Circular dichroism of hapten-antibody complexes

Immunochemistry. 1976, Vol. 13, pp. 509-515.

CIRCULAR STUDIES

OF

DICHROISM

SITES

SUBUNITS,

RESIDUE OF

MOPC-315

RICHARD

M. FREED,

Fv

JOHN

INVOLVEMENT PROTEIN

RECONSTITUTED ITS

Department

Printed in Great Britain

OF HAPTEN-ANTIBODY

TRYPTOPHANYL

COMBINING CHAIN

Pergamon Press.

IN

HEAVY

MOPC-315

COMPLEXES: THE

ANTIBODY

AND

LIGHT

PROTEIN

AND

FRAGMENT* H. ROCKEY?

and ROBERT

C. DAVIS

of Ophthalmology, University of Pennsylvania School of Medicine, Scheie Eye Institute. 51 North 39th St. and Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104, U.S.A.

(Receiwd 18 Drcernher 1975) Abstract-Circular dichroic spectra of the native MOPC-315 mouse IgA myeloma protein and the protein reformed from its isolated heavy and light chains, the amino-terminal variable region fragment (Fv-315)$ and the isolated subunits provide information about conformational features of the isolated chains, changes which occur upon subunit association and about local protein-protein interactions. A pair of negative peaks in the near-ultraviolet (at 285 and 293 nm) is present in both the MOPC-315 and Fv-3 I5 CD spectra, indicating that the structure responsible for the bands is located in the variable region of the intact immunoglobulin. These CD bands are absent from the CD spectra of the isolated heavy and light chain subunits but are regained in the process of subunit reassociation which results

in the formation of the intact antibody combining site. The near-u.v. circular dichroism of a model cyclic tryptophanyl dipeptide provides evidence that two or more closely juxtaposed tryptophanyl residues may be responsible for the protein aromatic chromophore transitions at 285 and 293 nm. Binding of chromophoric haptens (Dnp- and Tnp-aminocaproate) to the native and reformed MOPC-315 protein and to its Fv fragment generates an induced circular dichroism with unique features: (1) an increase of the negative CD at both 285 and 293 nm; (2) a perturbation (blue-shift) of the tryptophanyl band at 293 nm; and (3) an apparent reciprocal relationship between positive induced CD bands of haptenic chromophore (at 37&382nm) and the large negative induced CD band in the aromatic residue absorption region of the protein (near 300nm) where haptens display absorption minima. These spectral features imply that the induced CD arises from electronic interactions between the aromatic moiety of the bound hapten and tryptophan(s) in the protein. This pattern of induced CD furnishes additional evidence that the paired CD bands at 285 and 293 nm in the spectra of the intact immunoglobulin and its Fv fragment arise from closely situated tryptophans and indicates that these residues lie in or near the intact antibody combining site. Induced CD bands generated by binding Tnp-aminocaproate to the isolated light chains also exhibit reciprocal relations and may be due to electronic interactions between the aromatic moiety of the bound hapten and tryptophan(s) in the protein subunit. Data available from primary amino acid sequence and affinity-label studies are consistent with the suggestion that variable region tryptophans from different subunits are close neighbors in or near the intact antibody combining site. The negative 293 and 285 nm CD bands of the MOPC-3 15 and Fv-3 15 proteins may therefore represent short range electronic interactions occurring within an antibody combining site between chromophoric residues contributed respectively by separate polypeptide chains

notable successes (Amzel et al., 1974; Poljak et al., 1974; Segal et al., 1974; Edmunson et al., 1974), its use is neither always practical nor possible. Magnetic resonance techniques show considerable promise but their application to the determination of complex biological structures is still in the process of development (Dwek et al., 1975). Circular dichroic (CD) measurements constitute a unique tool for the analysis of protein structure in solution. They are highly sensitive to variations in tertiary structure and are capable of demonstrating small differences in stereochemical relationships (Schellman, 1968; Rockey et al., 1972; Orin et al., 1976). In addition, the recording of CD spectra of proteins has become fairly routine. CD spectral interpretation in terms of quantitative geometrical relationships and in terms of the assignment of CD bands

INTRODUCTION

Given the diversity of antigenic determinants with which specific antibodies associate, and the corresponding variety of tertiary structures which constitute their active sites, the problem of delineating the 3-dimensional structures of antibody combining sites poses a formidable challenge. While X-ray diffraction analysis of immunoglobulin fragments has achieved * Presented, in part, at the American Association of Immunologists Meeting, April 1974. ~To whom reprint requests should be sent, at the Department of Ophthalmology, University of Pennsylvania School of Medicine, Scheie Eye Institute. $ Abbreviations: Vn, VL, heavy and light chain variable regions respectively; Fv, variable region fragment; Dnp, 2,4-dinitrophenyl group; Tnp, 2,4,6_trinitrophenyl group. 509

510

RICHARD

M. FREED,

JOHN

H. ROCKEY

to specific amino acid residues is facilitated by a rapidly expanding body of experimental data and advances in the theory of optical activity (Sears & Beychok. 1973; Bayley, 1973; Strickland, 1974). Induced CD bands arising from the non-covalent association of a symmetric haptenic chromophore and specific antibody offer a potent means for investigating the topology of amino acid residues in the vicinity of bound ligand (Rockey et al., 1972; Gollogly & Cathou, 1974). The theoretical basis for the gain in CD by a haptenic chromophore such as a dinitrophenyl (Dnp) or trinitrophenyl (Tnp) group, whose elements of symmetry preclude optical activity in the isolated ligand, is well-established (Schellman, 1968; Sears & Beychok, 1973; Strickland, 1974). The induced CD bands of the non-covalently bound haptenic chromophore reflect quantitatively the asymmetric stereochemistry in its immediate environment. The induced CD bands of protein chromophores, e.g. aromatic amino acid residue side chains which participate in the asymmetry created upon binding, also give information on the local geometry of their surroundings. In previous reports, the CD generated by associating Dnp and Tnp haptens with mouse, goat and rabbit immunoglobulins having substantial affinity for nitrophenylated ligands was shown to display distinctive spectral features dependent on the structure of the hapten and on molecular characteristics of the protein (Rockey et al., 197la, b, 1972). The occurrence of multiple CD bands in each induced spectrum provides a means for assessing combining site heterogeneity, for demonstrating small structural differences in antibody-hapten complexes and for probing the reformed combining site resulting from reassociation of antibody heavy and light chains. Circular dichroism associated with the absorption bands of the immunoglobulins and their subunits (intrinsic CD bands) are equally sensitive to stereochemical relationships and may also be used to probe the native and reformed antibody combining site (Rockey et al., 1972). The intrinsic CD of a protein derives primarily from electronic perturbation of the symmetric amino acid side chain, e.g. aromatic chromophore, and the peptide chromophore. The CD deriving from a symmetric aromatic chromophore is strongly dependent on the effects of tertiary structure, i.e. the specific 3-dimensional structure within which the aromatic amino acid side chain occurs (Imahori et al., 1973). The presence of near-u.v. CD in the spectrum of an immunoglobulin whose hypervariable regions contain optically active aromatic residues therefore may provide information on stereospecific interactions occurring within the antibody combining site itself. An increasing body of information about the primary structure and activity of the MOPC-3 15 protein (e.g. Eisen et al., 1968; Haimovich et al., 1970, 1971, 1972; Goetzl & Metzger, 1970a, b; Bridges & Little, 1971; Givol et al., 1971; Underdown et al., 1971; Glaser & Singer, 1971; Inbar et al., 1971, 1972, 1973; Rockey et al., 197la, b, 1972; Strausbauch et al., 1971; Litman & Good, 1972; Pecht et al., 1972; Dugan et al., 1973; Hsia et al., 1973a, b, c; Hochman et al., 1973; Johnston et al., 1974; Haselkorn et al., 1974; Merz et al., 1974; Francis et al., 1974; Roholt et al.,

and ROBERT C. DAVIS

1974; Hoessli et al., 1974; Dwek et al., 1975) makes it well suited for CD studies of the antibody combining site. Absorption spectral, fluorescence quenching, affinity-label and primary amino acid sequence studies of the immunoglobulin have been interpreted to indicate the presence of an indole ring in or near the MOPC-315 combining site and to imply its interaction with non-covalently associated haptenic chromophore. In the present report, extrinsic CD bands generated upon binding chromophoric Dnp and Tnp haptens to the MOPC-315 IgA myeloma protein, its variable region (Fv-315) fragment and its isolated chains (Rockey et al., 1971a, b, 1972; Glaser & Singer, 1971; Inbar et al., 1973; Merz et al., 1974; Orin et al., 1976) are examined for the information which they provide about stereospecific interactions which occur between haptenic chromophore and neighboring Trp(s) in the proteins. The intrinsic CD of the intact immunoglobulin, its Fv fragment and its isolated subunits provide evidence for changes which occur in the environment of Trp(s) upon subunit association. Corroborative evidence for the stereochemistry deduced from the protein and hapten-induced CD spectra is furnished by the near-uv. CD of a model Trp dipeptide compound. An important result of these CD spectral studies of the MOPC-315 protein is that their data serve as a basis for the calculation of geometries which may exist between the Trp contact residue(s) and ligand chromophore. Such calculations are presented in the accompanying paper (Orin et al., 1976).

MATERIALS

AND METHODS

MOPC-315 tumor (Potter et al., 1967; Eisen et al., 1968) was obtained from Dr. Herman N. Eisen and maintained in Balb/c mice by intraperitoneal transfer of ascitic fluid. The MOPC-315 protein was purified from harvested MOPC-315 ascitic fluid and blood by utilizing a solid immunoabsorbant of Dnp-lysine covalently linked to Sepharose-2B (Dnp-lysine-Sepharose) as previously described (Rockey et al., 1971a;b, 1972). In -some instances the MOPC-315 nrotein was mildlv reduced (1 hr. 25°C. 10 mM dithiothreitoi) and alkylated i4”C, 11 mM iodoacetamide) prior to affinity chromatography. The MOPC-315 protein specifically bound to the Dnp-Sepharose was eluted with 1 M acetic acid, exhaustively dialyzed against borate buffered saline (pH 8.4) at 4”C, and concentrated by negative pressure ultrafiltration, Protein concentrations were determined from the absorbance at 280 nm (corrected for light scattering), employing an extinction coefficient, Ei$““, of 1.44and a mol. wt of 153,000. Light and heavy chains MOPC-315 IgA myeloma protein at a concentration of 2 mg/ml was reduced (17 hr, 25°C 1OmM dithiothreitol) and alkylated (4°C 11mM iodoacetamide), transferred at 4°C to 1 M propionic acid by Sephadex G-25 gel filtration, immediately applied to 4 in-series columns (each column 4 x 60cm) of Sephadex G-100 equilibrated with I M propionic acid and eluted with the same solvent (Rockey et al., 1971 b, 1972). The resolved monomeric heavy and light chain fractions were immediately and exhaustively dialyzed at 4°C against 5 mM sodium acetate buffer (nH 5.4) and concentrated when necessary by negative pressure ulnafiltration at 4°C. Heavy and light chain protein concentrations were determined from their peak absorbance at

511

CD of Trp in the MOPC-315 Antibody Combining Site 27X-280 nm (corrected for light scattering) employing extinction coefficients, E:‘$“‘, of 1.5 and 1.1 (Underdown et al., 1971) and mol. wt of 53,000 and 23,000, respectively. Reassociation

of MOPC-315

protein subunits

Heavy and light chain fractions in 5 mA4 sodium acetate buffer (pH 5.4) were recombined in the same solvent (Stevenson, 1968; Stevenson & Dorrington, 1970). The recombined protein was dialyzed against 0.1 M NaCl-10 mM phosphate buffer (pH 7.5) and filtered at 4°C through 4 in-series columns (each column 2 x 60 cm) of Sephadex G-150 in the same solvent, and the 7s fraction was used for CD studies. Fu-315 fragment The preparation of the Fv fragment has been described in detail (Inbar et al., 1972; Hochman et al., 1973). The MOPC-315 protein was incubated with pepsin for 6 hr at pH 4.5 and 37°C. Further digestion with pepsin was then performed at pH 3.7 for 4 hr. The digest was subjected to affinity chromatography on Dnp-lysine-Sepharose from which the Fv fragment was eluted with Dnp-glycine. The yellow fractions were applied to a column of Sephadex G-75 equilibrated in 0.05 M NaCl, 3 mM sodium phos-

phate buffer, pH 7.4, and the Fv-315 protein was eluted in the same solvent. Fv-315 protein concentration was determined at 280 nm employing an extinction coefficient, EfF$“‘, of 1.5 and a mol. wt of 25,000. Chemicals

Dnp-aminocaproate (W-2,4-dinitrophenylaminocaproate) was obtained from Sigma Chemical Co. Tnpaminocaproate (NE-2,4,6-trinitrophenylaminocaproate) was synthesized as previously described (Rockey et al., 1971b). L-Tryptophanyl-L-tryptophan diketopiperazine was obtained from Cycle Chemical. Circular

dichroism

Circular dichroism was measured at 25°C with a Durrum-Jasco model J-10 circular dichrometer or a Cary 61 recording spectropolarimeter. The instruments were calibrated with d-lo-camphorsulfonic acid and d-camphor (Cassim & Yang, 1969). Cell pathlengths of 0.5 cm and 1.0 cm were used in determining the CD spectra of protein and protein-ligand complexes. Data are presented in terms of molar ellipticities, [tIli, in deg cm2 dmole-‘, where [0], = (2.303)(4500/~)(~, - E,), the molar dichroic difference absorptivity, (E, - E,) = (A, - A,)/lc, I is the cell path length in centimeters, c is the molar concentration of protein or bound ligand and (A, - A,) is the observed difference in absorption between left and right circularly polarized light at wavelength i,. [B],(Apparent) refers to the circular dichroism of a protein-ligand complex, measured at high hapten excess and calculated by assuming that the molar concentration of protein equals the molar concentration of binding sites. Data are also presented in terms of the observed ellipticity, .!Yj. (Rockey et al., 1972).

RESULTS The CD of the isolated heavy and light chains of the MOPC-315 protein differed from that obtained

with the intact protein. The CD spectrum of the isolated heavy chain (Fig. 1A) displayed negative CD in the near-u.v. region of protein aromatic amino acid absorptivity (24&330 nm), but lacked the distinct banded structure of the native MOPC-315 protein. The CD spectrum of the light chain showed little CD at wavelengths greater than 260nm. An equal molar combination of the CD spectra of the isolated heavy and light chains does not sum to the equivalent molar

(L-H chain half molecule) spectrum of the intact MOPC-315 protein. Recombination of the isolated heavy and light chains of the MOPC-315 protein, however, led to a total regain of the CD of the native protein (Fig. 1B). The prominent 293 nm and 285 nm CD bands of the MOPC-315 protein were absent from the spectra of the isolated subunits but the ellipticity and i,,, of each were recovered upon subunit association. The induced CD generated by binding Dnp-aminocaproate to reconstituted MOPC-3 15 was identical to that obtained with native protein (Fig. 1B). A blueshift of the 293 nm CD band to 292 nm was observed at saturation with hapten in the spectra of both the native and reconstituted MOPC-315 protein. The induced CD difference spectrum of Tnpaminocaproate non-covalently bound to the MOPC-315 light chain (Fig. 1C) was similar to that obtained with the intact protein, but differed in peak values for CD maxima or minima, e.g. 375 nm &,,, obtained with light chain vs 370nm i,,,, obtained with native protein, in crossover points, e.g. 409 nm with light chains vs 389nm with native protein, and in the ratios of ellipticities (@j.l/0>,2)at different wavelengths. The induced CD difference spectrum generated with Tnp-aminocaproate and MOPC-315 heavy chains showed only negative ellipticity between 500 and 320nm (Fig. 1C). The induced CD difference spectrum obtained with Tnp-aminocaproate and native MOPC-315 protein again cannot be reproduced in detail by a combination of the hapten-light and hapten-heavy chain CD spectra. The CD spectrum of the Fv-315 (Fig. 1D) retained the prominent twin negative near-u.v. region bands at 293 and 285 nm characteristic of the intact MOPC-315 protein spectrum. The Fv 293nm CD again was distinctly blue-shifted by the addition of the hapten Tnp-aminocaproate (Fig. 1D). The blueshift is also apparent in induced CD difference spectra (CD of ligand bound protein minus CD of protein alone) of Dnp and Tnp haptens bound to the Fv-315 or to the native or reassociated MOPC-315 protein (Rockey et al., 1972; Inbar et al., 1973). In the CD difference spectra, the blue-shift of the 293 nm band results in a negative peak at 290 nm.

DISCUSSION Assignment

of CD bands

to tryptophan

The location in the near-u.v. of the negative CD bands at 293 and 285nm in the native or reformed MOPC-315 spectrum and in the CD spectrum of Fv-315 suggests that the electronic transitions responsible for these bands lie in the aromatic type of chromophore. The only other type of amino acid moiety capable of absorbing near-u.v. radiation is the disulfide bridge whose CD bands exhibit no fine structure and are considerably broader than those due to aromatic groups in amino acid side chains (Strickland, 1974). An electronic transition at or near 293 nm is highly distinctive of the indole chromophore. It has been successfully utilized, in spectroscopic techniques dependent upon the absorption of light, for the analysis of Trp in proteins and in similar compounds

-44

280

290

Wavelength,

250

270

290

Wavelength,

300 nm

310 nm

29’3 nm

1

Wavelength,

I

550

450

350

255

295

275 Wavelength,

nm

nm

Fig. I. (A) CD spectra of native MOPC-315 mouse IgA myeloma protein ( ~~ ). and isolated MOPC-315 protein heavy (H, -.-) and light (L, ----) chains, in 5 mM Na acetate buffer (pH 5.4). Prcsentcd in terms of molar ellipticities. [@I, = IO’ 0, I-’ c-’ in deg cm’ dmolc-‘. where 0, is the ohservcd ellipticity. I is the cell path length in cm, and c is the protein molar concentration. CD spectrum (Oj) of L-tryptophanyl-L-tryptophan diketopiperazinc (Tmp) () in dioxane. (B) Cary 61 circular dichrometer tracings of the CD spectra of: (I) reassociated MOPC-315 protein: and (2 and 3) protein- hapten complexes obtained upon sequential addition of Dnp-aminocaproatc to the reassociated protein (tracing 3 at antibody combining site saturation). Solvent 0.1 A4 NaCl-10 mM phosphate buffer (pH 7.5). Note the blue-shift of the protein 293 nm CD band in the protein-hapten complex spectra. (C) CD difference spectra of Tnp-aminocaproate complexed with MOPC-315 protem (- -), and with isolated MOPC-315 protein heavy (-.-) and light (----) chains. Solvent, 5 mM Na acetate buffer (pH 5.4). Heavy and light chain difference spectra were determined in presence of high conccntrations of free hapten. and are presented in terms of the apparent molar ellipticities. [O],, (apparent). calculated by letting E equal the molar concentration of the MOPC-315 protein subunit. A CD peak also is seen at 495 nm in the spectrum of NY-Tnp-tryptophan (Davis, Freed & Rockey, unpublished observations). (D) Jasco J-IO circular dichromcter tracings of the CD spectra of MOPC-315 protein Fv fragment. and Fv fragment plus Tnp-aminocaproate. Solvent. 0.15 M NaCI-IOmM phosphate buffer (pH 7.4). The induced CD difference spectrum obtained with the Fv fragment Tnp-aminocaproate complex above 3 IO nm (3 It& 550 nm. not illustrated) was the same as that obtained with the intact and reassociated MOPC-i 15 proteins. 512

CD of Trp in the MOPC-315 Antibody Combining Site (Donovan, 1969). The indole band near 293nm in magnetocircular dichroic (MCD) spectra permits the unambiguous identification and accurate quantitation of Trp in proteins (Barth et al., 1971; Holmquist et al., 1973). In the CD of proteins, only the Trp residue exhibits spectral fine structure in the region between 290 and 305 nm (Strickland, 1974). The 293 nm CD band is also characteristic of a Trp residue transition perturbed by dissymmetrically situated vicinal moieties (Rockey et al., 1972; Strickland, 1974). Dissymmetrically situated Trp residues may be responsible for both the 293 and the 285 nm CD bands. The analagous negative CD bands in the CD spectrum of the model compound, L-tryptophanyl+tryptophan diketopiperazine (Edelhoch et al., 1968), in which two Trps are constrained by a dipeptide ring to lie in close proximity, suggest that closely situated variable region Trp residues may be responsible for the 293 and 285 nm CD bands of the native and reformed MOPC-315 protein and its Fv fragment. In the CD spectra of proteins, a 285nm CD band may arise from either a Trp or Tyr electronic transition (Strickland, 1974). In the CD of proteins having one or more Trps, however, the 290 nm region CD band is characteristically accompanied by a companion CD band located 6-S nm on the short wavelength side and with the same sign (Strickland, 1974). Induced CD evidence for tryptophan The blue-shift. Non-covalently bound chromophoric hapten may cause the perturbation of Trp(s) located in or near the intact combining site and effect the distinct hapten-induced blue-shift of the 293 nm band seen in both the MOPC-315 protein and the Fv-315 CD spectra. Variations in the micro-environment of the individual Trp residues in different proteins, at different positions in a single protein, and at a single position of a given protein with local conformational perturbations (e.g. following temperature, solvent or pH changes) characteristically result in variations of the La, values of the Trp 290-295 nm CD bands (Strickland, 1974). The distinctive 290nm region peaks in the model diketopiperazine CD spectrum (cf. Fig. 1A) are displaced in wavelength simply by altering the polarity of the solvent (Edelhoch et al., 1968). Reciprocal relations. For two neighboring chromophoric groups such as a Trp and a Tnp moiety, having strong electronic absorption bands (molecular extinction coefficients greater than lOOO), the mutual interaction (coupling) of two electronic transitions located in different chromophores may be expected to play a major role in the development of induced CD (Schellman, 1968). The rotational strength (a quantity proportional to the area under the CD curve) of the transition will be equal in size but opposite in sign to the rotational strength generated in the transition to which it is coupled. The two induced CD bands coupled in this way must vary in a reciprocal nlanner. An apparent reciprocal relationship occurs in the induced CD spectra of Dnp and Tnp haptens complexed with MOPC-315 protein, between the positive *Numbering is according to protein Eu (see Francis et al., 1974).

513

370-382 nm CD band of the haptenic chromophore and the negative CD band centered in the protein aromatic residue absorption region near 300 f 15 nm where haptens display minimal absorption (Rockey et al., 1972). The corresponding bands in the CD difference spectrum of Tnp-aminocaproate complexed to the light chain (Fig. 1C) also may represent the induced optical activity of a Trp chromophore in a subunit combining site, generated by an intermolecular coupled oscillator mechanism. Calculations of the theoretical CD derived from a dynamic coupling of electronic transitions in a Trp group and a closely situated Tnp moiety as a function of the 3-dimensional arrangement of the transition vectors (Orin et al., 1976) simulate in large part the shape and magnitude of the CD induced in both the MOPC-315 protein and its isolated light chain by Tnp-aminocaproate. Tryptophan in the 315 combining site In addition to the CD data, there is a variety of evidence which implies the presence of Trp(s) in or around the antibody combining site of the MOPC-3 15 protein. Spectroscopic evidence includes the quenching of Trp fluorescence by bound hapten and changes in absorbance (red-shift, hypochromism) of bound haptenic chromophore (Eisen et al., 1968; Haimovich et al., 1972; Rockey et al., 1972).’ More recently it has been reported that in the proton magnetic resonance spectrum of Fv-315, nuclear spins relaxed by a paramagnetic (spin-labeled) hapten are characteristic of Trp and other aromatic residues in or near the combining site (Dwek et al., 1975). Amino acid sequence studies (Dugan et al., 1973; Francis et al., 1974) also indicate that each of the five variable region Trp residues of the MOPC315 protein lies in or near a hypervariable region (Wu & Kabat, 1970; Kabat & Wu, 1971; Capra & Kehoe, 1975) where the amino acid side chains of an antibody are considered most likely to make non-covalent contact with antigenic determinant. Two variable region Trps on the light chain and three on the heavy chain may thus be available to form paired combining site Trps in the intact MOPC-315 protein or its Fv fragment and to interact with haptenic chromophore in the antibody combining site. By analogy with the combining site of a human myeloma protein (IgG New), mapped from X-ray diffraction studies, it has been suggested that two Trps (V,_Trp,s and V,Trp,,)* may contribute to the sides or limits which delineate the MOPC-315 combining site (Poljak et al., 1974). Induced CD-subunits

us intact protein

The induced circular dichroic bands generated with Tnp-aminocaproate and MOPC-315 light chains resemble those observed in the induced CD difference spectrum of Tnp-aminocaproate complexed to the native or reformed MOPC315 protein. Each induced CD spectrum can in fact be substantially reproduced by coupling the electronic transitions of closely situated Tnp and Trp moieties (Orin et al., 1976). The differences in these induced CD spectra may result from the highly sensitive geometric dependence of circular dichroism on small variations in stereochem-

514

RICHARD

M. FREED,

JOHN

H. ROCKEY

ical relationships (Schellman, 1968). The coupled oscillator model suggests that alterations in the orientation of haptenic chromophore in relation to interacting combining site Trp(s) are responsible for observed differences in the induced CD spectra obtained with isolated subunits and the intact immunoglobulin. Constraints imposed by combining site contact residues of the heavy chain in the intact immunoglobulin on, for example, the non-chromophoric portion of hapten, could effect a difference in orientation of the haptenic chromophore because of their absence from the complex consisting of ligand and light chains alone. The stereochemical relationship between ligand chromophore and a combining site residue in an isolated subunit, such as Trp(s) in the isolated light chain, may, however, be the same as that obtained in the native or reformed protein. The CD generated in the intact site might then arise from multiple interactions. In addition to a dynamic coupling between the haptenic Tnp group and light chain Trp(s), perturbations could come from combining site contact residues, e.g. from still other Trps and/or from charged amino acid side chains emanating static electric fields (Schellman, 1968) contributed by the complementary heavy-chain subunit. It may also be that perturbation by solvent in an isolated subunit differs from that in the intact protein because of the influence of the complementary subunit on the access of solvent to the combining site region. The observed differences in the induced CD spectra obtained with isolated subunits and intact proteins also might result from protein conformational changes which occur within the binding site regions upon association of the light and heavy chains. Strickland (1974) however, has pointed out that it is unwise to attribute conformational significance to any CD change accompanying interactions between two molecules without considering the other factors which might bring it about. Local interactions in the binding regions can induce new CD or alter existing CD in either molecule without the occurrence of conformational change. The 3-dimensional protein conformation of combining site contact residues may thus remain unaltered upon chain recombination. It has been argued from thermodynamic calculations of free energies of binding that the active site regions of the heavy and light chains of rabbit IgG antibodies are conformationally similar in the native and isolated states (Painter et al., 1972a, b). The occurrence in the isolated subunits of a pseudosite formed by the pairing of two heavy or two light chain combining site regions, would offer additional mechanisms for generating different induced CD spectra. The induced CD of hapten bound to the heavy chain dimers of rabbit anti-fluorescein antibody differs strikingly from that obtained with intact antibody (Gollogly et al., 1973). Variable region binding sites in the human Bence Jones dimer Meg are formed from chemically identical i. chain monomers which differ from each other in protein conformation (Edmundson et al., 1974). Appreciable binding of hapten by isolated heavy and light chain fractions of rabbit antibody molecules specific for the Dnp or the 4-azobenzene-1-sulfonate apparently group requires the dimeric state (Stevenson, 1973). This was

and ROBERT

C. DAVIS

taken to imply that the binding of antigens by isolated chains is largely fortuitous, i.e. it depends on the ways in which chains may come together to form pseudosites. Dimerization, however, does not appear to be a completely random process. Binding of hapten by the separated chains reflects the specificities of the parent antibody molecules and therefore probably preserves structural features found in the original intact combining site. Intrinsic

CD-subunits

t’s intact protein

The aromatic chromophore(s) responsible for the negative 290nm region CD bands of the intact protein or Fv-315 can gain CD only from neighboring electronic perturbations because the aromatic ring possesses a plane of symmetry and is not inherently optically active. The gain of near-u.v. CD upon subunit association therefore results from the juxtaposition of amino acid side chains either on the same subunit or on complementary adjacent subunits. The protein backbone of a single polypeptide chain may undergo, for example, a change in conformation upon subunit association, which causes an aromatic, e.g. Trp or Tyr, chromophore to be perturbed by an adjacent residue on the same subunit in the native or reformed MOPC-3 15 immunoglobulin. The electronic transitions observed at 285 and 293 nm upon subunit association may result, however, simply from the close approach of amino acid residues on different polypeptide chains which come together without the occurrence of any change in the conformation of either chain. Combining site Trps on complementary

chains

Evidence suggesting a topology of the combining site in which closely situated variable region Trps are contact residues capable of interacting with haptenic chromophore has already been cited. That Trps from both chains may be involved is suggested by affinitylabel studies of the MOPC-315 protein (Haimovich et al., 1971) which have established that its combining site is a region to which both Vu and VL sequences contribute contact residues. It may therefore be that variable region Trps from complementary subunits become juxtaposed upon subunit association by the close approach of heavy and light chains. Amino acid sequence data (Dugan et al., 1973; Francis et al., 1974) and affinity-label studies impose constraints upon the topology of the MOPC-315 combining site which are fully consistent with this type of stereochemistry. A model of the MOPC-315 combining site in which V,_Trp,, and VuTrp,, are closely juxtaposed by an anti-parallel association of the Vn and V, amino acid sequences in the vicinity of these residues has been described (Freed & Rockey, 1974). In this arrangement of the subunits, VnLyssZ can be affinity-labeled with the bromoacetyl derivative of Dnp-lysine (BADL), or V,Tyr,4 can be affinity-labeled with bromoacetyl-Dnp-ethylenediamine (BADE) or with metanitrobenzene-diazonium fluoride (MNBDF), while the paired Trps are sufficiently close to the haptenic chromophore to be able to participate in dynamic coupling with it (Weinstein et al., 1969; Goetzl & Metzger, 1970h; Haimovich et al., 1970). The model of the MOPC-315 binding site that can be built by comparison to IgG new also contains

CD of Trp in the MOPC-315 Antibody Combining Site complementary Trps (V,Trp,, and V,Trp,,) in close proximity (Poljak et al., 1974). The pairing of Trps from different subunits could neatly account for the observed 285-295nm region negative CD bands in the intact MOPC-315 and Fv-315 proteins, which are absent in isolated subunit spectra but are regained upon subunit association. The 293 and 285 nm bands of the MOPC-315 protein therefore assume special interest since they may represent short range electronic interactions occurring within an antibody combining site between chromophoric residues contributed respectively by separate polypeptide chains. Acknowledgements-Supported by USPHS NIAID GRANT AI 11719 (J.H.R.). R.M.F. is the recipient of a Woodward Fellowship in Physiological Chemistry, Dept. of Pathology, University of Pennsylvania.

Hochman J., Inbar D. & Givol D. (1973) Biochemistry

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