Participation of amino groups in the active sites of antibodies

Participation of amino groups in the active sites of antibodies

Immunochemistry. Pergamon Press 1968. Vol. 5, pp. 367-381. Printed in Great Britain PARTICIPATION OF AMINO GROUPS IN THE ACTIVE SITES OF ANTIBODIES*...

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Immunochemistry. Pergamon Press 1968. Vol. 5, pp. 367-381.

Printed in Great Britain

PARTICIPATION OF AMINO GROUPS IN THE ACTIVE SITES OF ANTIBODIES* M. H. FREEDMAN,A. L. GaOSSBzRO and D. P m m u , ~ Department of Biochemistry Research, Rolwell Park Memorial Institute, Buffalo, New York 14203 (P,~¢e/va/27 November 1967 ; in revis#dform 2 January 1968) Almtraet--The effect of modification of amino groups on the binding activity of various antihapten antibodies was studied. The antibodies were prepared agaimt positively charged, negatively charged, and neutral haptem. The lysyl residues in the antibody preparatious were modified to an ex'ent of 65-70 per cent with N-carboxy-nL-alanine anhydride. Modification of amino groups in antibodies prepared agaimt two negatively charged haptem (#-azobenzenearmnate and p-azobenzenephosphonate) resulted in a decrease in the number of antibody combining rites, while the average combining constant for these antibodies was unaffected by the modifi~.ation procedure. The Ices of antibody combining sites could be protected against by tl-e presence of hapten during the alanylation reaction. The active sites of antilxxiies agair~t two other negatively charged haptem 0bazobenzoate and #-azobenzenemlfonate) were little affected by modification of protein amino groups. The extent of partic/pation of amino groups in the antibody sites ~etm to vary to a large extent within a population of antibody molecules made agaimt a ~agle hapten. Anti-lemmbermenearaonate antibody after papain digestion was aeparated into four Fab fractions with different properties by column chromatography, and the effect of amino group modification on these four fractions varied considerably. The Ices in binding activity was not due to conformational changes following modification of amino groups outside of the antibody combining sites since lint of activity could be prevented by the pretence of hapten during the alanylation, and since modification of amino groups in antibodies prepared agaimt a neutral hapten (3-azopyridine) and agaimt a pmitively charged hapten (p-azophenyltrimethylammonium) resulted in no change in either the average bindin8 constant or the number of antibody combining sites. These results show that amino groups contribute to the positive charge in the combining sites ofat least some antibody molecules directed against certain negatively charged haptem and appear to be absent or not available for alanylation in the active sites of antibody molecules directed against rome other negatively charged, pmitively charged, or neutral haptem. INTRODUCTION Tt-mm~ is considerable evidence which indicates that antibody sites directed against positively charged haptens are different from antibody sites directed against negatively charged groups. Antibodies directed against positively charged groups have a negative charge in their combining regions, while antibodies directed a r l m t negatively charged haptens have a positive charge in their combining regions. T h e presence of these charges has been shown by the specific binding activities of various inorganic cations[l] and anions[2]. Orossberg and Pressman [3] demonstrated the presence of carboxylate groups in the combining sites of antibodies against the pmi, tively charged p-azophenyltrimethylammonium group (anti-Ap). T h e positive charge in the combining sites of antibodies directed against negatively charged groups could * Supported in part by Grant No. AI-02342 from the National Imtitute of Allergy and Infectious Diseases. Presented in part at the 154th Meeting ofthe American Obemical Society, Chicago, Illinois, September 1967. 367

368

M. H. FREEDMAN,A. L. GRossszao and D. PRgssw~a~

be contributed by the guanidinium group of arginine, or by an amino group such as the ~-amino group of lysine or the a-amino at the N-terminal end of the polypeptide chains. From the recent studies of Grossberg and Pressman[4, 5], it appears that the positive charge in the active sites of antibodies directed against certain negatively charged haptens (p-azobenzoate and p-azobcnzenearsonate) is contributed to a large extent by a guanidinium group. However, the presence of amino groups in a portion of such sites has been suggested by previous studies utilizing carbamylation[6], amidination[7, 8], and carboxymethylatioq[8]. This report describes the effect on the antibody activity following chemical modification of amino groups, using the N-carboxya:lhydride of t~L-alanine under mild conditions, on antibodies made against different haptens. The results demonstrate the participation of amino groups in the combining sites of antibodies prepared against certain negatively charged haptens and suggest the absence of amino groups in the combining sites of antibodies prepared against a positively char~ed and a neutral hapten. MATERIALS AND METHODS Antigens. Azoproteins ,,,ere prepared by coupling bovine ~,-giobulin (BGG) with diazotized 3-aminopyridine to give BGG-P3, with diazotized p-aminophenyltrimethylammonium chloride to give BGG-A,, with diazotized p-aminobenzoate to give BGG-X,, with diazotized p-arsanilate to give BGG-R,, with di~otized p-sulfanilate to give BGG-Sulf,, and with diazotized p-phosphanilate to give BGG-P, according to the procedures described by Grossberg et al.[1] and Kreiter and Pressman[9]. Preparation ofan~era. The methods for preparing and testing rabbit antisera against 3-azopyridine (anti-P,), p-azophenyltrimethylammonium (anti-A,), p-azobenzoate (anti-X,), p-azobenzenearsonate (anti-R,), p-azobenzenesulfonate (anti-Sulf,), and p-azobenzenephosphonate (anti-P,) have been previously described by Gro~berg et al.[l] and Kreiter and Pressman[9]. Purification and characterization of antibodies. Anti-Pa, anti-A,, and anti-X, antibodies were purified by specific precipitation and ion-exchange column chromatography according to the methods described by Grossberg and Pressman[5]. Anti-R, and anti-Sulfa antibodies were prepared using solid immunoadsorbents as described by Onoue et al.[10]. The adsorbed antibodies were eluted with 1 ,~I propionic acid in the cold, according to the procedure described by Tanigaki et al.[l 1]. The purity of the eluted antibody preparations was estimated by the method of equilibrium dialysis using 1"I-labeled haptens according to the method described by Grossberg and Pressman[3]. All specifically purified antibody preparations were calculated to be greater than 90 per cent pure (determined by measuring the number of binding sites per mole of protein). In the case of anti-P, antibodies, the immunoglobulin fraction of the antiserum was prepared by three sodium sulfate precipitations at room temperature according to the method of Kekwick[12 ]. Pooled rabbit antisera were the source of all antibody preparations studied. Alanylation of the rabbit antibodies. Rabbit anti-hapten antibody preparations were treated with N-carboxy,DL-alanine anhydride (Pilot Chemicals, Inc., Watertown, Mass.), at weight ratios of anhydride to protein of 5 : 1 (96 moles of alanine anhydride

Amino Groups in Antibody Sites

369

per mole of lysine) according to the methods described by Fucks and Sela[13] and Karush and Sela[14]. The mixtures were allowed to react for 24 hours at 4 °. A/an/he ow/dmunt. The number of additional ~l*nyl residues acquired by the polyalanylated antibody preparations was determined by amino acid analyses. The samples were hydrolyzed in constant boiling HCI (6 N) in sealed ampoules flttlhed with nitrogen gas, for 20 hours at 107 °. Amino acid analyses were performed on a Technicon automatic amino acid analyse.r, using a 61 hour accelerated gradient[15]. Norleucine was used as the internal standard. The number of *lanyl residues per molecule of polyalanylated antibody was estimated from the analytical data asauming that antibodymolecules contain 90 residues ofleucine. Eztmt of l.ysine mad~f~alion. Polyal~nylated antibody samples were deaminated by treatment with sodium nitrite in glacial acetic acid according to the method of Anfinsen et a1.[16]. The deamination procedure altered the amino groups which were not modified by the alanylation procedure. The dcaminated samples, after extensive dialysis in distilled water and lyophylization, were hydrolyzed in 6 N HCI as described above, and the number of lysyl residues per mole of antibody was determined by amino acid analysis. The number oflysyl residues which had been modified by alanylation is then equal to the number of lysyl residues which had not been deaminated. Sp~oificity of ~ alan.ylatioa procedure. It is known that the N-carboxyamino acid anhydride reacts with free e- and a-amino groups. In order to determine ifa possible reaction of N-carboxy anhydride with hydroxyl groups affects antibody activity, the polyalanylated antibody preparations were treated with hydroxylamine (at a final concentration of 0.8 M) at pH 9.5. The reaction mixture was stirred at room temperature for 15 minutes and then left at 4 ° overnight. The mixture was then dialyzed against 3 or 4 changes of 1000 volumes of 0.1 M Tris-HCl, 0.002 M EDTA buffer, pH 8.0. The antibody activity of the polyalanylated antibodies before and after treatment with hydroxylamine was determined by equilibrium dialysis. The binding activity of the polyalanylated antibodies was identical before and after treatment with hydroxylamine. Therefore, any changes in antibody activity observed after the alanylation reaction were due to modification of amino groups and were not due to modification of hydroxyamino acids. Modification of amino groups in the presence and absency of hapten. In order to test for changes in the average binding constants and in the number of antibody combining sites after al~aylation of the antibody molecules, all experiments were performed using three different samples for each antibody preparation. The three samples included: (1) unprotected sample, (2) protected sample, and (3) control sample. The unprotected sample was treated directly with the N-carboxyamino acid anhydride. At the termination of the alanylation reaction, hapten was added to this sample to the same final hapten concentration of 0.04 M a s was used in the protected sample. The protected sample contained 0.04 M hapten (final concentration) added before the alanylation procedure. A slightly larger amount of N-carboxy-m.-al_anine anhydride was added to the protected samples to assure that the extent of lysine modification in the protected sample was at least equal to the extent of lysine modification in the unprotected sample. The control sample was exposed to 0.04 M hapten, and a volume of dioxane was added equivalent to that used to dissolve the anhydride for the unprotected and protected samples. The N-carboxyamino acid

370

M. H. FREEDMAN,A. L. GROSSBERGand D. PRESSMAN

anhydride was not added to the control sample. All three preparations were allowed to stand for 24 hours at 4 °. Then the samples (unprotected, protected and control) were exhaustively dialyzed at 4 ° first against 1000 volumes of0.15 M sodium chloride, 0.01 M tris-HCI, 0.002 M EDTA, pH 8.0, and finally against 0.1 M tris-HOl, 0.002 M EDTA, pH 8.0. The extent of lysine modification for the unprotected and protected samples was determined by amino acid analyses following deamination as described above. The lysine content of these samples was compared with the l~ine content of the control sample. Determination of the antibody activity by equilibrium dialysis. The antibody activity of the unprotected, protected and control antibody preparations was determined by equilibrium dialysis using mI-labeled haptens at several different free hapten concentrations, as previously dcscribed by Gros,sberg and Pressman[3]. Samples to be compared at a given hapten concentration were dialyzed against a common hapten solution so that the free hapten concentration at equilibrium was identical for all. The methods of plotting the data to obtain values for the average binding constant (K.), antibody site concentration (A.), and heterogeneity index (a) have been previoudy described by Nisonoff and Pressman[l 7], and Eisen[18]. The reciprocal of the bound hapten concentration (l/b) is plotted against the reciprocal of the free hapten concentration (l/c). Extrapolation of such curves to infinite free hapten concentration (the ordinate intercept) provides an approximation of the antibody site concentration. The data were also plotted as log [(Ao/b) -- !] against log (1/c) using various values of A.[18]. The value of d. which gave a linear plot was taken to be the true antibody site concentration. The average binding constant (K.) is the value for 1/c when [(A./b) -- 1] equals unity. The heterogenei~" index (a) is equal to the dope of the linear log-log plot. Results are given as the per cent of antibody sites remaining after treatment or as relative binding activity. The latter is calculated as the concentration of hapten bound by the unprotected or protected antibody preparations divided by the concentration of hapten bound by a control antibody preparation at the same free hapten concentration, and the quotient so obtained is multiplied by 100. All values were corrected to the same protein concentration. Binding of hapten to the immunogiobulin fraction of antibody preparations was corrected for nonspecifie binding to pooled normal rabbit immunogiobulin G. Equilibrium dialysis experiments were performed in 0.1 M tris-HCl, 0.002 M EDTA buffer, pH 8.0, except for anti-P, antibody which was performed in 0.1 M tris-HCl, 0-002 M EDTA, pH 9.0. 1"'I-Labeled haptens, mI-Labeled haptens (3-iodopyridine, p-iodophenyltrimethylammonium, #.iodobenzoate, p-iodobenzenearsonate, and p-iodobenzenesulfonate) were prepared by isotope exchange utilizing carrier free luI iodide according to the procedure of Grossberg et al.[l] for the preparation of ~q-labeled haptens.

Hydrolysis of anti-R, with papain and separation of fragmeats by column chromatography. Papain digestion of anti-R, antibody was performed in a manner similar to that described by Porter[19]. Specifically purified and-R, antibody (170 rag) in 0.1 M tris buffer, pH 8-0, was brought to a final volume of 17 ml with 0.1 M" sodium phosphate buffer, pH 7.0, 0.01 M eysteine and 0-002 M EDTA. Papain (2.5 rag) was added, and the solution was maintained at 37 ° for 3 hours. After two hours, a portion (0.8 ml) was taken for ultracentrifugadon. No residual intact antibody (6.6S) could be seen, and the fragments had a sedimentation coefficient of 3"5S.

Amino Groups in Antibody Sites

371

Following incubation at 37 °, the papain digest was dialyzed at 4 ° against distilled water for 12 hours, then against 0.01 Msodium acetate buffer, pH 5 -4, for 24 hours. Chromatography of the digest was performed on a CM-cellulose column (1 "8 X 54 cm) using 0.01 M sodium acetate buffer, pH 5.4, until the first fragment fraction was eluted. The remaining fragments were then eluted with a "cone-sphere" gradient of 500 ml of 0.01 M sodium acetate, pH 5.4, and 250 ml of 0.9 M sodium acetate, pH 5.4, according to the procedure of Stelos a al.[20]. Following chromatography, the separated Fab fractions were dialyzed against 0.1 M tris-HCl, 0.002 M EDTA, pH 8.0, then concentrated by uhrafiltration[21], and finally alanylated with N-carboxy-z~L-alanine anhydride as described above. Modification of guanidinium groups. The guanidinium groups of intact anti-R, antibody and of the papain fractious of anti-R, antibody were modified with 2,3butanedione according to the procedure described by Grossberg and Pressman[5]. The extent of the modification of the guanidinium groups was determined by amino acid analyses after extensive dialysis of the reaction mixtures against 1000 volumes of tris buffer, pH 8.0. Sedimentaaon ana~s/s. All sedimentation velocity measurements were performed in a Spinco MOdeI-E analytical ultracentrifuge equipped with schlieren optics. The ultracentrifuge was operated at 59,780 rev/min, and the rotor temperature was maintained at 20 _+ 0.05 °. For S,0.,, calculations the partial specific volume for all immunoglobulins was assumed to be 0-72. Determination of#rotsin. An E~%- of 14.6 at 280 m/~ was used for determination of normal and alanylated antibody preparations. The attachment ofpolyalanyl peptides onto lysyl residues does not alter the ultraviolet absorption spectrum of the antibody preparations in the region from 240 m/~ to 320 m/~ as shown by Karush and Sela[14]. Protein determinations of the antibody preparations modified with 2,3-butanedione were performed using the Lowry modification of the Folin reaction[22]. The molecular weight of immunoglobulin G and antibody preparations used was taken to be 150,000.

RESULTS Alanylation of antibody preparations. ImmunospecificaUy purified anti-Ps, anti-A~ anti-Xp, anti-Rp, and anti-SulfB antibodies, and the immunoglobulin fraction of anti-P, antibodies were reacted at pH 7.0 with N-carboxy-DL-alaninc anhydride (96 moles of alanine anhydride per mole of lysine) for 24 hours at 4 °. The alanine enrichment of the polyalanylated antibodies was 800 4- 80 residues per mole. Under these conditions 65--70 per cent of the lysyl residues were alanylated as determined by amino acid analyses following deamination of unaltered ly~/l residues.

The ~'ect of modification of amino groups on the antibody activi~ of purif~d anti-R, antibodies. In order to ascertain the effect of modification of amino groups on the average binding constant and on the antibody site concentration of antibodies made against negatively charged haptens, purified anti-R, antibodies were modified in the presence of 0.04 M p-nitrobenzenearsonate (protected sample). A similar preparation of anti-R, antibody was modified in the absence of hapten (unprotected sample). Amino acid analyses of the protected and unprotected anti-Rp preparations indicated

372

M. H. FREEDMAN,A, L. GamDsno and D. p=m=U,,N

that 69 per cent of the lysyl residues were modified in both samples when compared with the control sample. Binding curves for the unprotected, protected, and control anti-K, preparation are illustrated in Fig. 1, and the binding data are summarized in Table 1. T h e eurvalinear nature of the binding curves reflects the heterogeneity of the antibody population with respect to its affinity for the p-iodobenzenearsonate hapten. T h e unprotected anti-R, antibody sample lost 25 per cent of its binding sites, while the protected 4.0

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[FREE HAPTEN] x'O "S M" FIG. 1. The effect of alanylation on the binding of/bLUI-benzenearsonate by specifically purified anti-Rp antibodies. Sixty-nine per cent of the lysyl residues in the unprotected and protected samples were modified. The protected sample contained 0.04 Mp-nitrobenzenearsonate during alanylation. Each point represents the mean of duplicate determinations with an average deviation from the mean of 4-_ 0"8%. T x n ~ 1, T i m

~'*'Rer

o F M O D ~ I C A T I O N O F A M I N O G R O U P S ON T H E B I N D I N G S ~ ANTI-Pp ANTIBODIES

Sample Purified anti-Rp antibody Controll: Protected§ (0.04 Mp-nitrobenzenearsonate) Unprotected§ Immunoglobulin fraction of anti-P, antibody Control~; Protectedil (0" 04 M benzeneph0aphonate) Unprotectedll

OF A N T I - I ~ p A N D



Binding sites remaining* (% of control)

K , x 10-~ (l/mole)

at

100 94

1.1 I. 1

0- 7 0.7

75

I. 0

0.7

100 82

4.6 5.1

I. 0 O. 9

68

4.8

0.9

* Values obtained by extrapolation of the binding curves. t Heterogeneity index. ~, The control samples were exposed to hapten and dioxane as described in the Methods section. § Sixty-nine 4. three per cent of the lysyl residues were modified in both samples. II Sixty-seven 4- three per cent of the l~yl residues were modified in both samples.

Amino Groupl in Antibody Sites

373

sample, modified to the ~ m e extent as the unprotected sample, lost only 6 per cent of its antibody combining sites. The average binding constants and the heterogendty indices were almost indistinguishable for the unprotected, protected, and control anti-R, samples. Other preparations of specifically purified anti-R, antibody, when modified to the same extent without protection, lost 10-30 per cent of their antibody combining sites when compared with the control samples. The loss of antibody sites could be prevented almost completely by the presence ofp-nitrobenzenearsonate during the alanylation procedure.

The elect of modification of amino groups on the antibody actioi~ of anti-P, antibodies. The amino groups of an immunoglobulin fraction of anti-P, antiserum were also modified by alanylation. Anti-P, antibodies were modified in the presence and absence of 0.04 M benzenephosphonate, resulting in the modification of 67 per cent

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FIo. 2. The effect of alanylation on the binding ofp-~'~I-benzenearsonate by the immunoglobulin fraction of anti-P, antibodies. Sixty-seven per cent of the lysyl residues were modified in the unprotected and protected samples. The protected sample contained 0.04 M benzenephcaphonate during alanylation. Each point represents the mean of duplicate determinatiom with an average deviation from the mean of + 1.1%. of the lysyl residues in both the unprotected and protected samples as compared with the control sample. To test for antibody activity in the unprotected, protected and control anti-P, preparations, equilibrium dialysis was performed using several concentrations of p-iodobenzenearsonate in 0. I M tris-HOl, 0.002 M EDTA, pH 9.0, instead of p-iodobenzenephosphonate. Kreiter and Pressman[23] demonstrated that anti-P, antibodies bind the structurally similar benzenearsonate haptem. Since anti-P, antibodies contain primarily antibody formed in response to the doubly charged ion, equilibriu m dialysis was performed at pH 9.0 so that the p-iodobenzenearsonate hapten would also be present largely in the doubly charged form. The binding curves for the protected, unprotected, and control samples are shown in Fig. 2 and the binding data are summarized in Table 1. The linearity of these binding curves reflects a greater degree of homogeneity for these antibodies than observed with the anti-K, antibodies (Fig. 1). The unprotected anti-Pp sample lost 32 per cent of its binding sites, while the protected sample lost 18 per cent of its

mmJNo5/4-~o

M. H. i:REEDMAN,A. L. GROSSBERGand D. I'gEssstxy

374

antibody combining sites. The unprotected anti-Pp sample lost a somewhat larger proportion of its antibody sites than did the unprotected anti-R, sample, but the protection of antibody sites during alanylation was somewhat greater with anti-R, than with anti-P,. The average binding constants and heterogeneity indices are practically identical for the unprotected, protected, and control anti-P, samples.

The effect of modification of amino groups on the antibody activity of anti-Xp amt anti-Sulfp antibodies. Other purified antibodies, prepared against negatively charged haptens, were similarly modified by alanylation. The anti-X, and anti-Surf, antibody preparations studied failed to show significant losses of antibody sites when 65-70 per cent of their lysyl residues were modified. Thus, unprotected anti-X, samples lost 10 per cent of their combining sites, while protected samples lost 5 per cent of their 1.6 T

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[FREEHAPTEN]xIO'SM"l FIG. 3. The lack of effect of alanylation on the binding of p-tWI-phenyltrimethylammonium by specificallypurified anti-Ap antibodies. Sixty-eight per cent of the lysyl residues in the unprotected and protected samples were modified. The protected sample contained 0.04 M phenyltrimethylammonium during alanylation. Each point represents the mean of duplicate determinations with an average deviation from the mean of + 0-9%. antibody combining sites. Unprotected anti-SuE, samples lost 5 per cent of their combining sites on alanylation, while the protected samples showed no loss of their antibody combining sites. The unprotected, protected, and control preparations showed no differences in the average combining constants and heterogeneity indices.

The lack of effect of modification of amino groups on the antibody activity of aati-A~ and anti-P, antibodi~. Specifically purified anti-A, and anti-P, antibodies were also treated with the N-carboxyanhydride of DL-alanine in the presence and absence of hapten. Amino acid analyses of the unprotected and protected samples showed that 68 per cent of the lysyl residues were modified in anti-A, antibodies and 70 per cent of the lysyl residues were modified in the anti-Pa antibodies. The binding curves for anti-A, antibodies are shown in Fig. 3, and the binding curves for anti-Ps antibodies are shown in Fig. 4. The binding data for these antibodies are presented in Table 2. The binding curves for the protected, unprotected,

Amino Groups in Antibody Sites

375

and control anti-A, samples were practically indistinguishable from one another. The average binding constants, the number of antibody combining sites and heterogeneity indices were unaffected by extensive modification of the amino groups. Protected, unprotected and control samples of anti-P= antibody also failed to show differences in their average binding constants, number of antibody combining sites, and heterogeneity indices. A direct comparison of the effect of amino group modification of amino groups on anti-R, antibodies and on anti-A, or on anti-Ps antibodies was made by treating a _

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FIo. 4. The lack of effect of alanylation on the binding of 3-mI-pyridine by specifically purified anti-P, antibodies. Seventy per cent of the lysyl residues in the unprotected and protected samples were modified. The protected sample contained 0.04 M pyridine during alanylation. Each point represents the mean ofduplicate determinations with an average deviation from the mean of :1: 1.2%. mixture of anti-Ap and anti-R, antibodies and a mixture of anti-P= and anti-R, antibodies with N-carboxyanhydride of Dr-alanine. With anti-A, and anti-P, antibodies, there was no decrease in antibody combining sites, and no alteration of the average binding constants. However, under the identical conditions prevailing in each mixture, alanylation of the amino groups of anti-R, antibodies resulted in a loss of antibody combining sites with little or no change in the average combining constants of remaining sites.

The effect of modification of amino and ~anidiniurn groups in ths Fab fiagraents of anti-R, antibodies. Several purified samples of anti-R, antibody were digested with papaln and subjected to column chromatography on CM-ceUulose. A typical elution chromatogram is illustrated in Fig. 5. Fractions A-D contain the Fab fragments, while Fraction E contains the Fc fragment. The relative concentrations of the different Fab Fractions (A-D) varied considerably from one antibody preparation to another. Table 3 presents typical results of the relative antibody activity of intact anti-Rp antibody and the various Fab fractions after modification of amino groups. ODe fraction (Fraction B) lost more activity on modification than the others. The lysyl residues of intact

376

FREEDMAN,

M.H.

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A, L. G R O m B ~ O

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ANTI-A~AND ANTI-Ps Abrrll~ODIF.....~

Sample

Purified anti-A, antibody Controlt Protected§ (0.04 M phenyltrimethylammonium chloride) Unprotected§ Purified anti-Ps antibody Controlt Protected [I (0-04 M pyridine) Unprotected l[

Binding sites remaining* (% of control)

K . × 104 (I/mole)

at

100 100

4.9 5.0

0- 6 0.6

100

4.6

0" 6

100 100

1.4 1.4

0- 8 0.8

100

1.4

0.8

* Values obtained by extrapolation of the binding curves. t Heterogeneity index. The control samples were exposed to hapten and dioxane as de#cribed in the Methods g~on.

§ Sixty-eight + three per cent of the lywl residues were modified in both samples. Jl Seventy ± three per cent of the lysyl residue# were modified in both samples.

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Fro. 5, Chromatography on C,M-cellulme of the l ~ p ~ digest of specifically p u r i f ~ mlti-Rp antibody. Column, 1"8 × 54cm; elmmt~ sodium acetate buffer, pH 5.4, 0.01 to 0.9 M. Fractiom .% ]3, C and D conU6n the Fab fragmenu and Frgctioa E contaim the Fc Fragment. anti-R, aatibody and of the various Fab fractions were modified to an extent of 68 + 3 per cent for both the unprotected and protected ~ m p l e s as compared with the control ~ x a p l ~ . T h e unprotected intact anti-R, antibody sample lost 17 per cent of its antibody activity, while the protected s~mple lint 7 per cent of im antibody

Amino Groups in Antibody Sites

377

TABLE 3. THZ EI~ECT oy MODNICATION 01~ AMINO GROUPS ON T H E BINDING A C ' I I V r l ~ OF ~TX-Rp ANTIBODY AND OP THE F a b ~ ' ~ C T m N S OBTAINED A F I ~ I t PAPAIN DIGI~'IION OF ANTIoRp ANTIBODY

Sample*

Relative content (%)

(1) Intact purified anti-R, andbody (2) Fab fragments of purified anti-R, antibody§: Fraction A Fraction B Fraction C Fraction D

100

62 II 8 11 19

Relative binding activityf (% of control) Protected:~ Unprotected 93

83

93 86 94 93

85 66 91 82

Weighted average ----93

Weighted average ==64

* Sixty-eight + three per cent of the lysyl residues in all samples were modified. t Measured at a free hapten concentration of 8-5 x 10.4 M. 2~Protected with 0.04 M benzenearsonate. § See Fig. 5. fl Per cent of the total Fab content.

TABLE 4. THE

Zm,ZCT OY MODIFICATION OF OUANIDINIUM OitOUPS ON THZ BINDINO ACTIVITY OP

A N T I - R e ANTIBODY AND OF THE

Fab

FRACTIONll OBTAINZD AFFIgit PAPAIN DIOIgITION OF A N ' r I - R p ANTIBODY

Sample*

(1) Intact purified anti-Rp antibody (2) Fab fragments ofpurified anti-R, antibody§: Fraction A Fraction B Fraction C Fraction D

Relative content (%) 100 62 ;I 8 I1 19

Relative binding activityt (% of control) Protected~ Unprotected 84

64

88 81 89 86

68 40 70 71

Weighted average --87

Weighted average = 6 7

*Forty-nine _ three per cent of the arginyl residues in all samples were modified. t Measured at a free hapten concentration of 8.9 x 10 .4 M. Protected with 0.04 34 benze~learsonate. § See Fig. 5. II Per cent of the total Fab content.

378

M. H. FREEDMAN, A. L. GROSSBERG and D. PRESSMAN

activity. Papain Fraction B, which comprised 6--14 per cent of the total Fab content depending upon which anti-R, antibody preparation was used, lost a significantly larger proportion of its antibody activity than the other Fab fractions (Table 3). Fraction B lost 34 per cent of its antibody activity in the Unprotected sample, while the protected sample lost only 14 per cent of its antibody activity. The weighted average antibody activity of the four Fab fractions was equivalent to the antibody activity of the intact anti-R, antibody for both the unprotected and protected samples (Table 3). Table 4 presents typical results of the relative antibody activities of intact anti-R, antibody and the various Fab fractions after modification of guanidinium groups. Once again, Fraction B lost more activity on modification than the others. The guanidinium groups of intact anti-Rp antibody and of the various Fab fractions were modified to an extent of 49 +_ 3 per cent for both the unprotected and protected samples as compared with the control samples. The unprotected whole anti-R, antibody lost 36 per cent of its antibody activity, while the protected sample lost 16 per cent of its antibody activity. Papain Fraction B lost 60 per cent of its antibody activity in the unprotected sample and lost only 19 per cent of its antibody activity in the protected sample. The weighted average antibody activity of the four Fab fractions is very similar to the antibody activity of the intact antibody preparation for both the unprotected and protected samples (Table 4).

DISCUSSION The amino groups in the specifically purified anti-hapten antibodies were modified by alanylation using the N-carboxyanhydride Of DLoalanme at pH 7.0 in an aqueous medium. The ~-amino groups of lysine and the N-terminal a-amino groups on the polypeptide chains served as initiators for the polymerization reaction. The lysyl residues in the polyalanylated anti-hapten antibody preparations were modified to an extent of 65-70 per cent. Since more than 30 per cent of the amino groups were unreacted, it may be that they represent groups buried in the molecule or groups unavailable .to the reagent for other reasons such as steric effects of polyalanylated chains already formed on adjacent groups. Another possible factor is that the free a-amino group's of the alanyl residues already polymerized onto the lysine groups compete too effectively as initiators for the alanylation reaction. The a-amino groups are better initiators of the polymerization reaction than the E-amino groups. Therefore, it may be difficult to get complete modification of all lysyl residues in antibodies with the N-carboxy amino acid anhydrides, even though all the lysines in other proteins such as ribonuclcase[24] and pancreatic trypsin inhibitor[25] have been reported to be reactive toward alanylation. Figures 1 and 2 and Table I clearly demonstrate the effect of modification of amino groups on the binding activity of antibodies prepared against two negatively charged hapteus. Anti-R, and anti-P, antibodies daowcd a significant loss of antibody combining sites when their amino groups were modified. The antibody sites remaining did not differ in their average combining constants from those present M o r e amino group modification. This is in accord with previous studies on extreme alanylation of anti-p-azophenyl-~.lactoside antibodies by Karush and Sela[14] which had shown

Amino Groul~ in Antibody Sites

379

that such a modification had tittle effect on the average binding commnt of t h ~ antibodies. In the present work, the loss of antibody sites following alanylation could be almost completely prevented in the case of anti-R, antibody by the presence ofhapten during the alanylation reaction. The anti-P, antibody preparation studied lost a somewhat greater fraction of its antibody combining sites than did the anti-R, antibodies, but the protection of antibody sites with hapten was somewhat less effective in the case of anti-P, antibodies than in the case ofanti-R, antibodies. The above observations strongly suggest that the loss of antibody activity was not due to conformational changes due to modification of amino groups far removed from the antibody combining sites. It seems unlikely that only some anti-K, and anti-P, antibody sites should have their binding constants reduced to very low values (i.e., sites lost) by alanylation outside of these sites, whereas the majority of sites on the molecules which were equally extensively alanylated should not be affected at alL Furthermore~ the most direct interpretation of the protective effect of hapten is that hapten prevents modification of a residue in the site rather than prevents modification of a residue outside the site whose alanylation could cause a conform&tional change affecting the site. However, it is possible that the alanylation of an amino group adjacent to the site may cause the site to lose ability to bind because of steric interference by long polyalanyl side chains. However, we would expect that such an amino group would have to be so close to the site that hapten would sterically interfere with its alanyhtion. Not all antibodies directed against negatively charged haptem showed evidence for involvement of amino groups in their antibody combining sites. AntioX, and anti-Snlf, antibodies failed to demonstrate significant 1_o~___of antibody sites when 65-70 per cent of their lysyl residues were modified. Lysyl residues or a-amino groups may still be present in some of the sites of these antibody molecules, but be unavailable for alanylation. Antibodies directed against these haptens from individual rabbits might differ in the degree of involvement of amino groups in their antibody combining sites. Antibodies against the positively charged A, hapten failed to lose antibody sites after alanylation, and the average binding constants were practically unaltered in all three samples (Fig. $ and Table 2). Antibodies against the neutral P, hapten also failed to demonstrate losses either in average combining constants or in the number of antibody combining sites following alanylation (Fig. 4 and Table 9). These results show that lysyl residues or a-amino end groups are either absent or not available for alanylation in the sites of these antibodies. The results with anti-A, and anti-P, antibodies also emphasize that the loss ofantibody combining sites in anti-R, and antiP, is due to modification of amino groups within the antibody: sites rather than modification elsewhere in the antibody molecule to produce conformational changes affecting the site; any such changes might be expected in the antibodies against neutral or positively charged haptens as well. The positive charge in the combining site of most antibody molecules[5] ag!imt negatively charged haptens appears to be contributed by the arginyl rosidue. Only some of the anti-R, antibody sites contain an amino group. Some of the molecules with such a group in the site have other properties different from the

380

M. H. FRltEDMAN,A. L. GROgmEROand D. PRESSMAN

molecules having no amino group in the site which permits their Fab fragments to bc separated from the Fab fragments of the other molecules by chromatography on CM-ceUUlme. T h e fact that a fraction of Fab fragments could be isolated (Fraction B, Fig. 5 and T a b l e 3) which had a significantly larger proportion of sites containing an amino group than did the total antibody population provided a way of showing the effect of amino group alteration more clearly. Whether the presence of an amino group in the site is related to antibodies directed against the singly or the doubly charged forms of the p-azobenzenearsonate group[26] remains to be determined. Modification of guanidinium groups in the same fraction (B) of Fab fragments caused loss of activity, and the loss was twice as great as when the amino groups were modified. It still remains to be determined whether the amino and guanidL-dum groups observed as present in the antibody sites in this fraction are present within the same antibody site, or whether they are present in different antibody sites ~vithin this heterogeneous population of antibody molecules. Contribution of ~syl residues to the binding sites. I n the experiments carried out here, amino groups are exclusively modified. However, the amino groups could be either an a-amino group of the N-terminal amino acid of a polypcptide chain or the e-amino group of a lysyl residue. Where no effect is observed, we say that an easily modified lysine is not involved. Where there is an effect, we cannot say with certainty that a lysyl residue is involved since the N-terminal residues of the polypeptide chains m a y be involved. Nevertheless, since the N-terminal residues of the polypeptide chains account for only 3 per cent of the amino groups present in the molecule, it is likely that the amino groups in the sites are on lysyl residues.

AcknowlM£onsnts--The able technical assistance of Mr. Fred Maenza is gratefidly acknowledged. The authors are indebted to Mr. R. Chr-s~nowaki for amistance with the amino acid analyses. REFERENCES 1. GRoe~zno A. L., RADZrmZ, G. and Pazum,a~ D., Biodunnist~, N.Y. 1, 391 (1962). 2. ~ N D., NlSONO~ A. and RADZlmm G., J. lramun.86135 (1961). 3. GRommmo A. L. and P t ~ s s ~ D., J. Am. ¢Asra.$oc. I ~ 5478 (1960). 4. G R o s n n o A. L. and P R ~ J s ~ D., Fain Prec. Fain Am. Soes exp. Biol. 25, 339 (1967). 5. GRom~RO A. L. and PU~aUAN D., Bioehsm/s~, N.Y. 7, 272 (1968). 6. CtmN C. C., GRo~ssRo A. L. and ~ D.,Bioctumistry, N.Y. 1, 1025 (1962). 7. W o o L. and Smoza S.J., Biochomstry, N.Y. 2, 104 (1963). 8. Pm~M,~ D. and GRmsezRo A. L., The Strtc,~a/ Bas~ of Ant/body Sp~f~ty, p. 189. W. A. Benjamin, New York (1960) 9. KxtErrzR V. P. and Pa~niAN D., Bioehsmistry, N.Y. 2, 97 (1963). I0. ONotm K., YAO! Y. and PnESMAN D., I m ~ 2, 181 (1965). II. TAmOAm N., KrrAoAWA M., YAOXY. and P ~ m u ~ D., Cancer R#s. 2"1, 747 (1967). 12. Kexwxcx R. A., B i o a ~ . J. St, 1248 (1940). 13. Fucm S. and SSLA M., J. biol. Chem. 24~ 3558 (1965). 14. KARUSHF. and SELX M., Immunochemistr) 4, 259 (1967). 15. Instruction manual AAA-1 for Technicon Amino Acid Analyzer, p. 38. 16. Am,ltcszN C. B., SELA M. and Cooxs J. P., J. biol, Chem. 2 ~ 1825 (1962). 17. NtSONO~ A. and IhtSUMAN D., J. Immun. 80, 417 (1958). 18. F~szN H. N., M#thods in Medical Res~rch 1O, 115 (1964). 19. Pop,mR R. R., Bioehsm. J. 73, 119 (1959). 20. STELOSP., ROHOLTO. and PRXSSMAND., J. Immun. 89, 113 (1962).

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21. H o r s ~ B. V. and FoLrmRrNo S. C., Analyt. Bioelmn. I, 437 (1960). 22. LOwRy O. H., Ros~eaOVOH N. J., F ~ t A. L. and RANDALLR. J., J. biol. Chem. 153, 265 (1951). 23. KaErrER V. P. and Pl~SSUAN D., Biochemistry, N.Y. 3, 274 (1964). 24-. Am~s~r~ C. B., SELA M. and CoorJ J. P . , J . biol. Chem. 237, 1825 (1962). 25. EPSTmNC.J., ANI'm~N C. B. and SELA M., J. biol. Chem. 237, 3458 (1962). 26. KrrAGAWAM., GRosssaao A. L., YAoxY. and PRESSMAND., Immunoalmni.ary4, 197 (1967).