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Induced optical activity (circular dichroism) of antibody-hapten complexes
Journal of Immunological Methods 1 (1971) 67-82. North-Holland Publishing Company
INDUCED OPTICAL ACTIVITY (CIRCULAR DICHROISM) OF A N T I B O D Y - H A P T E N COMPLEXES * John H. ROCKEY, Keith J. DORRINGTON and Paul C. MONTGOMERY Department o f Ophthalmology, School o f Medicine, University of Pennsylvania, Philadelphia; Department o f Biochemistry, University o f Toronto, Canada; Department of Microbiology, School o f Dental Medicine and Center for Oral Health Research, University o f Pennsylvania; MRC Molecular Pharmacology Unit, Medical School, University of Cambridge, England
Received 23 March, 1971, in revised form 18 May, 1971 The induced optical activity (circular dichroism) originating from the interaction of dinitrophenylated and trinitrophenylated haptens with antibody has been used as a probe of the antibody combining site. A mouse IgA myeloma protein (MOPC-315) with high affinity for DNP- and TNP-ligands was employed as an example of a homogeneous antibody. The sign and the magnitude of circular dichroism bands differed when DNP- and TNP-haptens were bound, respectively, to the single homogeneous MOPC-315 protein. Differences in the relative magnitude of distinct circular dichroism bands also were seen when the DNP-group was incorporated into different haptens and complexed with the same homogeneous antibody. The circular dichroism spectra of hapten complexed with native MOPC-315 protein, mildly reduced and alkylated protein, and reassociate~l MOPC-315 protein were similar. The induced circular dichroism of e-DNP-lysine bound to a goat anti-DNP antibody differed markedly from the circular dichroism spectrum of the same ligand bound to the MOPC-315 prutein. Circular dichroism has been shown to offer a new and powerful parameter for studying and comparing the fine structure of the combining sites of antibodies of similar specificity, and to probe the reformed site resulting from the reassociation of heavy and light polypeptide chains.
1. INTRODUCTION A new method of studying the interactions of haptens and antibodies has been developed which offers a sensitive probe of the fine structure of the antibody combining site, We have observed that distinct circular dichroism (CD) bands may arise when the inherently symmetric chromophore of a small ligand is bound within * Supported in part by Research Grants AI-05305 from the National Institute of Allergy and Infectious Diseases, and DE-02623 from the National Institute of Dental Research, USPHS; and by the Medical Research Councils of Canada and Great Britain.
68
J.H.Rockey et al., Hapten -antibody interaction
the dissymmetric environment of a specific antibody combining site (Rockey et al., 1971). Distinct circular dichroism spectra have been noted when 2,4-dinitrophenylated (DNP) or 2,4,6-trinitrophenylated (TNP) haptens were bound to a homogeneous mouse IgA myeloma protein with high affinity for DNP- and TNP-ligands. The induced optical activity of antibody-hapten complexes may differ significantly when a single chromophore is incorporated into different haptens and specifically bound to a single homogeneous antibody. Distinct circular dichroism spectral characteristics also may be evident when a single ligand is bound by two different antibodies. The magnitude and sign of the induced circular dichroism bands are sensitive to the relative orientation of the ligand chromophore and the amino acid residues within the antibody combining site. The analysis of circular dichroism arising from hapten-antibody interactions has been found to offer a powerful tool with which to monitor ligand binding, to study and compare the fine structure of the combining sites of antibodies which possess similar specificities, and to probe the reformed antibody combining site obtained upon reassociation of heavy and light chains.
2. MATERIALS AND METHODS 2.1. Purification of a n ti-DNP antibody The mouse MOPC-315 plasma cell tumor line of Potter and Lieberman (1967) was maintained in 5 - 8 month old BALB/c mice by the intraperitoneal transfer of 0.5 ml of ascitic fluid from a BALB/c mouse containing the tumor. The harvested ascitic fluid was allowed to stand overnight at 4°C and centrifuged to remove insoluble materials. The MOPC-315 IgA myeloma protein, which displays a high affinity for DNP- and TNP-ligands (Eisen et al., 1968), was purified by affinity chromatography (ibid.; Goetzl and Metzger, 1970). A solid immunoabsorbent of DNP-lysine covalently linked to Sepharose (DNP-Sepharose) (Porath et al., 1967) was prepared by mixing 200 mg of Sepharose 2B (Pharmacia) and 500 mg of cyanogen bromide in 40 ml of water. The pH of the mixture was adjusted to and maintained at 11.5 for 5 min by the addition of 2 N NaOH. The activated Sepharose then was washed on a fritted disc Buchner funnel with 500 ml of cold water, 500 ml of 0.1 M Na2CO3, dried by vacuum, added to 20 ml of 5.45 mM DNP-lysine (e-N-2,4-dinitrophenyl-L-lysine, British Drug House) in water, and the mixture was allowed to stand at 4°C overnight in the dark. Residual coupling activity was neutralized by the addition of ethanolamine until a final pH of 9.0 was obtained. The DNP Sepharose was washed on a Buchner funnel with 500 ml of 0.15 M NaC1-50 mM borate buffer (pH 8.4), 500 ml of 0.15 M NaC1, 500 ml of 1 M acetic acid, and 500 ml of 0.15 M NaCI, and dried by vacuum. A slurry was prepared by adding 25 ml of 0.15 M NaC1-50 mM borate buffer (pH 8.4), and columns (packed volume, 10 ml) were prepared in disposable syringes containing glass wool plugs and glass bead bases. Each column was washed with 200 ml of
J.H.Rockey et al., Hapten-antibody interaction
69
0.15 M NaC1-50mM borate buffer (pH 8.4), 10ml of 1% (w/v)bovine serum albumin, 200 ml of 1 M acetic acid, and 200 ml of borate-buffered saline immediately prior to use. Fifty ml of MOPC-315 tumor ascitic fluid was dialysed against borate-buffered saline (pH 8.4) overnight at 4°C, reduced with 10mM dithiothreitol (Calbiochem) for 1 hr at 25°C, and alkylated at 0°C by the addition of solid iodoacetamide (British Drug House, 2× recrystallized from ethanol, 2× recrystallized from water) to give a final molarity of 11 mM. The reduced and alkylated MOPC-315 protein was dialysed against borate-buffered saline (pH 8.4) at 4°C overnight, diluted with an equal volume of borate-buffered saline, and applied to a DNP-Sepharose column. In some instances, the reduction and alkylation of the MOPC-315 protein prior to affinity chromatography was omitted. The MOPC-315 protein was specifically bound to the DNP-Sepharose column, while other ascitic fluid proteins were eluted with a borate-buffered saline wash. The eluate was set aside for recycling. Sequential elution of the column with 1.0 M and 4.4 M acetic acid removed two protein fractions. These were dialysed immediately and exhaustively against borate-buffered saline at 4°C, and insoluble materials were removed by centrifugation. When necessary, the samples were concentrated by negative pressure ultrafiltration at 4°C. Both the 1 M and the 4 M eluates were found to contain only a single MOPC-315 IgA myeloma protein component when examined by immunoelectrophoresis with rabbit anti-whole mouse serum and anti-MOPC-315 protein antisera. Between 60 and 80% of the recovered MOPC-315 protein was present in the fraction eluted with 1 M acetic acid, and this material was used for further study. MOPC-315 IgA myeloma protein concentrations were determined from the absorbance at 280 nm (corrected for light scattering), measured with a Zeiss PM QII spectrophotometer, employing an extinction coefficient, Elmm~ml, of 1.44, and a molecular weight of 153,000 (Eisen, 1970; Haimovich et al., 1970; Underdown et al., 1970). Polydispersed anti-DNP antibody (Montgomery and Williamson, 1970)was isolated from rabbits and goats hyperimmunized with 2,4-dinitrophenylated antigens, by affinity chromatography or by specific immunoprecipitation (Eisen et al., 1967). 2.2. DNP- and TNP-haptens DNP-lysine (e-N-2,4-dinitrophenyl-L-lysine), DNP-aminocaproate (e-N-2,4-dinitrophenyl-aminocaproate) and DNP-glycine (tx-N-2,4-dinitrophenyl-glycine) were obtained from Sigma Chemical Company and used without further purification. TNP-aminocaproate (e-N-2,4,6,-trinitrophenyl-aminocaproate) was synthesized by reacting 20 g of e-amino-n-caproic acid (Sigma) with 40 g of picryl chloride (British Drug House, 3X recrystallized from ethanol-water) and 10 g of di-sodium tetraborate in 1.25 L of H20 at room temperature in a light-tight reaction vessel (Benacerraf and Levine, 1962). The pH was maintained at 9.5-10.0 by the addition of 3 N NaOH. After 90min, the pH was brought to 2 with HC1, and the precipitated TNP-aminocaproic acid was removed by filtration. TNP-aminocaproate
70
J.H.Rockey et al., Hapten-antibody interaction
was recrystallized 3 times from 95% ethanol, and dried in a light-tight container in vacuo over P 2 0 s . The molar extinction coefficients employed to determine hapten concentrations were: DNP-lysine, 6362 - 1.75 X 104; DNP-aminocaproate, e362 = 1 . 7 5 × 104; DNP-glycine, e36o = 1.59X 104; TNP-aminocaproate, e348 = 1.57 × 104 (Eisen et al., 1968; Goetzl and Metzger, 1970; Benacerraf and Levine, 1962). 2.3. Circular dichroism
Circular dichroism (CD) was measured with a Jasco model ORD/UV-5 spectropolarimeter equipped with a circular dichroism attachment, or with a Durrum-Jasco model J-10 circular dichrometer. The temperature of the sample in the model ORD/UV-5 spectropolarimeter was maintained at 25 + 0.5°C by cycling water from a thermostatically-controlled constant-temperature circulator through the cell housing. Thermostatting of the sample in the model J-10 circular dichrometer was accomplished by placing the cylindrical cuvette in a specially constructed brass cell holder which was in contact with both thermoelectric heating modules (used for both heating and cooling) and a sensing thermometer for a Hallikainen Resistotrol model 1215 temperature regulator. Fluid (water or methanol) was circulated through the cell housing from a cooling bath, and the temperature was maintained at 25°C with the temperature regulator in the heating mode. The relationship between the thermoregulator settings and the sample temperature was determined by directly measuring the temperature of liquid in the cell with a thermocouple over a range of thermoregulator settings. The accuracy of the temperature measurement was +- 0.3°C, and the fluctuation of the temperature at each thermoregulator setting was less than -+ 0.03°C. The circular dichrometers were calibrated with d-10-camphorsulfonic acid (Eastman Organic Chemicals) in water, and d-camphor (Eastman Organic Chemicals, further purified by repeated sublimations) in methanol, according to the method of Cassim and Yang (1969). The agreement between the two independent standardizations consistently was within 1% of the anticipated ratios for the molar dichroic absorption ((e 1 - er)max) or molar ellipticity ([0] max) values (ibid.). For the d-10-camphorsulfonic acid, which had not been specially dried, (e 1 er)ma x and [0] max at 291 -+ 1 nm were taken as +2.20 and +7260, respectively (ibid). De Tar (1969) has reported a (e 1 - er)29 o value of 2.49 for recrystallized d-10-camphorsulfonic acid dried at 50°C over P 2 0 s . The discrepancy between the two (e 1 - e r ) m a x values (Cassim and Y a n g 1969; De Tar, 1969) may largely be accounted for by a correction for hydration (De Tar, 1969). The consistency of the results of the calibrations with d-10-camphorsulfonic acid and d-camphor indicate that the use of the lower (e l - er)ma x value for hydrated d-10-camphorsulfonic acid (uncorrected for hydration) furnishes a satisfactory CD calibration if the d-10-camphorsulfonic acid is sufficiently free of impurities other than water. A d-10-camphorsulfonic acid standard CD spectrum was determined after each ligand-binding experiment and used to correct for any small variation in machine calibration.
J.H.Rockey et al., Hapten-antibody interaction
71
Cell path lengths of from 1.0 cm to 0.1 mm, and protein concentrations ranging from 4 × 10-3 to 1 g/100 ml were used ill determing the CD spectra of antibody and antibody ligand complexes between 600 and 190 nm. The data are presented in terms of molar ellipticity, [0]x , in deg cm 2 dmole -~, where [0]~, = (2.303) × (4500/70 (e 1-er)x, (e 1-er) x = ( A L - A R ) x / l c , and l is the call path length in cm, c is the molar concentration of bound ligand or antibody, and ( A L - A R ) x is the observed difference in absorption between left and right circularly polarized light at wavelength ~ (Cassim and Yang, 1969; Urry, 1970). The average residue weight Mo, taken as l lO for the antibodies, rather than the molecular weight was employed in defining protein [0Ix values. 2.4. Absorption spectroscopy
Absorption spectra and absorptivity difference spectra were determined at room temperature with either a Jasco ORD/UV-5 recording spectrophotometer, a Zeiss PM QII spectrophotometer or a Cary model 15 recording spectrophotometer. 2.5. Fluorescence quenching The quenching of antibody tryptophan fluorescence by bound hapten was measured at 25 + 1°C in a thermostatted cell compartment in either a Zeiss double M4 QIII monochromator spectrophotofluorometer equipped with a Farnell stabilized EHT power supply and a Keithley 610B electrometer, or an Aminco-Bowman spectrofluorometer (Eisen et al., 1968; Rockey, 1967). Proteins were excited at 280-295 nm and fluorescence was measured at 345 nm. The fluorescence data were corrected for dilution and internal quenching as previously described (Rockey, 1967). Average intrinsic association constants, Ko, were determined from the plots o f r / c 'versus r, where r is the moles of hapten bound per mole of antibody, and c ' is the free hapten concentration (Eisen et al., 1968; Rockey, 1967; Wu and Rockey, 1969). The valence of the antibody was taken as 2(n = 2) (Rockey et al., 1971; Underdown et al., 1970; Eisen, 1970; Haimovich et al., 1970). As Ko = [ r ] / c ' [ 2 - r ] , when r is 1, Ko = 1/c'. The binding data also were analyzed according to the Sips distribution function, in the form log[r/(n-r)] = a logc' + a logK0, where a is the index of heterogeneity (Eisen et al., 1968; Wu and Rockey, 1969; Sips, 1948). 2.6. Re-association o f MOPC-315 protein subunits Mildly reduced and alkylated MOPC-315 protein, previously utilized for CD DNP- or TNP-ligand binding studies, at a concentration of 2 mg/ml was transferred to 1 M propionic acid by Sephadex G-25 gel filtration and immediately applied to a Sephadex G-100 column equilibrated with 1 M propionic acid, and the column was eluted with the same solvent. The resolved heavy and light chain fractions were immediately and exhaustively dialysized at 4°C against 4 mM sodium acetate buffer (pH 5,4) and recombined in the same solvent (Stevenson and Dorrington, 1970). The recombined MOPC-315 protein was dialysized against 0.1 M N a C I - 1 0 m M phosphate buffer (pH 7.5) and used for CD ligand-binding studies.
J.H.Rockey et al., Hapten-antibody interaction
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J.H.Rockey et al., Hapten-antibody interaction
73
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Fig. 2. Circular dichroism spectrum o f a-N-2,4,-dinitrophenyl-glycine in free solution ( - - - ) , and bound to MOPC-315 protein ( ).
Fig. 1A. Circular dichroism spectrum of e-N-2,4-dinitrophenyl-aminocaproate in free solution (.... ), and bound to MOPC-315 IgA myeloma protein ( ). The [ 0 ] h values below 325 nm have been corrected for the negative CD contributed by the protein alone. B. Circular dichroism spectrum o f e-N-2,4,6-trinitrophenyl-aminocaproate in free solution ( - - - ) , and bound to MOPC-315 IgA myeloma protein ( ). C. Absorption spectra o f DNP-aminocaproate in free solution ( - - - ) , and an equal molar concentration b o u n d to the MOPC-315 protein ( ). The same concentration o f the MOPC-315 protein in the absence of DNP-aminocaproate was used as a reference to obtain the spectrum of the b o u n d DNP-aminocaproate. The DNP-aminocaproate-MOPC-315 versus MOPC-315 difference spectrum was examined at r = 1.1 where more than 99% of the total ligand was specifically bound to the protein. The spectra at maximum absorbance are plotted on an expanded wavelength scale in the upper right insert to illustrate that the spectra are composed o f more than one c o m p o n e n t near hma x. The absorption difference spectrum between b o u n d and free DNP-aminocaproate also is plotted( ..........). D. Absorption spectra o f TNP-aminocaproate in free solution ( - --) and bound at an equal molar concentration to MOPC-315 protein ( ), and the bound versus free ligand difference spectrum ( ............).
74
J.H.Rockey et al., Hapten antibody &teraction
3. EXPERIMENTAL RESULTS No circular dichroism was evident in the MOPC-315 IgA myeloma protein spectrum between 600 nm and 325 nm. DNP-lysine, DNP-aminocaproate, DNP-glycine and TNP-aminocaproate in free solution, at concentrations as great as 1.5 × 10-4 M, also did not display circular dichroism in this spectral region. In sharp contrast, the specific binding of the DNP- and TNP-ligands to the MOPC-315 protein resulted in the induction of distinct circular dichroism bands which were characteristic of the ligand involved (figs. 1A, 1B, 2). The CD spectra of DNP-lysine and DNP-aminocaproate complexed with the MOPC-315 protein were closely similar, but distinct spectral characteristics were apparent when DNP-glycine or TNP-aminocaproate were bound by the same antibody. The DNP-lysine- and DNP-aminocaproate-MOPC-315 protein spectra displayed a broad positive CD band centered near 420 nm, a sharper positive CD band centered at 380 nm and a large negative CD band centered at a wavelength less than 320 nm (fig. 1A). The ellipticity at 380 nm was approximately two times greater than the ellipticity at 420 nm in both the DNP-lyisne-MOPC-315 and DNP-aminocaproate-MOPC-315 CD spectra. The CD spectrum of DNP-glycine bound to the MOPC-315 protein also showed a broad positive CD band centered near 420 nm, and a broad negative band centered below 320 nm (fig. 2). Here, in contrast to the bound DNP-lysine and DNP-aminocaproate spectra, only a small shoulder centered near 380 nm was present in the bound DNP-glycine CD spectrum (fig. 2). The ellipticity at 380 nm in the DNP-glycine-MOPC-315 protein spectrum was less than the ellipticity at 420 nm (fig. 2). The CD spectrum of TNP-aminocaproate differed strikingly in that a large negative CD band was present at 420 nm (fig. 1B). A small positive CD band centered near 490 nm, a narrow positive CD band centered at 370 nm and a large negative band centered below 320 nm "also were evident in the TNP-aminocaproate-MOPC-315 protein spectrum (fig. 1B). Circular dichroism spectra of DNP- and TNP-ligands bound to non-reduced MOPC-315 protein, MOPC-315 protein isolated after mild reduction and alkylation, and re-associated antibody formed from isolated MOPC-315 protein heavy and light chains, were similar. The circular dichroism spectrum of the MOPC-315 protein between 325 nm and 200 nm displayed a large negative band centered at 217 nm, and smaller negative bands centered near 292,286 and 280 rim, in the region of high absorptivity by the protein aromatic amino acid residues. Determination of the circular dichroism of complexes of the MOPC-315 protein and DNP-lysine, DNP-aminocaproate, DNP-glycine or TNP-aminocaproate between 325 nm and 250 nm, subtracting the ellipticity of the protein alone from the ellipticity of the ligand-protein complex, demonstrated that the negative CD bands in the spectra of the ligand-protein complexes at the lower wavelength were centered near 295 -+ 15 nm. The low signal to noise ratio resulting from the high absorptivity of the ligand-MOPC-315 protein complexes made the determination of the induced ellipticity more difficult in this spectral region.
J.H.Rockey et al., Hapten-antibody interaction
75
The absorption spectra of free and bound DNP-aminocaproate and TNP-aminocaproate, and the difference spectra of bound versus free ligands, between 600 nm and 300 nm, are presented in figs. 1C and 1D. The absorption spectra of the free ligand and of the ligand bound to the MOPC-315 protein, at peak absorptivity are plotted on an expanded scale in the upper parts of figs. 1C and 1D to illustrate that the spectra are formed from more than a single component in this wavelength region. This is particularly evident in the free and bound DNP-aminocaproate spectra (fig. 1C). The spectra of DNP-lysine display a similar phenomena. Free DNP-aminocaproate and TNP-aminocaproate also showed absorption bands at 265 and 212 nm, and at 211 and near 250 nm, respectively. The circular dichroism spectrum of DNP-lysine specifically bound to a goat anti-DNP antibody (fig. 3) differed markedly from the spectrum of the same ligand bound to the MOPC-315 protein. Broad negative overlapping CD bands centered near 3 5 0 - 3 0 0 nm were evident in the goat antibody-ligand spectrum. We also have been able to detect induced circular dichroism in the spectrum of DNP-lysine complexed with polydispersed rabbit anti-DNP antibody. No induced circular
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WAVELENGTH (nm) Fig. 3. Circular dichroism spectrum of e-N-2,4-dinitrophenyl-L-lysine in free solution ( - - - - - ) and bound to goat anti-DNP antibody ( ). Antibody concentration, 5.36 × 10-SM. DNP-lysine concentration, 4.0 X 10-SM. The apparent molar eUipticity, [0]h (apparent), equals (2.303) (4500/~r)(AL-AR)h(l)-l(total DNP-lysine concentration) -1, where all of the ligand present is assumed to be bound to antibody. The ellipticity of the bound ligand has been corrected for the positive circular dichroism of the goat anti-DNP antibody at wavelengths less than 325 nm.
J.H.Rockey et al., Hapten-antibody interaction
76
dichroism was evident when human myeloma proteins and rabbit "),G-globulins lacking anti-DNP activity were mixed with the DNP- or TNP-ligands. Hapten-binding curves were constructed by serially adding the DNP- or TNPligand with a Hamilton microsyringe to a constant amount of antibody and measuring the change in circular dichroism at selected wavelengths (e.g., 420 nm, 3 7 0 - 3 8 0 rim, 325 nm). The measurement of induced circular dichroism at 325 nm, where both the protein and the DNP- or TNP-ligands showed a minimum of absorbance and a maximum ratio of induced ellipticity to absorbance (fig. 1), presented a highly useful optical window for following ligand binding. The circular dichroism data were plotted as (AL-A R)x versus (total ligand concentration); and as [0] x (apparent) versus (total ligand concentration), where [0] x (apparent) = 3300(A L - A R ) X / ( t o t a l ligand concentration)/. The molar ellipticity, [0]x , of bound ligand was obtained from the slope of the (AL--AR) x versus (total ligand concentration) plot where the percentage of ligand which was not bound was minimal (fig. 4). With a sufficient concentration (e.g., 5 X 10 -s M) of high affinity (K0 ~> 107 M- l ) antibody, the percentage of the total ligand that is free in solution
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J.H.Rockey et al., Hapten-antibody interaction
77
is less than 1% over a wide range of r values (r is the moles of hapten bound per mole of antibody). This applies here to the DNP-lysine, DNP-aminocaproate and TNP-aminocaproate haptens b o u n d to the MOPC-315 protein. For these ligands the (AL--AR) x versus (ligand concentration) data in the region below antibody site saturation have been treated by the method of least squares to give the (e I - er) x and [0] x values listed in table 1. With lower affinity l i g a n d - a n t i b o d y systems, where significant free hapten is present at higher r values (e.g., DNP-glycineMOPC-315 protein), the tangent of the slope of the (A L - A R)x versus (total ligand concentration) plot at infinite ligand dilution may be taken to give (e 1- er) x and [0] x for the b o u n d ligand. This procedure was used here to obtain the values for b o u n d DNP-glycine presented in table 1. The circular dichroism binding data intrinsically contain all of the information needed to define the binding characteristics. The molar concentration of antibody combining sites may be obtained from the ratio (Z t - A R ) x ( m a x ) / ( e 1 - er)x, where (A L - A R ) x ( m a x ) is the observed ( A L - - A R ) x value at antibody site saturation (fig. 4). This ratio gives the molar concentration of antibody combining sites and may be used, together with the protein concentration, to determine the valence of the antibody. This procedure has been used to demonstrate the bivalence of the MOPC-315 protein (Rockey et al., 1971). The value o f r at each dilution may
Table 1 Circular dichroism of antibody-hapten complexes. Ligand
Antibody
e-DNP-aminocaproate
MOPC-315
e-DNP-lysine
MOPC-315
a-DNP-glycine
MOPC-315
e-TNP-aminocaproate
MOPC-315
e-DNP-lysine
Goat anti-DNP
h (nm)
(e 1- er)h
325 380 420 325 380 420 325 380 420 325 370 420 325 360
- 6.37 + 5.09 + 2.45 - 6.46 + 5.03 + 2.44 - 2.37 + 1.44 + 2.06 -11.0 + 4.02 - 6.48 - 3.64 * - 3.64 *
[0 ]?, -21,000 +16,800 + 8080 -21,300 +16,600 + 8030 - 7800 + 4770 + 6800 -36,250 +13,250 -21,350 -12,000 * -12,000 *
Molar circular dichroic absorption (el-er)h, and molar eUiptieity [0]~, at wavelength h, of DNP- and TNP-ligands bound to MOPC-315 protein and goat anti-DNP antibody. [O]h = (2.303)
× (4500/n)(el-er). * Apparent (e1- er) h and [0] h values, calculated by assuming that all of the ligand present was bound to antibody (5.36 × 10-s M antibody, 4 × 10-s M e-DNP-lysine).
78
J.H.Rockey et al., Hapten-antibody interaction
be obtained from the ratio 2(/IL -- A R)x(°bserved)/( A L - A R)X(max) for bivalent antibody. The concentration of bound ligand c = (.tl L -AR)7`/(e 1 --er)7`l, and the free ligand concentration, c ' = [(total concentration)-c]. With high-affinity antibody, however, where the percentage of the total ligand free in solution is very small (e.g., < 1%) at r = 1, the uncertainty of the data severely restricts usefulness of the method for obtaining accurate intrinsic association constants. Alternately, if the average intrinsic association constant for an antibody-ligand system is known from some other experimental parameter (e.g., equilibrium dialysis, fluorescence quenching) this value may be used to determine the free ligand concentration and the molar ellipticity of bound ligand at r = 1. This procedure also has been used here to determine [0] 7, values for bound DNP- and TNP-ligands, and the results were similar to those obtained from the (A L A R)7` versus (total ligand concentration) plots.
4. DISCUSSION The optical activity (circular dichroism) of the inherently symmetric dinitrophenyl and trinitrophenyl chromophores of the bound ligands is provided by interactions between the ligand chromophore and dissymmetrically placed vicinal moieties of the antibody combining site (Rockey et al., 1971; Urry, 1970; Perrin and Hart, 1970; Eyring et al., 1968). The vicinal perturbations that give rise to optical activity may be static in nature, and may result from dissymmetrically placed static charge, or from the polarizability and incomplete screening of nuclei of a vicinal group by the surrounding electrons such that the electron undergoing transition is able to sence the positive nuclei (Urry, 1970; Kauzmann et al., 1940; Condon et al., 1937; Hooker and Schellman, 1970; Hohn and Weigang, 1968). Circular dichroism also may arise from dynamic coupling of electronic transitions of the bound chromophore with electronic transitions of a vicinal chromophore in the antibody combining site. Optical activity may be induced by means of the coupled oscillator mechanism of Kuhn (1933), Kirkwood (1937) and Moffitt (1956) which involves the coupling of the electric transition dipole moments of two electronic transitions (Woody and Tinoco, 1967; Hohn and Weigang, 1968; Bayley et al., 1969; Hooker and Schellman, 1970; Urry, 1970). This mechanism is suggested when the ligand chromophore and a vicinal chromophore exhibit strong absorption bands (large electric transition dipole moments), and reciprocal relations are evident between a circular dichroism band centered at the ligand chromophore transition and a circular dichroism band of the opposite sign centered over the vicinal chromophore absorption band (Urry, 1970). Dynamic coupling between two chromophores also may give rise to optical activity by coupling an electrically allowed transition of one chromophore with a magnetically allowed transition of a second chromophore (Jones and Eyring, 1961 ;Woody and Tinoco, 1967; Hohn and Weigang, 1968; Bayley et al., 1969; Hooker and Schellman, 1970; Urry, 1970).
J.H.Rockey et al., Hapten antibody interaction
79
Circular dichroism arising by this mechanism again will display reciprocal relationships (Urry, 1970). There is an apparent reciprocal relationship between the positive CD band centered at 3 7 0 - 3 8 0 nm and the negative band centered over the aromatic amino acid absorption region in the spectra of DNP- and TNP-ligands complexed with the MOPC-315 protein. When a correction is made for the contribution at 3 7 0 - 3 8 0 nm of the broad band centered near 420 nm, it is seen that the ratio of the ellipticity of the 370-380 nm band to that of the 295 -+ 15 nm band is maintained for each ligand, even though the molar ellipticities vary widely between the different ligands. This reciprocal relationship, together with the large molar extinction coefficients of the DNP and TNP groups, suggests that these circular dichroism bands arise, at least in part, from dynamic coupling of electronic transitions of the ligand chromophore and a protein chromophore (e.g., tryptophan) in the antibody combining site. Both the magnitude and the sign of the rotational strength of a circular dichroism band arising by this mechanism will be dependent upon the relative orientation of the two interacting chromophores (Urry, 1970). The absorption spectra of DNP- and TNP-ligands bound to the MOPC-315 protein display hypochroism and a red-shift (Eisen et al., 1968; Rockey et al., 1971). Little and Eisen (1966) have studied the difference spectra resulting from the association of DNP- and TNP-ligands with anti-DNP and anti-TNP antibodies from a number of sources, and have suggested that a tryptophan residue is present in the combining site of anti-DNP and anti-TNP antibodies in general. The formation of a charge transfer comp! "" between the DNP or TNP group and the indole group of a tryptophan residue in the antibody combining site has been inferred (Little and Eisen, 1967). The DNP and TNP groups are good electron acceptors, indole is a good electron donor and there is substantial evidence from model systems which indicates these groups may form charge transfer complexes (Szent-Gyorgyi et al., 1961; Little and Eisen, 1967; Sigman and Blout, 1967; Shinitzky and Katchalski, 1968). The fluorescence of tryptophan residues of the MOPC-315 protein is quenched by bound DNP- and TNP-ligands (Eisen et al., 1968; Rockey et al., 1971). This is a general phenomenon observed with anti-DNP, anti-TNP and other antibodies (Little and Eisen, 1966; Rockey, 1967), and furnishes added support for the concept of the occurrence of a tryptophan residue in or near the antibody combining site of diverse antibodies. Circular dichroism furnishes a particularly powerful probe of the combining sites of anti-DNP and anti-TNP antibodies. While hypochromism and a red-shift of the spectrum of bound ligands, and quenching of antibody tryptophan fluorescence by ligand are general properties of anti-DNP and anti-TNP antibodies (Little and Eisen, 1966, 1967), the detailed structure of the CD spectra of bound DNP- and TNP-ligands may vary greatly between different antibodies. The CD spectra of DNP-haptens complexed with polydispersed rabbit anti-DNP antibodies may not display induced circular dichroism (Cathou et al., 1968; Ashman and Kaplan, 1970). The induced circular dichroism obtained when a single iigand (e.g.,
80
J.H.Rockey et al., Hapten-antibody interaction
DNP-lysine) is bound to two different antibodies (e.g., MOPC-315 protein, goat anti-DNP antibody) may differ markedly in the magnitude, sign and kmax of distinct circular dichroism bands. The complexing of a single chromophore, incorporated into two different ligands (e.g., e-N-DNP-L-lysine, a-N-DNP-glycine), with a single homogeneous antibody (e.g., MOPC-315 IgA protein) may result in induced circular dichroism which varies in the relative magnitude of the ellipticity (rotational strengths) at two different bands (e.g., 380 nm, 420 nm) in a manner dependent upon the ligand. The sign of a CD band at a single wavelength (e.g., 420 nm) of two related ligands (e.g., DNP-aminocaproate, TNP-aminocaproate) bound to the same homogeneous antibody may be reversed. It may be noted that the 2,4,6-trinitrophenyl group has an additional element of symmetry absent from the 2,4-dinitrophenyl group: the TNP-group has C2v symmetry for rotation about the N-C(phenyl) bond. This furnishes an added element of dissymmetry for interactions between the DNP-group and an antibody site chromophore such as a tryptophan indole group. The CD band centered at 420 nm arising from the DNP chromophore may vary in sign: in the circular dichroism spectra of ct-N-DNP-tryptophan and ct-N-DNP-glutamine a CD band is present at 420-440 nm, but it is negative (Rockey, Dorrington, Montgomery, unpublished observation). There is evidence from other systems that the magnitude and/or sign of extrinsic Cotton effects arising from complexing a single ligand to different macromolecules may differ (Blout, 1964; Perrin and Hart, 1970). The sign of the long wavelength CD bands seen on binding N-arylanthranilates (e.g., flufenamic acid) to different species of albumins were in some instances reversed (Chignell, 1969). The extrinsic CD spectra of cattle and squid rhodopsins, formed with the same chromophore, l l-cis-retinall, have been found to differ (Kito et al., 1968). Oppositely signed extrinsic Cotton effects have been observed when 2,4-dinitroanilines were complexed with RNA and DNA, respectively (Gabbay, 1969). In model systems (e.g., o-, m- and p-tyrosine) slight differences in the orientation of the coupling groups (e.g., the aromatic ring and the carboxylate group) can lead to a reversal of the sign of a CD band (Hooker and Schellman, 1970). Within the binding site of the MOPC-315 protein, the DNP group appears to be relatively rigidly fixed. Two related affinity-labeling reagents, bromoacetyl-N-DNPethylenediamine (BADE) and bromoacetyl-e-N-DNP-L-lysine (BADL) which differ in the separation of the reactive bromoacetyl group from the DNP group, react wholly with different residues (tyrosyl of the light chain, and lysyl of the heavy chain, respectively) of the MOPC-315 protein combining site (Haimovich et al., 1970). Our studies serve to establish a new method of assaying hapten-antibody interactions, and of probing the antibody combining site. Circular dichroism presents a powerful method to study and compare the fine structure of the combining sites of antibodies of related specificity, and to probe the reformed site resulting from the reassociation of antibody heavy and light chains. This method therefore may be of special use in our quest for two distinct molecular forms of
J.H.Rockey et ak, Hapten-antibody interaction
81
antibody of identical specificity, formed by the translocation of a single heavy chain variable (N-terminal) gene to two different heavy chain constant region (C-terminal) cistrons (Rockey et al., 1970).
ACKNOWLEDGEMENTS We wish to thank Dr. Robert C. Davis of the Department of Chemistry, University of Pennsylvania, for the use of his calibrated Jasco-Durrum model J-10 spectropolarimeter, and for many helpful discussions.
REFERENCES Ashman, R.F. and A.P. Kaplan, 1970, Federation Proc. 29, 773. Bayley, P.M., E.B. Nielsen and J.A. Schellman, 1969, J. Phys. Chem. 73, 228. Benacerraf, B. and B.B. Levine, 1962, J. Exptl. Med. 115, 1023. Blout, E.R., 1964, Biopolymer Symp., No. 1,397. Cassim, J.Y. and J.T. Yang, 1969, Biochemistry 8, 1947. Cathou, R.E., A. Kulczycki, Jr. and E. Haber, 1968, Biochemistry 7, 3958. Chignell, C.F., 1969, Mol. Phaxmacol. 5,455. Condon, E.V., W. Altar and H. Eyring, 1937, J. Chem. Phys. 5,753. De Tax, D.F., 1969, Anal. Chem. 41, 1406. Eisen, H., 1970, Abstracts of the 8th Internat. Congr. of Biochem. 309. Eisen, H.N., W. Gray, J.R. Little and E.S. Simms, 1967, in: Methods in immunology and immunochemistry, eds. C.A. Williams and M.W. Chase (Academic Press, New York), vol. 1, p. 351. Eisen, H.N., E.S. Simms and M. Potter, 17~"°, Biochemistry 7, 4126. Eyring, H., H.-C. Liu and D. CaldweU, 1968, Chem. Rev. 68, 525. Gabbay, E.J., 1969, J. Am. Chem. Soc. 91, 5136. Goetzl, E.J. and H. Metzger, 1970, Biochemistry 9, 1267. Haimovich, J., D. Givol and H.N. Eisen, 1970, Proc. Natl. Acad. Sci. US 67, 1656. Hohn, E.G. and O.E. Weigang, 1968, J. Chem. Phys. 48, 1127. Hooker, Jr., T.M. and J.A. Schellman, 1970, Biopolymers 9, 1319. Jones, L.L. and H. Eyring, 1961, Tetrahedron 13,235. Kauzmann, W., J.E. Walter and H. Eyring, 1940, Chem. Rev. 26, 339. Kirkwood, J.G., 1937, J. Chem. Phys. 5,479. Kito, Y., M. Azuma and Y. Maeda, 1968, Biochim. Biophys. Acta 154,352. Kuhn, W., 1933, in: Stereochemie, ed. K. Freudenberg (Deuticke, Leipzig) p. 317. Little, J.R. and H.N. Eisen, 1966, Biochemistry 5, 3385. Little, J.R. and H.N. Eisen, 1967, Biochemistry 6, 3119. Moffitt, W., 1956, J. Chem. Phys. 25,467. Montgomery, P.C. and A.R, Williamson, 1970, Nature 228, 1306. Perrin, J.H. and P.A. Hart, 1970, J. Phaxmaceut. Sci. 59,431. Porath, J., R. Axen and S. Ernback, 1967, Nature 215, 1491. Potter, M. and R. Lieberman, 1967, Advan. lmmunol. 7, 91. Rockey, J.H., 1967, J. Exptl. Med. 125,249. Rockey, J.H., P.C. Montgomery and K.J. Dorrington, 1970, Biochemistry 9, 4310. Rockey, J.H., K.J. Dorrington and P.C. Montgomery, 1971, Nature 232, 192.
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Shinitzky, M. and E. Katchalski, 1968, in: Molecular associations in biology, ed. B. Pullman (Academic Press, New York) p. 361. Sigman, D.S. and E.R. Blout, 1967, J. Am. Chem. Soc. 89, 1747. Sips, R., 1948, J. Chem. Phys. 16,490. Stevenson, G.T. and K.J. Dorrington, 1970, Biochem. J. 118, 703. Szent-Gyorgyi, A., I. Isenberg and J. McLaughlin, 1961, Proc. Natl. Acad. Sci. US 47, 1089. Underdown, B.J., E.S. Simms and H.N. Eisen, 1970, Federation Proc. 29, 437. Urry, D.W., 1970, in: Spectroscopic approaches to biomolecular conformation, ed. D.W. Urry (Am. Med. Assoc., Chicago) p. 33. Woody, R.W. and I. Tinoco, 1967, J. Chem. Phys. 46, 4927. Wu, W.-H. and J.H. Rockey, 1969, Biochemistry 8, 2719.
INDUCED OPTICAL ACTIVITY (CIRCULAR DICHROISM) OF A N T I B O D Y - H A P T E N COMPLEXES * John H. ROCKEY, Keith J. DORRINGTON and Paul C. MONTGOMERY Department o f Ophthalmology, School o f Medicine, University of Pennsylvania, Philadelphia; Department o f Biochemistry, University o f Toronto, Canada; Department of Microbiology, School o f Dental Medicine and Center for Oral Health Research, University o f Pennsylvania; MRC Molecular Pharmacology Unit, Medical School, University of Cambridge, England
Received 23 March, 1971, in revised form 18 May, 1971 The induced optical activity (circular dichroism) originating from the interaction of dinitrophenylated and trinitrophenylated haptens with antibody has been used as a probe of the antibody combining site. A mouse IgA myeloma protein (MOPC-315) with high affinity for DNP- and TNP-ligands was employed as an example of a homogeneous antibody. The sign and the magnitude of circular dichroism bands differed when DNP- and TNP-haptens were bound, respectively, to the single homogeneous MOPC-315 protein. Differences in the relative magnitude of distinct circular dichroism bands also were seen when the DNP-group was incorporated into different haptens and complexed with the same homogeneous antibody. The circular dichroism spectra of hapten complexed with native MOPC-315 protein, mildly reduced and alkylated protein, and reassociate~l MOPC-315 protein were similar. The induced circular dichroism of e-DNP-lysine bound to a goat anti-DNP antibody differed markedly from the circular dichroism spectrum of the same ligand bound to the MOPC-315 prutein. Circular dichroism has been shown to offer a new and powerful parameter for studying and comparing the fine structure of the combining sites of antibodies of similar specificity, and to probe the reformed site resulting from the reassociation of heavy and light polypeptide chains.
1. INTRODUCTION A new method of studying the interactions of haptens and antibodies has been developed which offers a sensitive probe of the fine structure of the antibody combining site, We have observed that distinct circular dichroism (CD) bands may arise when the inherently symmetric chromophore of a small ligand is bound within * Supported in part by Research Grants AI-05305 from the National Institute of Allergy and Infectious Diseases, and DE-02623 from the National Institute of Dental Research, USPHS; and by the Medical Research Councils of Canada and Great Britain.
68
J.H.Rockey et al., Hapten -antibody interaction
the dissymmetric environment of a specific antibody combining site (Rockey et al., 1971). Distinct circular dichroism spectra have been noted when 2,4-dinitrophenylated (DNP) or 2,4,6-trinitrophenylated (TNP) haptens were bound to a homogeneous mouse IgA myeloma protein with high affinity for DNP- and TNP-ligands. The induced optical activity of antibody-hapten complexes may differ significantly when a single chromophore is incorporated into different haptens and specifically bound to a single homogeneous antibody. Distinct circular dichroism spectral characteristics also may be evident when a single ligand is bound by two different antibodies. The magnitude and sign of the induced circular dichroism bands are sensitive to the relative orientation of the ligand chromophore and the amino acid residues within the antibody combining site. The analysis of circular dichroism arising from hapten-antibody interactions has been found to offer a powerful tool with which to monitor ligand binding, to study and compare the fine structure of the combining sites of antibodies which possess similar specificities, and to probe the reformed antibody combining site obtained upon reassociation of heavy and light chains.
2. MATERIALS AND METHODS 2.1. Purification of a n ti-DNP antibody The mouse MOPC-315 plasma cell tumor line of Potter and Lieberman (1967) was maintained in 5 - 8 month old BALB/c mice by the intraperitoneal transfer of 0.5 ml of ascitic fluid from a BALB/c mouse containing the tumor. The harvested ascitic fluid was allowed to stand overnight at 4°C and centrifuged to remove insoluble materials. The MOPC-315 IgA myeloma protein, which displays a high affinity for DNP- and TNP-ligands (Eisen et al., 1968), was purified by affinity chromatography (ibid.; Goetzl and Metzger, 1970). A solid immunoabsorbent of DNP-lysine covalently linked to Sepharose (DNP-Sepharose) (Porath et al., 1967) was prepared by mixing 200 mg of Sepharose 2B (Pharmacia) and 500 mg of cyanogen bromide in 40 ml of water. The pH of the mixture was adjusted to and maintained at 11.5 for 5 min by the addition of 2 N NaOH. The activated Sepharose then was washed on a fritted disc Buchner funnel with 500 ml of cold water, 500 ml of 0.1 M Na2CO3, dried by vacuum, added to 20 ml of 5.45 mM DNP-lysine (e-N-2,4-dinitrophenyl-L-lysine, British Drug House) in water, and the mixture was allowed to stand at 4°C overnight in the dark. Residual coupling activity was neutralized by the addition of ethanolamine until a final pH of 9.0 was obtained. The DNP Sepharose was washed on a Buchner funnel with 500 ml of 0.15 M NaC1-50 mM borate buffer (pH 8.4), 500 ml of 0.15 M NaC1, 500 ml of 1 M acetic acid, and 500 ml of 0.15 M NaCI, and dried by vacuum. A slurry was prepared by adding 25 ml of 0.15 M NaC1-50 mM borate buffer (pH 8.4), and columns (packed volume, 10 ml) were prepared in disposable syringes containing glass wool plugs and glass bead bases. Each column was washed with 200 ml of
J.H.Rockey et al., Hapten-antibody interaction
69
0.15 M NaC1-50mM borate buffer (pH 8.4), 10ml of 1% (w/v)bovine serum albumin, 200 ml of 1 M acetic acid, and 200 ml of borate-buffered saline immediately prior to use. Fifty ml of MOPC-315 tumor ascitic fluid was dialysed against borate-buffered saline (pH 8.4) overnight at 4°C, reduced with 10mM dithiothreitol (Calbiochem) for 1 hr at 25°C, and alkylated at 0°C by the addition of solid iodoacetamide (British Drug House, 2× recrystallized from ethanol, 2× recrystallized from water) to give a final molarity of 11 mM. The reduced and alkylated MOPC-315 protein was dialysed against borate-buffered saline (pH 8.4) at 4°C overnight, diluted with an equal volume of borate-buffered saline, and applied to a DNP-Sepharose column. In some instances, the reduction and alkylation of the MOPC-315 protein prior to affinity chromatography was omitted. The MOPC-315 protein was specifically bound to the DNP-Sepharose column, while other ascitic fluid proteins were eluted with a borate-buffered saline wash. The eluate was set aside for recycling. Sequential elution of the column with 1.0 M and 4.4 M acetic acid removed two protein fractions. These were dialysed immediately and exhaustively against borate-buffered saline at 4°C, and insoluble materials were removed by centrifugation. When necessary, the samples were concentrated by negative pressure ultrafiltration at 4°C. Both the 1 M and the 4 M eluates were found to contain only a single MOPC-315 IgA myeloma protein component when examined by immunoelectrophoresis with rabbit anti-whole mouse serum and anti-MOPC-315 protein antisera. Between 60 and 80% of the recovered MOPC-315 protein was present in the fraction eluted with 1 M acetic acid, and this material was used for further study. MOPC-315 IgA myeloma protein concentrations were determined from the absorbance at 280 nm (corrected for light scattering), measured with a Zeiss PM QII spectrophotometer, employing an extinction coefficient, Elmm~ml, of 1.44, and a molecular weight of 153,000 (Eisen, 1970; Haimovich et al., 1970; Underdown et al., 1970). Polydispersed anti-DNP antibody (Montgomery and Williamson, 1970)was isolated from rabbits and goats hyperimmunized with 2,4-dinitrophenylated antigens, by affinity chromatography or by specific immunoprecipitation (Eisen et al., 1967). 2.2. DNP- and TNP-haptens DNP-lysine (e-N-2,4-dinitrophenyl-L-lysine), DNP-aminocaproate (e-N-2,4-dinitrophenyl-aminocaproate) and DNP-glycine (tx-N-2,4-dinitrophenyl-glycine) were obtained from Sigma Chemical Company and used without further purification. TNP-aminocaproate (e-N-2,4,6,-trinitrophenyl-aminocaproate) was synthesized by reacting 20 g of e-amino-n-caproic acid (Sigma) with 40 g of picryl chloride (British Drug House, 3X recrystallized from ethanol-water) and 10 g of di-sodium tetraborate in 1.25 L of H20 at room temperature in a light-tight reaction vessel (Benacerraf and Levine, 1962). The pH was maintained at 9.5-10.0 by the addition of 3 N NaOH. After 90min, the pH was brought to 2 with HC1, and the precipitated TNP-aminocaproic acid was removed by filtration. TNP-aminocaproate
70
J.H.Rockey et al., Hapten-antibody interaction
was recrystallized 3 times from 95% ethanol, and dried in a light-tight container in vacuo over P 2 0 s . The molar extinction coefficients employed to determine hapten concentrations were: DNP-lysine, 6362 - 1.75 X 104; DNP-aminocaproate, e362 = 1 . 7 5 × 104; DNP-glycine, e36o = 1.59X 104; TNP-aminocaproate, e348 = 1.57 × 104 (Eisen et al., 1968; Goetzl and Metzger, 1970; Benacerraf and Levine, 1962). 2.3. Circular dichroism
Circular dichroism (CD) was measured with a Jasco model ORD/UV-5 spectropolarimeter equipped with a circular dichroism attachment, or with a Durrum-Jasco model J-10 circular dichrometer. The temperature of the sample in the model ORD/UV-5 spectropolarimeter was maintained at 25 + 0.5°C by cycling water from a thermostatically-controlled constant-temperature circulator through the cell housing. Thermostatting of the sample in the model J-10 circular dichrometer was accomplished by placing the cylindrical cuvette in a specially constructed brass cell holder which was in contact with both thermoelectric heating modules (used for both heating and cooling) and a sensing thermometer for a Hallikainen Resistotrol model 1215 temperature regulator. Fluid (water or methanol) was circulated through the cell housing from a cooling bath, and the temperature was maintained at 25°C with the temperature regulator in the heating mode. The relationship between the thermoregulator settings and the sample temperature was determined by directly measuring the temperature of liquid in the cell with a thermocouple over a range of thermoregulator settings. The accuracy of the temperature measurement was +- 0.3°C, and the fluctuation of the temperature at each thermoregulator setting was less than -+ 0.03°C. The circular dichrometers were calibrated with d-10-camphorsulfonic acid (Eastman Organic Chemicals) in water, and d-camphor (Eastman Organic Chemicals, further purified by repeated sublimations) in methanol, according to the method of Cassim and Yang (1969). The agreement between the two independent standardizations consistently was within 1% of the anticipated ratios for the molar dichroic absorption ((e 1 - er)max) or molar ellipticity ([0] max) values (ibid.). For the d-10-camphorsulfonic acid, which had not been specially dried, (e 1 er)ma x and [0] max at 291 -+ 1 nm were taken as +2.20 and +7260, respectively (ibid). De Tar (1969) has reported a (e 1 - er)29 o value of 2.49 for recrystallized d-10-camphorsulfonic acid dried at 50°C over P 2 0 s . The discrepancy between the two (e 1 - e r ) m a x values (Cassim and Y a n g 1969; De Tar, 1969) may largely be accounted for by a correction for hydration (De Tar, 1969). The consistency of the results of the calibrations with d-10-camphorsulfonic acid and d-camphor indicate that the use of the lower (e l - er)ma x value for hydrated d-10-camphorsulfonic acid (uncorrected for hydration) furnishes a satisfactory CD calibration if the d-10-camphorsulfonic acid is sufficiently free of impurities other than water. A d-10-camphorsulfonic acid standard CD spectrum was determined after each ligand-binding experiment and used to correct for any small variation in machine calibration.
J.H.Rockey et al., Hapten-antibody interaction
71
Cell path lengths of from 1.0 cm to 0.1 mm, and protein concentrations ranging from 4 × 10-3 to 1 g/100 ml were used ill determing the CD spectra of antibody and antibody ligand complexes between 600 and 190 nm. The data are presented in terms of molar ellipticity, [0]x , in deg cm 2 dmole -~, where [0]~, = (2.303) × (4500/70 (e 1-er)x, (e 1-er) x = ( A L - A R ) x / l c , and l is the call path length in cm, c is the molar concentration of bound ligand or antibody, and ( A L - A R ) x is the observed difference in absorption between left and right circularly polarized light at wavelength ~ (Cassim and Yang, 1969; Urry, 1970). The average residue weight Mo, taken as l lO for the antibodies, rather than the molecular weight was employed in defining protein [0Ix values. 2.4. Absorption spectroscopy
Absorption spectra and absorptivity difference spectra were determined at room temperature with either a Jasco ORD/UV-5 recording spectrophotometer, a Zeiss PM QII spectrophotometer or a Cary model 15 recording spectrophotometer. 2.5. Fluorescence quenching The quenching of antibody tryptophan fluorescence by bound hapten was measured at 25 + 1°C in a thermostatted cell compartment in either a Zeiss double M4 QIII monochromator spectrophotofluorometer equipped with a Farnell stabilized EHT power supply and a Keithley 610B electrometer, or an Aminco-Bowman spectrofluorometer (Eisen et al., 1968; Rockey, 1967). Proteins were excited at 280-295 nm and fluorescence was measured at 345 nm. The fluorescence data were corrected for dilution and internal quenching as previously described (Rockey, 1967). Average intrinsic association constants, Ko, were determined from the plots o f r / c 'versus r, where r is the moles of hapten bound per mole of antibody, and c ' is the free hapten concentration (Eisen et al., 1968; Rockey, 1967; Wu and Rockey, 1969). The valence of the antibody was taken as 2(n = 2) (Rockey et al., 1971; Underdown et al., 1970; Eisen, 1970; Haimovich et al., 1970). As Ko = [ r ] / c ' [ 2 - r ] , when r is 1, Ko = 1/c'. The binding data also were analyzed according to the Sips distribution function, in the form log[r/(n-r)] = a logc' + a logK0, where a is the index of heterogeneity (Eisen et al., 1968; Wu and Rockey, 1969; Sips, 1948). 2.6. Re-association o f MOPC-315 protein subunits Mildly reduced and alkylated MOPC-315 protein, previously utilized for CD DNP- or TNP-ligand binding studies, at a concentration of 2 mg/ml was transferred to 1 M propionic acid by Sephadex G-25 gel filtration and immediately applied to a Sephadex G-100 column equilibrated with 1 M propionic acid, and the column was eluted with the same solvent. The resolved heavy and light chain fractions were immediately and exhaustively dialysized at 4°C against 4 mM sodium acetate buffer (pH 5,4) and recombined in the same solvent (Stevenson and Dorrington, 1970). The recombined MOPC-315 protein was dialysized against 0.1 M N a C I - 1 0 m M phosphate buffer (pH 7.5) and used for CD ligand-binding studies.
J.H.Rockey et al., Hapten-antibody interaction
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WAVELENGTH
(nm)
Fig. 2. Circular dichroism spectrum o f a-N-2,4,-dinitrophenyl-glycine in free solution ( - - - ) , and bound to MOPC-315 protein ( ).
Fig. 1A. Circular dichroism spectrum of e-N-2,4-dinitrophenyl-aminocaproate in free solution (.... ), and bound to MOPC-315 IgA myeloma protein ( ). The [ 0 ] h values below 325 nm have been corrected for the negative CD contributed by the protein alone. B. Circular dichroism spectrum o f e-N-2,4,6-trinitrophenyl-aminocaproate in free solution ( - - - ) , and bound to MOPC-315 IgA myeloma protein ( ). C. Absorption spectra o f DNP-aminocaproate in free solution ( - - - ) , and an equal molar concentration b o u n d to the MOPC-315 protein ( ). The same concentration o f the MOPC-315 protein in the absence of DNP-aminocaproate was used as a reference to obtain the spectrum of the b o u n d DNP-aminocaproate. The DNP-aminocaproate-MOPC-315 versus MOPC-315 difference spectrum was examined at r = 1.1 where more than 99% of the total ligand was specifically bound to the protein. The spectra at maximum absorbance are plotted on an expanded wavelength scale in the upper right insert to illustrate that the spectra are composed o f more than one c o m p o n e n t near hma x. The absorption difference spectrum between b o u n d and free DNP-aminocaproate also is plotted( ..........). D. Absorption spectra o f TNP-aminocaproate in free solution ( - --) and bound at an equal molar concentration to MOPC-315 protein ( ), and the bound versus free ligand difference spectrum ( ............).
74
J.H.Rockey et al., Hapten antibody &teraction
3. EXPERIMENTAL RESULTS No circular dichroism was evident in the MOPC-315 IgA myeloma protein spectrum between 600 nm and 325 nm. DNP-lysine, DNP-aminocaproate, DNP-glycine and TNP-aminocaproate in free solution, at concentrations as great as 1.5 × 10-4 M, also did not display circular dichroism in this spectral region. In sharp contrast, the specific binding of the DNP- and TNP-ligands to the MOPC-315 protein resulted in the induction of distinct circular dichroism bands which were characteristic of the ligand involved (figs. 1A, 1B, 2). The CD spectra of DNP-lysine and DNP-aminocaproate complexed with the MOPC-315 protein were closely similar, but distinct spectral characteristics were apparent when DNP-glycine or TNP-aminocaproate were bound by the same antibody. The DNP-lysine- and DNP-aminocaproate-MOPC-315 protein spectra displayed a broad positive CD band centered near 420 nm, a sharper positive CD band centered at 380 nm and a large negative CD band centered at a wavelength less than 320 nm (fig. 1A). The ellipticity at 380 nm was approximately two times greater than the ellipticity at 420 nm in both the DNP-lyisne-MOPC-315 and DNP-aminocaproate-MOPC-315 CD spectra. The CD spectrum of DNP-glycine bound to the MOPC-315 protein also showed a broad positive CD band centered near 420 nm, and a broad negative band centered below 320 nm (fig. 2). Here, in contrast to the bound DNP-lysine and DNP-aminocaproate spectra, only a small shoulder centered near 380 nm was present in the bound DNP-glycine CD spectrum (fig. 2). The ellipticity at 380 nm in the DNP-glycine-MOPC-315 protein spectrum was less than the ellipticity at 420 nm (fig. 2). The CD spectrum of TNP-aminocaproate differed strikingly in that a large negative CD band was present at 420 nm (fig. 1B). A small positive CD band centered near 490 nm, a narrow positive CD band centered at 370 nm and a large negative band centered below 320 nm "also were evident in the TNP-aminocaproate-MOPC-315 protein spectrum (fig. 1B). Circular dichroism spectra of DNP- and TNP-ligands bound to non-reduced MOPC-315 protein, MOPC-315 protein isolated after mild reduction and alkylation, and re-associated antibody formed from isolated MOPC-315 protein heavy and light chains, were similar. The circular dichroism spectrum of the MOPC-315 protein between 325 nm and 200 nm displayed a large negative band centered at 217 nm, and smaller negative bands centered near 292,286 and 280 rim, in the region of high absorptivity by the protein aromatic amino acid residues. Determination of the circular dichroism of complexes of the MOPC-315 protein and DNP-lysine, DNP-aminocaproate, DNP-glycine or TNP-aminocaproate between 325 nm and 250 nm, subtracting the ellipticity of the protein alone from the ellipticity of the ligand-protein complex, demonstrated that the negative CD bands in the spectra of the ligand-protein complexes at the lower wavelength were centered near 295 -+ 15 nm. The low signal to noise ratio resulting from the high absorptivity of the ligand-MOPC-315 protein complexes made the determination of the induced ellipticity more difficult in this spectral region.
J.H.Rockey et al., Hapten-antibody interaction
75
The absorption spectra of free and bound DNP-aminocaproate and TNP-aminocaproate, and the difference spectra of bound versus free ligands, between 600 nm and 300 nm, are presented in figs. 1C and 1D. The absorption spectra of the free ligand and of the ligand bound to the MOPC-315 protein, at peak absorptivity are plotted on an expanded scale in the upper parts of figs. 1C and 1D to illustrate that the spectra are formed from more than a single component in this wavelength region. This is particularly evident in the free and bound DNP-aminocaproate spectra (fig. 1C). The spectra of DNP-lysine display a similar phenomena. Free DNP-aminocaproate and TNP-aminocaproate also showed absorption bands at 265 and 212 nm, and at 211 and near 250 nm, respectively. The circular dichroism spectrum of DNP-lysine specifically bound to a goat anti-DNP antibody (fig. 3) differed markedly from the spectrum of the same ligand bound to the MOPC-315 protein. Broad negative overlapping CD bands centered near 3 5 0 - 3 0 0 nm were evident in the goat antibody-ligand spectrum. We also have been able to detect induced circular dichroism in the spectrum of DNP-lysine complexed with polydispersed rabbit anti-DNP antibody. No induced circular
6
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-15 I
I
I
I
I
I
300
350
400
450
500
550
WAVELENGTH (nm) Fig. 3. Circular dichroism spectrum of e-N-2,4-dinitrophenyl-L-lysine in free solution ( - - - - - ) and bound to goat anti-DNP antibody ( ). Antibody concentration, 5.36 × 10-SM. DNP-lysine concentration, 4.0 X 10-SM. The apparent molar eUipticity, [0]h (apparent), equals (2.303) (4500/~r)(AL-AR)h(l)-l(total DNP-lysine concentration) -1, where all of the ligand present is assumed to be bound to antibody. The ellipticity of the bound ligand has been corrected for the positive circular dichroism of the goat anti-DNP antibody at wavelengths less than 325 nm.
J.H.Rockey et al., Hapten-antibody interaction
76
dichroism was evident when human myeloma proteins and rabbit "),G-globulins lacking anti-DNP activity were mixed with the DNP- or TNP-ligands. Hapten-binding curves were constructed by serially adding the DNP- or TNPligand with a Hamilton microsyringe to a constant amount of antibody and measuring the change in circular dichroism at selected wavelengths (e.g., 420 nm, 3 7 0 - 3 8 0 rim, 325 nm). The measurement of induced circular dichroism at 325 nm, where both the protein and the DNP- or TNP-ligands showed a minimum of absorbance and a maximum ratio of induced ellipticity to absorbance (fig. 1), presented a highly useful optical window for following ligand binding. The circular dichroism data were plotted as (AL-A R)x versus (total ligand concentration); and as [0] x (apparent) versus (total ligand concentration), where [0] x (apparent) = 3300(A L - A R ) X / ( t o t a l ligand concentration)/. The molar ellipticity, [0]x , of bound ligand was obtained from the slope of the (AL--AR) x versus (total ligand concentration) plot where the percentage of ligand which was not bound was minimal (fig. 4). With a sufficient concentration (e.g., 5 X 10 -s M) of high affinity (K0 ~> 107 M- l ) antibody, the percentage of the total ligand that is free in solution
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Fig. 4. Hapten binding curve constructed by serially adding DNP-aminocaproate to MOPC-315 IgA protein, and measuring the change in circular dichroism at 325 nm. The (AL-A R) data (o o) have been plotted as a function of the total ligand concentration. The values of [0] 325 nm (apparent), which equals (3300) (l) -1 [(AL-AR)/(total DNP-aminocaproate concentration)], also are plotted as a function of the total ligand concentration (o e).
J.H.Rockey et al., Hapten-antibody interaction
77
is less than 1% over a wide range of r values (r is the moles of hapten bound per mole of antibody). This applies here to the DNP-lysine, DNP-aminocaproate and TNP-aminocaproate haptens b o u n d to the MOPC-315 protein. For these ligands the (AL--AR) x versus (ligand concentration) data in the region below antibody site saturation have been treated by the method of least squares to give the (e I - er) x and [0] x values listed in table 1. With lower affinity l i g a n d - a n t i b o d y systems, where significant free hapten is present at higher r values (e.g., DNP-glycineMOPC-315 protein), the tangent of the slope of the (A L - A R)x versus (total ligand concentration) plot at infinite ligand dilution may be taken to give (e 1- er) x and [0] x for the b o u n d ligand. This procedure was used here to obtain the values for b o u n d DNP-glycine presented in table 1. The circular dichroism binding data intrinsically contain all of the information needed to define the binding characteristics. The molar concentration of antibody combining sites may be obtained from the ratio (Z t - A R ) x ( m a x ) / ( e 1 - er)x, where (A L - A R ) x ( m a x ) is the observed ( A L - - A R ) x value at antibody site saturation (fig. 4). This ratio gives the molar concentration of antibody combining sites and may be used, together with the protein concentration, to determine the valence of the antibody. This procedure has been used to demonstrate the bivalence of the MOPC-315 protein (Rockey et al., 1971). The value o f r at each dilution may
Table 1 Circular dichroism of antibody-hapten complexes. Ligand
Antibody
e-DNP-aminocaproate
MOPC-315
e-DNP-lysine
MOPC-315
a-DNP-glycine
MOPC-315
e-TNP-aminocaproate
MOPC-315
e-DNP-lysine
Goat anti-DNP
h (nm)
(e 1- er)h
325 380 420 325 380 420 325 380 420 325 370 420 325 360
- 6.37 + 5.09 + 2.45 - 6.46 + 5.03 + 2.44 - 2.37 + 1.44 + 2.06 -11.0 + 4.02 - 6.48 - 3.64 * - 3.64 *
[0 ]?, -21,000 +16,800 + 8080 -21,300 +16,600 + 8030 - 7800 + 4770 + 6800 -36,250 +13,250 -21,350 -12,000 * -12,000 *
Molar circular dichroic absorption (el-er)h, and molar eUiptieity [0]~, at wavelength h, of DNP- and TNP-ligands bound to MOPC-315 protein and goat anti-DNP antibody. [O]h = (2.303)
× (4500/n)(el-er). * Apparent (e1- er) h and [0] h values, calculated by assuming that all of the ligand present was bound to antibody (5.36 × 10-s M antibody, 4 × 10-s M e-DNP-lysine).
78
J.H.Rockey et al., Hapten-antibody interaction
be obtained from the ratio 2(/IL -- A R)x(°bserved)/( A L - A R)X(max) for bivalent antibody. The concentration of bound ligand c = (.tl L -AR)7`/(e 1 --er)7`l, and the free ligand concentration, c ' = [(total concentration)-c]. With high-affinity antibody, however, where the percentage of the total ligand free in solution is very small (e.g., < 1%) at r = 1, the uncertainty of the data severely restricts usefulness of the method for obtaining accurate intrinsic association constants. Alternately, if the average intrinsic association constant for an antibody-ligand system is known from some other experimental parameter (e.g., equilibrium dialysis, fluorescence quenching) this value may be used to determine the free ligand concentration and the molar ellipticity of bound ligand at r = 1. This procedure also has been used here to determine [0] 7, values for bound DNP- and TNP-ligands, and the results were similar to those obtained from the (A L A R)7` versus (total ligand concentration) plots.
4. DISCUSSION The optical activity (circular dichroism) of the inherently symmetric dinitrophenyl and trinitrophenyl chromophores of the bound ligands is provided by interactions between the ligand chromophore and dissymmetrically placed vicinal moieties of the antibody combining site (Rockey et al., 1971; Urry, 1970; Perrin and Hart, 1970; Eyring et al., 1968). The vicinal perturbations that give rise to optical activity may be static in nature, and may result from dissymmetrically placed static charge, or from the polarizability and incomplete screening of nuclei of a vicinal group by the surrounding electrons such that the electron undergoing transition is able to sence the positive nuclei (Urry, 1970; Kauzmann et al., 1940; Condon et al., 1937; Hooker and Schellman, 1970; Hohn and Weigang, 1968). Circular dichroism also may arise from dynamic coupling of electronic transitions of the bound chromophore with electronic transitions of a vicinal chromophore in the antibody combining site. Optical activity may be induced by means of the coupled oscillator mechanism of Kuhn (1933), Kirkwood (1937) and Moffitt (1956) which involves the coupling of the electric transition dipole moments of two electronic transitions (Woody and Tinoco, 1967; Hohn and Weigang, 1968; Bayley et al., 1969; Hooker and Schellman, 1970; Urry, 1970). This mechanism is suggested when the ligand chromophore and a vicinal chromophore exhibit strong absorption bands (large electric transition dipole moments), and reciprocal relations are evident between a circular dichroism band centered at the ligand chromophore transition and a circular dichroism band of the opposite sign centered over the vicinal chromophore absorption band (Urry, 1970). Dynamic coupling between two chromophores also may give rise to optical activity by coupling an electrically allowed transition of one chromophore with a magnetically allowed transition of a second chromophore (Jones and Eyring, 1961 ;Woody and Tinoco, 1967; Hohn and Weigang, 1968; Bayley et al., 1969; Hooker and Schellman, 1970; Urry, 1970).
J.H.Rockey et al., Hapten antibody interaction
79
Circular dichroism arising by this mechanism again will display reciprocal relationships (Urry, 1970). There is an apparent reciprocal relationship between the positive CD band centered at 3 7 0 - 3 8 0 nm and the negative band centered over the aromatic amino acid absorption region in the spectra of DNP- and TNP-ligands complexed with the MOPC-315 protein. When a correction is made for the contribution at 3 7 0 - 3 8 0 nm of the broad band centered near 420 nm, it is seen that the ratio of the ellipticity of the 370-380 nm band to that of the 295 -+ 15 nm band is maintained for each ligand, even though the molar ellipticities vary widely between the different ligands. This reciprocal relationship, together with the large molar extinction coefficients of the DNP and TNP groups, suggests that these circular dichroism bands arise, at least in part, from dynamic coupling of electronic transitions of the ligand chromophore and a protein chromophore (e.g., tryptophan) in the antibody combining site. Both the magnitude and the sign of the rotational strength of a circular dichroism band arising by this mechanism will be dependent upon the relative orientation of the two interacting chromophores (Urry, 1970). The absorption spectra of DNP- and TNP-ligands bound to the MOPC-315 protein display hypochroism and a red-shift (Eisen et al., 1968; Rockey et al., 1971). Little and Eisen (1966) have studied the difference spectra resulting from the association of DNP- and TNP-ligands with anti-DNP and anti-TNP antibodies from a number of sources, and have suggested that a tryptophan residue is present in the combining site of anti-DNP and anti-TNP antibodies in general. The formation of a charge transfer comp! "" between the DNP or TNP group and the indole group of a tryptophan residue in the antibody combining site has been inferred (Little and Eisen, 1967). The DNP and TNP groups are good electron acceptors, indole is a good electron donor and there is substantial evidence from model systems which indicates these groups may form charge transfer complexes (Szent-Gyorgyi et al., 1961; Little and Eisen, 1967; Sigman and Blout, 1967; Shinitzky and Katchalski, 1968). The fluorescence of tryptophan residues of the MOPC-315 protein is quenched by bound DNP- and TNP-ligands (Eisen et al., 1968; Rockey et al., 1971). This is a general phenomenon observed with anti-DNP, anti-TNP and other antibodies (Little and Eisen, 1966; Rockey, 1967), and furnishes added support for the concept of the occurrence of a tryptophan residue in or near the antibody combining site of diverse antibodies. Circular dichroism furnishes a particularly powerful probe of the combining sites of anti-DNP and anti-TNP antibodies. While hypochromism and a red-shift of the spectrum of bound ligands, and quenching of antibody tryptophan fluorescence by ligand are general properties of anti-DNP and anti-TNP antibodies (Little and Eisen, 1966, 1967), the detailed structure of the CD spectra of bound DNP- and TNP-ligands may vary greatly between different antibodies. The CD spectra of DNP-haptens complexed with polydispersed rabbit anti-DNP antibodies may not display induced circular dichroism (Cathou et al., 1968; Ashman and Kaplan, 1970). The induced circular dichroism obtained when a single iigand (e.g.,
80
J.H.Rockey et al., Hapten-antibody interaction
DNP-lysine) is bound to two different antibodies (e.g., MOPC-315 protein, goat anti-DNP antibody) may differ markedly in the magnitude, sign and kmax of distinct circular dichroism bands. The complexing of a single chromophore, incorporated into two different ligands (e.g., e-N-DNP-L-lysine, a-N-DNP-glycine), with a single homogeneous antibody (e.g., MOPC-315 IgA protein) may result in induced circular dichroism which varies in the relative magnitude of the ellipticity (rotational strengths) at two different bands (e.g., 380 nm, 420 nm) in a manner dependent upon the ligand. The sign of a CD band at a single wavelength (e.g., 420 nm) of two related ligands (e.g., DNP-aminocaproate, TNP-aminocaproate) bound to the same homogeneous antibody may be reversed. It may be noted that the 2,4,6-trinitrophenyl group has an additional element of symmetry absent from the 2,4-dinitrophenyl group: the TNP-group has C2v symmetry for rotation about the N-C(phenyl) bond. This furnishes an added element of dissymmetry for interactions between the DNP-group and an antibody site chromophore such as a tryptophan indole group. The CD band centered at 420 nm arising from the DNP chromophore may vary in sign: in the circular dichroism spectra of ct-N-DNP-tryptophan and ct-N-DNP-glutamine a CD band is present at 420-440 nm, but it is negative (Rockey, Dorrington, Montgomery, unpublished observation). There is evidence from other systems that the magnitude and/or sign of extrinsic Cotton effects arising from complexing a single ligand to different macromolecules may differ (Blout, 1964; Perrin and Hart, 1970). The sign of the long wavelength CD bands seen on binding N-arylanthranilates (e.g., flufenamic acid) to different species of albumins were in some instances reversed (Chignell, 1969). The extrinsic CD spectra of cattle and squid rhodopsins, formed with the same chromophore, l l-cis-retinall, have been found to differ (Kito et al., 1968). Oppositely signed extrinsic Cotton effects have been observed when 2,4-dinitroanilines were complexed with RNA and DNA, respectively (Gabbay, 1969). In model systems (e.g., o-, m- and p-tyrosine) slight differences in the orientation of the coupling groups (e.g., the aromatic ring and the carboxylate group) can lead to a reversal of the sign of a CD band (Hooker and Schellman, 1970). Within the binding site of the MOPC-315 protein, the DNP group appears to be relatively rigidly fixed. Two related affinity-labeling reagents, bromoacetyl-N-DNPethylenediamine (BADE) and bromoacetyl-e-N-DNP-L-lysine (BADL) which differ in the separation of the reactive bromoacetyl group from the DNP group, react wholly with different residues (tyrosyl of the light chain, and lysyl of the heavy chain, respectively) of the MOPC-315 protein combining site (Haimovich et al., 1970). Our studies serve to establish a new method of assaying hapten-antibody interactions, and of probing the antibody combining site. Circular dichroism presents a powerful method to study and compare the fine structure of the combining sites of antibodies of related specificity, and to probe the reformed site resulting from the reassociation of antibody heavy and light chains. This method therefore may be of special use in our quest for two distinct molecular forms of
J.H.Rockey et ak, Hapten-antibody interaction
81
antibody of identical specificity, formed by the translocation of a single heavy chain variable (N-terminal) gene to two different heavy chain constant region (C-terminal) cistrons (Rockey et al., 1970).
ACKNOWLEDGEMENTS We wish to thank Dr. Robert C. Davis of the Department of Chemistry, University of Pennsylvania, for the use of his calibrated Jasco-Durrum model J-10 spectropolarimeter, and for many helpful discussions.
REFERENCES Ashman, R.F. and A.P. Kaplan, 1970, Federation Proc. 29, 773. Bayley, P.M., E.B. Nielsen and J.A. Schellman, 1969, J. Phys. Chem. 73, 228. Benacerraf, B. and B.B. Levine, 1962, J. Exptl. Med. 115, 1023. Blout, E.R., 1964, Biopolymer Symp., No. 1,397. Cassim, J.Y. and J.T. Yang, 1969, Biochemistry 8, 1947. Cathou, R.E., A. Kulczycki, Jr. and E. Haber, 1968, Biochemistry 7, 3958. Chignell, C.F., 1969, Mol. Phaxmacol. 5,455. Condon, E.V., W. Altar and H. Eyring, 1937, J. Chem. Phys. 5,753. De Tax, D.F., 1969, Anal. Chem. 41, 1406. Eisen, H., 1970, Abstracts of the 8th Internat. Congr. of Biochem. 309. Eisen, H.N., W. Gray, J.R. Little and E.S. Simms, 1967, in: Methods in immunology and immunochemistry, eds. C.A. Williams and M.W. Chase (Academic Press, New York), vol. 1, p. 351. Eisen, H.N., E.S. Simms and M. Potter, 17~"°, Biochemistry 7, 4126. Eyring, H., H.-C. Liu and D. CaldweU, 1968, Chem. Rev. 68, 525. Gabbay, E.J., 1969, J. Am. Chem. Soc. 91, 5136. Goetzl, E.J. and H. Metzger, 1970, Biochemistry 9, 1267. Haimovich, J., D. Givol and H.N. Eisen, 1970, Proc. Natl. Acad. Sci. US 67, 1656. Hohn, E.G. and O.E. Weigang, 1968, J. Chem. Phys. 48, 1127. Hooker, Jr., T.M. and J.A. Schellman, 1970, Biopolymers 9, 1319. Jones, L.L. and H. Eyring, 1961, Tetrahedron 13,235. Kauzmann, W., J.E. Walter and H. Eyring, 1940, Chem. Rev. 26, 339. Kirkwood, J.G., 1937, J. Chem. Phys. 5,479. Kito, Y., M. Azuma and Y. Maeda, 1968, Biochim. Biophys. Acta 154,352. Kuhn, W., 1933, in: Stereochemie, ed. K. Freudenberg (Deuticke, Leipzig) p. 317. Little, J.R. and H.N. Eisen, 1966, Biochemistry 5, 3385. Little, J.R. and H.N. Eisen, 1967, Biochemistry 6, 3119. Moffitt, W., 1956, J. Chem. Phys. 25,467. Montgomery, P.C. and A.R, Williamson, 1970, Nature 228, 1306. Perrin, J.H. and P.A. Hart, 1970, J. Phaxmaceut. Sci. 59,431. Porath, J., R. Axen and S. Ernback, 1967, Nature 215, 1491. Potter, M. and R. Lieberman, 1967, Advan. lmmunol. 7, 91. Rockey, J.H., 1967, J. Exptl. Med. 125,249. Rockey, J.H., P.C. Montgomery and K.J. Dorrington, 1970, Biochemistry 9, 4310. Rockey, J.H., K.J. Dorrington and P.C. Montgomery, 1971, Nature 232, 192.
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Shinitzky, M. and E. Katchalski, 1968, in: Molecular associations in biology, ed. B. Pullman (Academic Press, New York) p. 361. Sigman, D.S. and E.R. Blout, 1967, J. Am. Chem. Soc. 89, 1747. Sips, R., 1948, J. Chem. Phys. 16,490. Stevenson, G.T. and K.J. Dorrington, 1970, Biochem. J. 118, 703. Szent-Gyorgyi, A., I. Isenberg and J. McLaughlin, 1961, Proc. Natl. Acad. Sci. US 47, 1089. Underdown, B.J., E.S. Simms and H.N. Eisen, 1970, Federation Proc. 29, 437. Urry, D.W., 1970, in: Spectroscopic approaches to biomolecular conformation, ed. D.W. Urry (Am. Med. Assoc., Chicago) p. 33. Woody, R.W. and I. Tinoco, 1967, J. Chem. Phys. 46, 4927. Wu, W.-H. and J.H. Rockey, 1969, Biochemistry 8, 2719.