by affinophoresis. This discrepancy can be largely attributed to the difference in temperature used in the different determinations and the positive H values for the dissociation of the pea lectin–sugar complexes, that is, 10 and 13.6 kJ=mol kJ=mol for d-glucose and methyl--d-glucoside, respectively.21 The dissociation constants of recombinant human galectin-1 (rhGal-1), its C2S variant,22 and recombinant human galectin-3 (rhGal-3)23,24 for 23 24
Y. Oda, H. Leffler, Y. Sakakura, K. Kasai, and S. H. Barondes, Gene 99, 279 (1991). J. Hirabayashi, Y. Sakakura, and K. Kasai, in ‘‘Lectins and Glycobiology’’ (H.-J. Gabius and S. Gabius, eds.), p. 474. Springer-Verlag, New York, 1993.
[28]
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capillary affinophoresis
Fig. 5. Determination of the dissociation constants of pea lectin for neutral sugars (I) through competition experiments of affinophoresis using the mannoside affinophore. The results are plotted according to Eq. (13), (d) p-Aminophenyl--d-mannoside; ( ) methyl-d-mannoside; (m) d-mannose; (4) maltose; (&) methyl--d-glucoside; (h) d-glucose. Reproduced from Ref. 12 .
various neutral sugars, as determined by capillary affinophoresis, are summarized in Fig. 6. The striking preference of rhGal-1 and its C2S variant for N-acetyllactosamine [Gal(1–4)GlcNAc] and lactose [Gal(1–4)Glc] is clear, as has been repeatedly reported.25,26 The affinity of the two lectins for N-acetyllactosamine, lactose, and galactose is hardly distinguishable, indicating that the effect of the substitution of the second cysteine residue of rhGal-1 to a serine residue on affinity is negligible, as has been previously noted.22 The most relevant work that can be compared with the present results for rhGal-1 is that reported by Lee et al.26 They have reported the concentrations, I50, for various sugars that cause a 50% reduction in the binding of 125I-labeled galectin-1 to lactose-immobilized agarose beads. The values of I50, with relative values of 1=I50 to lactose in parentheses, are as follows: N-acetyllactosamine, 0.13 mM (350%); lactose, 0.46 mM (100%); methyl--galactoside, 26 mM (1.8%); melibiose [Gal(1–6)Glc], 25 26
C. P. Sparrow, H. Leffler, and S. H. Barondes, J. Biol. Chem. 262, 7383 (1987). R. T. Lee, Y. Ichikawa, H. J. Allen, and Y. C. Lee, J. Biol. Chem. 265, 7864 (1990).
414
[28]
general techniques TABLE 1 Affinity Constants of Pea Lectin for Neutral Sugars
Sugar
Affinophoresisa (mM)
Calorimetryb
p-Aminophenyl--d-mannoside Methyl--d-mannoside d-Mannose Maltose Methyl--d-glucoside d-Glucose
0.25 0.65 1.6 2.1 2.4 3.6
Not determined 0.61 mMc 1.3 mMd Not determined 1.3 mMe 2.8 mMf
a
0.1 M Tris–acetic acid buffer (pH 7.9) containing 0.02% NaN3, 25 (Ref. 12). 0.02 M sodium phosphate buffer (pH 7.4) containing 0.15 M NaCl (Ref. 21). c 25 . d 24.3 . e 16 . f 14.2 . b
30 mM (1.5%); galactose, 63 mM (0.7%) and methyl--galactoside, 85 mM (0.5%). The Ki values of C2S and its relative affinities determined by affinophoresis are as follows, in the same order as above: N-acetyllactosamine, 0.067 mM (352%); lactose, 0.23 mM (100%); methyl--galactoside, 16 mM (1.5%); melibiose, 21 mM (1.1%); galactose, 30 mM (0.78%); methyl--galactoside, 39 mM (0.6%). Although the I50 values are about twice as large as the Ki values determined by capillary affinophoresis, the consistency of the relative magnitude of the affinity is remarkable. When lactose-immobilized beads are used at a concentration equivalent to the Kd value for the lectin and the modified beads, as was the case in their experiments,26 the I50 value for each sugar should be twice as large as the corresponding Ki value. This theory may simply explain the difference, although there are some additional factors to be considered, that is, the divalency of the lectin in the interaction with the lactose-immobilized beads and the difference in temperature between their experiments, 25 , and ours, 20 . rhGal-3 also has an exclusively high affinity for N-acetyllactosamine and lactose. rhGal-3 was shown to have a 2 to 10 times higher affinity than rhGal-1 for sugars containing galactose at their nonreducing ends, whereas the affinities for sucrose [Glc(1-2)Fru] are almost the same. This result indicates a higher preference of galectin-3 for galactosides than galectin-1. The preference for methyl--galactoside over methyl--galactoside is not observed for rhGal-3. Sparrow et al. report I50 values for a variety of sugars
[28]
capillary affinophoresis
415
Fig. 6. Dissociation constants of recombinant human galectins for neutral sugars determined by capillary affinophoresis. The Ki values (M) are the dissociation constants for a single binding site of lectins. rhGal-1, Recombinant human galectin-1; C2S, a C2S variant of rhGal-1; rhGal-3, recombinant human galectin-3; Gal, d-galactose; Man, d-mannose; Glc, d-glucose; Fru, d-fructose; GalNAc, N-acetyl-d-galactosamine; GlcNAc, N-acetyl-d-glucosamine; IPTG, isopropyl--d-thiogalactoside; me-, methyl. Original data were taken from Ref. 13 .
and oligosaccharides in the binding of human galectin-3 (called HL-29 at that time) to asialofetuin–agarose beads.25 A portion of the data that are relevant to our results is as follows (with a relative value of 1=I50 to lactose in parentheses): lactose (100%), N-acetyllactosamine (1130%), galactose (1.8%), methyl--galactoside (2.6%), and methyl--galactoside (1.6%). Overall agreement in the relative affinity data based on the I50 values and Ki values of our determinations by capillary affinophoresis
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is apparent, although the affinity for N-acetyllactosamine has been emphasized in their data. Divalency Revisited
The monoliganded affinophore was developed to circumvent difficulty related to the divalent nature of lectins. After the determination of affinity constants for neutral sugars had been established by capillary affinophoresis, we attempted a new approach for estimating the degree of contribution of divalent interactions between lectins and polyliganded matrices. The interactions between a divalent pea lectin and polyliganded affinophores, succinylpolylysine bearing a p-aminophenyl--d-mannoside at different ligand densities, were investigated.11 The overall dissociation constant between the lectin and the affinophore was determined by capillary affinophoresis to show a higher affinity for the affinophore with higher ligand densities. The overall interactions should comprise both monovalent and divalent interactions. When a neutral sugar, methyl -d-mannoside, interferes with these interactions, the contribution of divalent interactions should decrease more rapidly than monovalent interactions. The reason for this is that one binding site is already occupied with the neutral sugar, and the lectin should behave as a monovalent molecule. The ratio of the temporary monovalent lectins to the divalent lectins with two free binding sites increases with increasing degree of saturation by the neutral sugar. When affinophoresis using a polyliganded affinophore was interfered with by a neutral sugar, the plot according to Eq. (13) became nonlinear with a gradual increase in the slope with increasing concentration of neutral sugar, indicating the decreasing contribution of divalency11: the more curvature, the higher the contribution of divalent interactions. Because the Ki value of the neutral sugar had already been determined, nonlinear regression allows the determination of two elemental equilibrium constants, one for the monovalent binding (K1 ¼ [L][A]=[LA], where LA denotes the monovalent complexes) and the other for the divalent interactions (K2 ¼ [LA]=[L ¼ A], where L ¼ A denotes the divalent complexes). The divalency value, representing the mole fraction of the divalent species in the complex, can be calculated from K2 values. Polyliganded affinophores bearing a mannoside ligand at different ligand densities from 4.2 to 17.5% of mannoside over a succinyllysine residue were found to interact with pea lectin with divalency values of 24 to 62%.10 This principle, based on competition with a monovalent ligand, should be applicable to the analysis of interactions with polyliganded cell surface or extracellular matrices in conjunction with a wide variety of techniques for analyzing molecular interactions.
[29]
bound ligand topology: tr noe and modeling
417
Conclusion
Capillary affinophoresis, performed with an automated electrophoresis instrument, is a useful technique for determining the affinity constants of divalent lectins for neutral sugars. The affinity constants obtained are strictly for a single binding site and are free from the components related to multivalent interactions. These values should be useful in comparing affinities of various lectins and sugars. Once a suitable affinophore is established for a target lectin, the method can be carried out with a relatively small amount of lectin sample and time for a variety of sugars with good precision and reproducibility.
[29] Describing Topology of Bound Ligand by Transferred Nuclear Overhauser Effect Spectroscopy and Molecular Modeling By Hans-Christian Siebert, Jesu´ s Jime´ nez-Barbero, Sabine Andre´ , Herbert Kaltner, and Hans-Joachim Gabius Introduction
The perspective needed to translate progress in unraveling the role of protein–carbohydrate interactions in clinically relevant processes (e.g., initiation of infections by pathogen attachment or lectin-dependent endocytosis) into therapeutic concepts is one reason for the structural analysis of lectin–ligand complexes.1–4 Despite the exquisite sensitivity of powerful X-ray diffraction techniques, concerns about whether the crystal structures fully reflect structural details in solution cannot be ignored.5,6 To address this issue, nuclear magnetic resonance (NMR) spectroscopy can provide salient input on ligand topology even without meeting the requirement for a complete signal assignment. This is especially helpful when the size 1
Y. C. Lee and R. T. Lee, J. Biomed. Sci. 3, 221 (1996). K.-A. Karlsson, Mol. Microbiol. 29, 1 (1998). 3 H. Ru¨ dı´ger, H.-C. Siebert, D. Solı´s, J. Jime´ nez-Barbero, A. Romero, C.-W. von der Lieth, T. Dı´az-Maurin˜ o, and H.-J. Gabius, Curr. Med. Chem. 7, 389 (2000). 4 N. Yamazaki, S. Kojima, N. V. Bovin, S. Andre´ , S. Gabius, and H.-J. Gabius, Adv. Drug Deliv. Rev. 43, 225 (2000). 5 G. Wagner, S. G. Hyberts, and T. F. Havel, Annu. Rev. Biophys. Biomol. Struct. 21, 167 (1992). 6 M. W. MacArthur, P. C. Driscoll, and J. M. Thornton, Trends Biotechnol. 12, 149 (1994). 2
METHODS IN ENZYMOLOGY, VOL. 362
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