Continuous titration of difference absorption upon binding of 4-methylumbelliferyl glycosides to concanavalin A and peanut agglutinin

ANALYTICAL

BIOCHEMISTRY

K&308-3

13 ( 1982)

Continuous Titration of Difference Absorption upon Binding of 4-Methylumbelliferyl Glycosides to Concanavalin A and Peanut Agglutinin G.LOONTIENS,ANDCLEMENT

K. DE BRUYNE

Laboratory of Biochemistry, University of Gent, Ledeganckstraat

35, B-9000 Gent, Belgium

HILDEDE

BOECK,FRANK

ReceivedJanuary 22, 1982 A continuous titration of absorption differences is described. Equal volumes of the titration fluid are dispensed from two micrometer-driven Hamilton gas-tight syringes into two 1 X 1 X 4.5cm cuvettes. These are placed in the reference and sample beam. Each cuvette stopper is equipped with a capillary inlet connected to a syringe and with a minimotor for continuous stirring. Details of the stirring device are given. The delivered volumes of titration fluid are sufficiently reproducible to allow titration of absorption differences as a function of chromophore concentration. The usefulness of this approach is tested with the binding of 4-methylumbelliferyl cY-rr-mannopyranoside and concanavalin A as a well-characterized system. It is applied to the binding of similarly labeled anti-T disaccharide with the lectin from peanuts. With both lectins, the change in molecular extinction coefficient of the ligand and the association constant, valid for the entire protein saturation range, were obtained. The results are identical to those from other methods. including equilibrium dialysis.

Difference absorption spectrometry is a useful tool in the study of the interaction of macromolecules and ligands, for example, when changes in intrinsic or extrinsic absorption are measured. Pairs of two-compartment mixing cells (1) are widely used for this purpose. For the determination of binding parameters, however, their application results in time-consuming and tedious measurements as a function of concentration, even at a single wavelength; this technique can also be demanding on the amount of available material, and the reproducibility of cuvette blanks can be problematic. In view of these drawbacks, we prefer the continuous titration procedure described here. MATERIAL AND METHODS

With concanavalin perimental conditions

A (Con A)’ the exwere the same as in

’ Abbreviations used: Con A, concanavalin A; MeUmb-Manp, 4-methylumbelliferyl a-D-ITtanIIOpyranoside; PNA, peanut agglutinin; MeUmb-GalpS( 1 3)GalNAcp, Cmethylumbelliferyl2-acetamido-2-deoxy3-0-(,!I-D-galactopyranosyl)+D-galactopyranoside.

0003-2697/82/120308-06$02.00/O Copyright Q 1982 by Academic Press, Inc. All rights of reproduction in any form rcservcd.

previous equilibrium studies (2,3). Con A was essentially free of nicked polypeptide chains (4), and its concentration was expressed as binding sites. Concentrations were determined spectrophotometrically at 280 nm for Con A with t = 2.91 X lo4 M-' cm-‘, corresponding to iki, 25,500 of its polypeptide chain; at 3 18 nm for 4-methylumbelliferyl a-D-mannopyranoside (MeUmbManp) with c = 1.36 X lo4 M-’ cm-;‘. The solutions were made up in 0.05 M NaOAcHOAc, 1 M NaCl, 1 m&i NiC&, and 1 mM CaClz (pH 5.5). Peanut agglutinin (PNA), prepared by affinity chromatography on Sepharose-N-(6aminohexanoyl) - j3 - D - galactopyranosyla mine (5) and a gift from N. Sharon (Rehovot, Israel), was determined at 280 nm and its concentration expressed as binding sites with t = 2.64 X lo4 M-' cm-’ (6), corresponding to M, 27,500 for its polypeptide chain (6). 4-methylumbelliferyl 2-a&amido - 2 - deoxy - 3 - 0 - (/3 - D - galactopy ranosyl)+D-galactopyranoside (MeUmbGalp/3( 1 - 3)GalNAcp), a gift from K. L. 308

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DIFFERENCE

Matta (Buffalo, N. Y.), was determined at 3 16 nm with an experimental value of E = 1.38 X lo4 M-’ cm-‘. The solutions were made up in 0.05 M 4-( 2-hydroxyethyl)- 1-piperazineethanesulfonic acid (Hepes), 0.1 M MgC& (pH 6.9). All solutions were passed through 0.45-pm-pore filters. Differences in absorption of MeUmbManp, caused by a blue shift of its absorption spectrum upon binding to Con A (2), were measured at 334 nm in a single-beam spectrophotometer (Zeiss M4QIII-PMQII) with sensitivity of A = 0.001. At this wavelength, Efor MeUmb-Manp equals 7.9 X lo3 M--’ cm-‘. Upon binding of MeUmbGalp@( 1 - 3)GalNAcp to PNA, the ligand absorption spectrum shows a red shift that is maximal at 322 nm (De Boeck et al., unA I 0 -

-

C

I \.

[II ’0

ABSORPTION

b

1 cm

I

1

I

1. The motor housing (A) of the cuvette stirrer consists of three parts (made of PVC) that are screwed together. The cuvette stopper C is made of polyacetal; the stirrer D (made of stainless steel) is connected to the motor shaft by a sleeve of silicone tubing (B). FIG.

309

published). At this wavelength, c for MeUmb-Galp/?( 1 3)GalNAcp equals 1.26 X lo4 M-’ cm-‘. With this system the PMQII unit (Zeiss) was exchanged for an analog amplifier and digital processing unit (Brock & Michelsen, Denmark) that allows measurement of absorption differences hA 2 0.0002. The reference and sample cuvettes were thermostated in an all-copper cuvette changer. To accommodate the cuvette stirrers, the cuvette compartment was increased in height, and it contained an opening for the capillary tubings and electric contacts for the stirrers. Details of a stirrer are given in Fig. 1. The motor housing consists of three parts that are screwed together. It is sufficiently narrow to allow the stirrers to be mounted on cuvettes at a minimum distance of 2 cm. The motors used are 15 mm wide; they are 1516 E 004 S DC micromotors (System Faulhaber) purchased from Minimotor S.A. (Agno, Switzerland) with an idling speed of 12,000 rpm when operated at 4 V. The stirrer shaft and blade were constructed from a single piece of a 6-mm-thick bar of stainless steel. The larger part was reduced to a thickness of 2 mm to form the shaft, leaving a 7-mm part to form the blade, obtained by filing it to a thickness of 0.3 mm, rounding the corners to an overall oval shape, polishing, and tortioning. The stirrer did not cause frothing of protein solutions. The two cuvettes were filled with an equal volume (2141 ~1) of the two appropriate solutions using a calibrated syringe. To both continuously stirred cuvettes, equal portions of the common titration fluid (either ligand or protein) were dispensed manually and in succession. Two micrometer-driven 1-ml Hamilton gas-tight syringes were used for this purpose. The reproducibility of the delivered volumes was better than 0.1% since any difference in absorbance did not exceed 0.001 when, e.g., 0 to 250 ~1 of 1.57 mM MeUmb-Manp was diluted into two cuvettes, yielding absolute values of A up to 1.3 at 336 nm. After each addition of the

4 21 I

TITRATION

310

DE

BOECK, LOONTIENS,

AND DE BRUYNE

0 0

200

400 J.II Con

A (549pM)

FIG. 2. Difference absorption titration of 18.8 PM MeUmb-Manp with 0 to 90 PM Con A at 16.4”C. The sample and reference cuvettes contained equal volumes (2141~1) of the ligand and of the nonbinding 4-methylumbelliferyl fi-D-galactopyranoside, respectively, each with A = 0.151 at 334 nm. The experimental values -A& represent the decrease in absorption due to formation and dilution of the MeUmbManp-Con A complex. They are plotted against the volume of Con A that was dispensed from the two micrometer-driven Hamilton syringes in the two continuously stirred cuvettes. The curve through the data is the binding isotherm plus dilution, simulated with the parameters obtained from the inset. The inset is the linearized titration curve in which the signal change (-ti) and the concentration of free protein sites [P] have been corrected for dilution. Vertical lines represent an experimental uncertainty of A = 0.001. A 1:l complex was assumed to calculate [P] by iterative approximation till the slope K became constant; the starting value of K corresponded to the slope of -A-4/[P0] plotted against [PO], [PO] being the concentration of free plus occupied sites. Linear regression, with omission of the lowest eight concentration points, yields 10e3 (-A,4/[P]) = (2.173 + 0.018) - (51.9 + 0.8)(-u) with correlation coefficient -0.9972. The intercept on the -AA axis corresponds to the maximal difference 0.0418 + 0.0007 which is the maximal change for 18.8 PM MeUmb-Manp, corresponding to -AC = (2.22 + 0.03) lo3 M-’ Cm-’ at 334 nm.

titration fluid to the two cuvettes, zero absorption was set with the reference cuvette in all titrations, keeping the slit width constant at 0.3 mm (3.3 nm at 334 nm, 3.0 nm at 322 nm). RESULTS

AND

DISCUSSION

The titrations were performed at a single wavelength. In order to determine the change in extinction coefficient AC at 334 nm due to a decrease of MeUmb-Manp absorption upon binding to Con A, equal volumes of a ligand solution in the sample cuvette and of the nonbinding 4-methylumbelliferyl B-Dgalactopyranoside, with an equal absorption

in the reference cuvette, were both titrated with a concentrated solution of Con A. Such a difference absorption titration is given in Fig. 2. The binding parameters at 16.4”C were calculated from the inset as the association constant K = (5.19 +- 0.08)104 M-’ and, assuming a 1: 1 complex per protomer (2), as -AC = (2.22 * 0.03)103 M-t Cm-’ at 334 nm. The latter value is identical to 2.21 X lo3 M-’ cm-’ as obtained with Yankeelov mixing cuvettes (2). The number of binding sites per protomer of Con A was verified by titrating a protein solution against buffer in the reference cuvette with a concentrated solution of chro-

CONTINUOUS

DIFFERENCE

ABSORPTION

TITRATION

311

0.06

200

100

yl MeUmb-Manp

(157mM)

FIG. 3. Difference absorption titration of 40.7 PM Con A with concentrations of MeUmb-Manp ([La]) increasing from 0 to 170 pM. Equal volumes (2141 ~1) of protein and buffer solutions were contained in the sample and reference cuvettes, respectively, and 1.57 mM MeUmb-Manp was added to both cuvettes from the two syringes. This resulted in near saturation of the protein. The curve through the experimental values is simulated for the combined effect of MeUmb-Manp-Con A complex formation and its dilution. The parameters used were calculated from the inset, which is valid for the dilutioncorrected data. Lr is the concentration of free ligand calculated by iteration as in Fig. 2; and r is the binding ratio of ligand and protomer ([L,] - Lr)/[P,,]) calculated on the basis of -AZ = 2.2 X lo3 M-' cm-’ from Fig. 2. The straight line corresponds to lo-’ (r/L,) = (4.69 f 0.02) - (5.07 + 0.04)r with correlation coefficient -0.9992. The number of binding sites equals 0.924 + 0.008 per Con A protomer.

mophoric MeUmb-Manp being dispensed in the two cuvettes. Such a difference titration is given in Fig. 3. From the inset, the number of binding sites per protomer, as based on -AC = 2.22 X lo3 M-’ cm-i, is calculated as 0.924 f 0.008. The corresponding K value, obtained over the entire saturation range, equals (5.07 + 0.04)104 M-t. Within error limits this is identical to the value from Fig. 2, obtained at low saturation. For the binding of MeUmb-Manp to Con A, the experimental data obtained by continuous titration of difference absorption are in excellent agreement with the simulated binding curves in Figs. 2 and 3. The errors of the corresponding binding parameters are smaller than those obtained with Yankeelov cuvettes (2). The binding parameters obtained here are entirely consistent with the results of independent methods, such as

equilibrium dialysis or titrations of ligand fluorescence (2,3). We have also applied this technique to the binding of MeUmb-Galp@( 1 - 3)GalNAcp and PNA. This system was characterized by equilibrium dialysis (one binding site per PNA protomer, without interaction of sites and without binding to ancillary sites) and by titration of ligand fluorescence (De Boeck et al., unpublished). An example of a difference absorption titration obtained by adding a concentrated solution of MeUmbGalpa( 1 - 3)GalNAcp into dilute PNA and buffer is given in Fig. 4. It represents the increase of ligand absorption upon binding to PNA. The results are consistent with those obtained by the independent methods: simple binding to one site per protomer with K = (9.28 + 0.06)104 M-’ at 14.5”C assuming AE = 2.46 X lo3 M-’ at 322 nm.

312

DE BOECK, LGGNTIENS,

I OO

AND DEBRUYNE

I 0.5 t

Y

r

1 100 f ;j

80 s

~1 MdJmb-Galp

Oh33)GalNAcp

&89#4)

FIG.4. Ditference absorption titration of 21 PMPNA with MeUmb-Galpfi (1 - 3)GalNAcp ([&I) concentrations from 0 to 73.9 PM at 14S”C. Ligand (469 PM) was added from the two syringes into both sample and reference cuvette, containing equal volumes (2141 pl) of protein and buffer solutions, respectively. The experimental increase in absorption difference at 322 nm (A&) is plotted with curve A, which is a simulation for the combined effect of complex formation and its dilution, The percentage of dilution is given as curve B for comparison. The parameters used were calculated from the inset, which is valid for the dilution-corrected data. The symbols are the same as in Fig. 3; r was calculated with AC= 2.46 X 10’ M-’ cm-’ on the basis of a 1:1 complex demonstrated by equilibrium dialysis (data not shown). The straight line corresponds to lo-’ (r/Lr) = (9.29 f 0.04) - (9.28 k 0.06)r with correlation coefficient = -0.994. Curve C shows that the absorption difference between experimental and simulated data is smaller than 0.0005.

The results obtained by continuous titration of absorption differences as a function of chromophore concentration depend critically on the reproducibility of the volumes delivered by the two syringes. Widely used commercial all-glass syringes were systematically found to be unreliable due to leakage of the titration fluid between barrel and wall. Hamilton gas-tight syringes with a Teflon barrel tip were satisfactory. The delivered volumes yielded cuvette concentrations with a reproducibility that was better than 0.1%. Therefore, it was unnecessary in this work to calibrate this difference by a blank titration. The method, as illustrated by the results of carbohydrate binding to two lectins, seems

to be of general application for the determination of binding parameters by continuous titration of absorption differences. In comparison with the use of two-compartment mixing cells, it has distinct advantages. These are no cleaning of the cuvettes between measurements, no manual mixing of the cuvette content during a titration, a fixed difference in cuvette blanks, the possibility to obtain any number of data points, simplicity, and speed. The method is particularly suited for use in a double-beam instrument, preferably at a constant slit width. The two syringes can also be actuated with a single motor-driven micrometer provided that equilibrium establishes rapidly. This condition is fulfilled by the MeUmb-ManpCon

CONTINUOUS

DIFFERENCE

A system since at the lowest concentrations used, 99% of the equilibrium was reached within less than 1 s (7,8). Due to a slowly establishing equilibrium between PNA and its ligand used here, readings were made 1 min after operating the micrometers manually. ACKNOWLEDGMENTS H. D. B. is an IWONL bursar. The authors thank K. Matta and N. Sharon for samples of MeUmbGal@( 1 - 3)GalNAcp and of PNA, and Etienne Wauters for his technical contribution.

REFERENCES 1. Yankeelov, J. A., Jr. (1963)Anal. 289.

Biochem. 6,287-

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2. Loontiens, F. G., Clegg, R. M., and Jovin, T. M. ( 1977) Biochemis:ry 16, 159- 166. 3. Van Landschoot, A., Loontiens, F. G., and De Bruyne, C. K. (1978) Eur. J. Biochem. 83,277285. 4. Cunningham, B. A., Wang, J. L., Pflumm, M. N., and Edelman, G. M. (1972) Biochemistry 11, 3233-3239.

5. Lotan, R., Skutelsky, E., Danon, D., and Sharon, N. (1975) J. Biol. Chem. 250, 8518-8523. 6. Fish, W. W., Hamlin, L. M., and Miller, R. L. (1978) Arch. Biochem. Biophys. 190, 693-698. 7. Clegg, R. M., Loontiens, F. G., and Jovin, T. M. (1977) Biochemistry 16, 167-175. 8. Loontiens, F. G., Clegg, R. M., Van Landschoot, A., and Jovin, T. M. (1977) Eur. J. Biochem. 78,465-469.