lmmunochemistry, 1977, Vol. 14, pp. 269 276. Pergamon Press. Printed in Great Britain
POSITION OF A LIGHT CHAIN TYROSYL RESIDUE IN THE COMBINING SITE OF A RABBIT
ANTI-p-AZOBENZOATE ANTIBODY* Y. LEE, O. A. ROHOLT, E. APPELLA and D. PRESSMAN Department of Immunology Research, Roswell Park Memorial Institute,5" 666 Elm Street, Buffalo, NY 14263; and Laboratory of Cell Biology, National Institutes of Health, Bethesda, MD 20014, U.S.A. (Received 20 September 1976)
Abstrac~The isolation and characterization of chymotryptic peptides containing a tyrosyl residue from the combining site of a rabbit anti-p-azobenzoate antibody preparation of restricted heterogeneity has been achieved by the paired-iodination procedure. The sequence of the first 38 residues from the N-terminus of the light chain was determined and the combining site tyrosyl residue is shown to be at position 30 of the light chain, i.e. in the first hypervariable region.
INTRODUCTION X-ray crystallographic analyses of Fab' fragments of some myeloma proteins have shown that the hypervariable (hv):~ regions of the VL and VH domains of the fragment are in close proximity at one end of the fragment as a consequence of the folding and the interaction of the heavy and light chains (Poljak et al., 1973; Segal et al., 1974). It is generally accepted that the complex of hv regions constitutes the antibody site and it is the steric relation between particular residues in hv regions, i.e. the complementarityforming regions, that give a particular antibody its ligand-binding specificity. Binding sites of different specificities result from a difference of residues and steric relations in the complementarity-determining regions. The analysis of Wu and Kabat (1970) clearly established the presence and location of the hv regions in light chains. That analysis was based on Bence Jones and light chain sequences of human and mouse proteins and showed three regions of hypervariability, viz. in the vicinities of residues 32, 55 and 96. Recently it has been reported (Margoties et al., 1975; Thunberg & Kindt, 1976; Poljak, 1975) that the second region is not hypervariable in rabbit light chains, but instead is relatively invariant in that species. Reference to the crystal structure of mouse McPC 603 phosphorylcholine-binding protein shows that the second region is actually apart from the site (Padlan et al., 1974). This may still effect the steric arrangement of the contact residues of the site. Thus it may be general that, in the light chain, only the first and third regions are involved as contact residues in ligand binding. * This investigation was supported by grant No. AI12100, awarded by the National Institute of Allergy and Infectious Diseases, DHEW, and by grant No. P30-CA17609, awarded by the National Cancer Institute, DHEW. ~"A unit of the New York State Department of Health. :~Abbreviations used: MIT, monoiodotyrosine; DIT, diiodotyrosine; PTH, phenylthiohydantoin; Xp, p-azobenzoate. 269
In the heavy chain V-region there appear to be four hv regions, in the vicinities of positions 34, 58, 87 and 105, on the basis of human sequences (Capra & Kehoe, 1974). Again, as has been noted (Margolies et al., 1975), the third hv region, that around residue 87, is not in the binding site based on examination of crystallographic models (Poljak et al., 1973; Segal et al., 1974). Chemical evidence that it is the hv regions that constitute the binding site has been obtained by a number of site-labeling procedures using raised antibody or ligand-binding myeloma proteins followed by isolation and characterization of a peptide containing the labeled residue and localizing it within the heavy or light chain sequences (Takeda et al., 1973; Appella et al., 1973; Friedenson et al., 1976; Friedenson et al., 1972; Grossberg et al., 1976; Franek, 1971 ; Goetzl & Metzger, 1970; Haimovich et al., 1972; Koo & Cebra, 1973). All of the labeled residues reported and localized as site residues have been in hv regions. Thus, chemical methods as well as crystallographic analyses have contributed substantial evidence that antibody specificity is derived from the hv regions. However, no pattern between structure and specificity has developed and to determine a pattern would appear to require the continued accumulation of VL and VH sequences and information on the ligandbinding role of hv region residues. In our laboratory several studies on the isolation and characterization of tyrosyl peptides from the combining sites of rabbit anti-hapten antibodies using the paired-iodination technique have been reported. The locations of these tyrosyl residues were at position 102 in the fourth hv region of the heavy chain of an anti-p-azophenyltrimethylammonium antibody (Takeda et al., 1973), at position 96 in the third hv region of the light chain of an anti-p-azobenzoate antibody (Appella et al., 1973), and at position 30 in the first hv region of the light chain sequence of an anti-p-azobenzoate antibody (Friedenson et al., 1976). In one case (Friedenson et al., 1972), the labeled tyrosyl was between heavy chain residues 85 and 120 of a rabbit anti-p-azopyridine antibody.
270
Y. LEE, O. A. ROHOLT, E. APPELLA and D. PRESSMAN
In this communication we report the isolation and location of chymotryptic peptides containing a (monoido)tyrosyl residue from the site of a rabbit anti-p-azobenzoate antibody preparation of restricted heterogeneity (No. 5690) and the sequence of the first 38 residues from the N-terminus of the light chain. The tyrosyl residue in this peptide has been shown to be at position 30 of the light chain, in the first hv region. MATERIALS AND METHODS Preparation of antibody Rabbit No. 5690 was injected with 1 mg of a conjugate of diazotized-p-aminobenzoic acid and bovine-~-globulin intravenously three times a week for four weeks. Serum was then collected at weekly bleedings for 35 weeks and pooled to give 460 ml. One milligram of the conjugate was injected intravenously after each bleeding. The anti-p-azobenzoate (Xp) antibody was specifically purified by means of a solid absorbent consisting of diazotized p-aminobenzoic acid coupled to a polymer of rabbit serum albumin (Onoue et al., 1965). The yield was 385 mg. Pairecl-iodination of antibody Two equal portions of the antibody were each iodinated to a level of 21 iodine atoms/mole (Roholt & Pressman, 1972). One portion of antibody (20 mg) was iodinated with 13q-labeled hypoiodite in the presence of hapten (0.05 M p-nitrobenzoate) and the other portion (20 rag) was iodinated with x25I-labeled hypoiodite in the absence of hapten. The two labeled portions were mixed for the next experiments.
Amino-terminal amino acid sequences of light chain These were determined by sequential Edman degradation (Edman, 1950) with a Beckman model 890B protein sequencer. The resulting phenylthiohydantoin derivatives were identified by gas chromatography (Pisano & Brozert, 1969) and high pressure liquid chromatography (Zimmerman et al., 1976), by HI hydrolysis followed by amino acid analysis (Smithies et al., 1971), and by mass spectrometry (Fairwell & Brewer, 1973). Measurement of association constant of antibody by equilibrium dialysis Equilibrium dialysis was carried out using 12~I-labeled p-iodobenzoate as the hapten (Grossberg & Pressman, 1960). RESULTS Isoelectric focusing In Fig. 1 is shown the electrofocusing pattern of the specifically purified anti-Xp antibody, visualized by protein staining. Four equi-spaced major bands and the presence of only a few minor bands indicate that the antibody preparation was of very restricted heterogeneity.
Separation of heavy and light chains The pair-iodinated anti-Xp antibody was reduced and alkylated (Birshtein et al., 1971) using iodoacetamide as an alkylating agent, and the heavy and light chains were isolated by gel filtration through Sephadex G-100 in 1 M propionic acid at 5°C. Amino acid analyses These were carried out according to Spackman et al. (1958). Two microliters of 0.2 M phenol was added to prevent the destruction of tyrosine. Amino acid analyses were performed on Beckman model 120 C automatic amino acid analyzer. Subtractive micro-Edman degradation This was performed on 5 nmole of sample using N-ethylmorpholine-pyridine acetate buffer, pH 8.6, for the coupling reaction. A Durrum model D-500 automatic amino acid analyzer was used to identify the amino acid removed at each cycle in the degradation. COOH-terminus analysis For carboxypeptidase A digestion, the peptide (3 nmole) was dissolved in 0.2M N-ethylmorpholine, pH 8.3, and enzyme added to give an enzyme to substrate ratio of 1:250; the mixture was incubated at 37°C for 3hr. Two drops of 30% acetic acid were added to stop the reaction and the digest was lyophilized. The released amino acid residues were identified on the Durrum model D-500 amino acid analyzer. E dman-dans y lat ion This was performed using the conditions described for small peptides (Gray & Smith, 1970). The dansyl-amino acid was identified by thin-layer chromatography (Morse & Horecker, 1966).
Fig. 1. Isoelectric focusing pattern of anti-p-azobenzoate antibody of rabbit No. 5690 according to the procedure of Williamson (1971), as modified by Keck et al. (1973). A 0.8 mm layer of 5% acrylamide gel containing 3 M urea and 2% ampholine was photopolymerized. The gel area was 15 cm wide x 20 cm long. The sample size was 200 #g. A constant current of 2 mA/plate at 250 V was applied for 20hr at 5°C. The protein bands were fixed in 18% Na2SO4 containing 0.1% (v/v) glutaraldehyde and stained with 0.2% bromphenol blue. The pH gradient is from pH 3 at the top to pH 10 at the bottom.
Tyrosyl Residue in the Site of a Rabbit Antibody
,.of
high-ratio spots were identified in the whole antibody map (Fig. 3a). One of these, found on the H-chain map (Fig. 3b), is designated as C-l-H, and the other two, found on the L-chain map (Fig. 3c), are designated C-2-L and C-3-L. The values of the ratios were 3.5, 6.7 and 12.0, respectively. In order to obtain the high-ratio peptides, C-I-H, C-2-L, and C-3-L, in sufficient quantities for sequence analysis, larger amounts of chymotryptic digests of heavy and light chains separated from the pairiodinated antibody were fractionated by the same two-dimensional system as described in the legend of Fig. 3. The peptides eluted from three high-ratio spots were further purified by two-dimensional analysis using a different HVPE and descending chromatography system as described in the legend of Fig. 4.
! ! ! ! !
I
I
it
!
I
I
i
I I/"
I I
//
I
iI
/
/ I
/
!
i/ j~
/
271
/
Characterization of peptide C-1-H /
0
I
t
2
3
4
5
6
?
8
b x 10 6 M
Fig. 2. Plot of bindingofp-iodobenzoate by specificallypurified anti-Xp-antibody (No. 5690) measured by equilibrium dialysis (curve III). Curves I and II represent a resolution of curve III into the binding by two populations, one of high affinity (I) and the other of lower affinity (II). For population I, K = 3 × 106 M -1 with a site concentration of 5.8 x l 0 - 6 M and for population lI, K =0.6 x 10SM -1 with a site concentration of 1.6 x 10-6 M. The concentration of antibody was 0.6 mg per ml, equivalent to an antibody site concentration of 7.5 x 10-6M (taking the mol. wt of antibody as 160,000). Each dotted line has the slope, l/c, where c is the free hapten concentration at which the binding indicated by the points falling on a particular dotted line was measured. The resolution is such that the sum of the lengths of the segments marked off on a particular line 1/c by curve I and curve II is equal to the length of the segment marked off by curve III on that line 1/c.
Equilibrium dialysis The extent of binding results obtained by equilibrium dialysis are shown in Fig. 2 on a Scatchard plot. The binding curve can be resolved into two lines as described for another antibody preparation (Roholt et al., 1972). One line represents a high binding population of about 80% of the sites having a K of 3 x 106 and the other line represents a population of about 20~o of the sites having a K of 0.6 x 105. These data are consistent with the apparent restricted heterogeneity of the antibody preparation as indicated by the isoelectric focusing data above.
Identification of tyrosyl peptide of the antibody site Samples of the pair-labeled whole antibody, and the heavy chains and light chains derived therefrom were digested with ~-chymotrypsin and the digest subjected to mapping followed by radioautography (Fig. 3). The radioactive spots on the maps were assayed for the ratio of 1251/131I using a dual channel y-ray spectrometer. The 1251/131I ratio for each spot on the maps was normalized to the ratio in the unfractionated digest of whole antibody. Three distinct
This peptide was derived from the heavy chain of the pair-iodinated antibody and the amino acid analysis gave aspartic acid, serine, tyrosine, and three glycines (Table 1). According to the electrophoretic pattern at pH 6.5 (see Fig. 4), peptide C-1-H is acidic, indicating that the aspartic acid is present as Asp in the peptide. Electrophoresis of a pronase-pancreatic digest of this peptide (Dube et al., 1972) and the radioactive counts of a known quantity of peptide showed that the peptide contained monoidotyrosyl as the sole radioactive residue. For the sequence study of the peptide we carried out the Edman-dansylation technique on 12.5 nmole of the sample which was divided into five portions. One portion was used for each step. Thus the peptide was analyzed through the 5th cycle and its sequence was found to be Gly-GlyGly-Asp-Ser-MIT. The MIT was placed at the C-terminal end since this peptide was released by chymotryptic hydrolysis and also 65% of the radioactivity still remained in the water phase after the 5th cycle of Edman degradations. In an attempt to locate the position of this peptide in the heavy chain sequence, its sequence was compared with published sequences of rabbit heavy chains (Dayhoff, 1972). No similar sequence was found in either framework or hypervariable regions. Thus, the peptide appears to come from one of the hv regions of the heavy chain but the exact position remains unknown.
Characterization of peptide C-2-L Upon amino acid analysis this peptide was found to yield threonine, two serines, and one each of glutamic acid, valine, and tyrosine (Table 1). Since the electrophoretic pattern of this peptide at pH 6.5 showed the peptide to be neutral, this peptide must contain glutamine and not glutamic acid (Fig. 4). The radioactive amino acid of this peptide was identified as DIT by electrophoresis of a pronase-pancreatin digest of the peptide (Dube et al., 1964). The sequence analysis of this peptide was carried out following the procedures of substractive microEdman degradation through the 5th position. Because of the small quantity of this peptide, it was difficult for us to estimate very precisely the exact amount of amino acid removed after each cycle of degradation. However, the analysis clearly revealed the
m
rig. 3. Radioautograms of chymotryptic peptide maps of whole anti-p-azo~enzoate antibody from rabbit No. 5690 (a), separated heavy chains (b), and ight chains (c). Chymotryptic digestion of the anti-Xp-antibody, the heavy :hains, and the light chains was carried out at pH 8.0 at 37°C for 20 hr with ubstrate to enzyme weight ratio of 25. Two-dimensional analyses of the digests vere performed by high-voltage paper electrophoresis on Whatman 3MM )aper at 2 kV for 2.5 hr in 1 M formic acid. pH 1.8. followed by descending :hromatography in butanol-acetic acid water (4:1:5) for 10hr. The amount tpplied at the origin (X) was 1 mg, 0.75 mg and 0.25 mg, respectively, for digests ff the antibody, the heavy chain and the light chain. Radioautographs of the peptide maps were prepared with Kodak No-screen Medical X-ray film.
z
7~
...]
.
POSITION OF A LIGHT CHAIN TYROSYL RESIDUE IN THE COMBINING SITE OF A RABBIT
ANTI-p-AZOBENZOATE ANTIBODY* Y. LEE, O. A. ROHOLT, E. APPELLA and D. PRESSMAN Department of Immunology Research, Roswell Park Memorial Institute,5" 666 Elm Street, Buffalo, NY 14263; and Laboratory of Cell Biology, National Institutes of Health, Bethesda, MD 20014, U.S.A. (Received 20 September 1976)
Abstrac~The isolation and characterization of chymotryptic peptides containing a tyrosyl residue from the combining site of a rabbit anti-p-azobenzoate antibody preparation of restricted heterogeneity has been achieved by the paired-iodination procedure. The sequence of the first 38 residues from the N-terminus of the light chain was determined and the combining site tyrosyl residue is shown to be at position 30 of the light chain, i.e. in the first hypervariable region.
INTRODUCTION X-ray crystallographic analyses of Fab' fragments of some myeloma proteins have shown that the hypervariable (hv):~ regions of the VL and VH domains of the fragment are in close proximity at one end of the fragment as a consequence of the folding and the interaction of the heavy and light chains (Poljak et al., 1973; Segal et al., 1974). It is generally accepted that the complex of hv regions constitutes the antibody site and it is the steric relation between particular residues in hv regions, i.e. the complementarityforming regions, that give a particular antibody its ligand-binding specificity. Binding sites of different specificities result from a difference of residues and steric relations in the complementarity-determining regions. The analysis of Wu and Kabat (1970) clearly established the presence and location of the hv regions in light chains. That analysis was based on Bence Jones and light chain sequences of human and mouse proteins and showed three regions of hypervariability, viz. in the vicinities of residues 32, 55 and 96. Recently it has been reported (Margoties et al., 1975; Thunberg & Kindt, 1976; Poljak, 1975) that the second region is not hypervariable in rabbit light chains, but instead is relatively invariant in that species. Reference to the crystal structure of mouse McPC 603 phosphorylcholine-binding protein shows that the second region is actually apart from the site (Padlan et al., 1974). This may still effect the steric arrangement of the contact residues of the site. Thus it may be general that, in the light chain, only the first and third regions are involved as contact residues in ligand binding. * This investigation was supported by grant No. AI12100, awarded by the National Institute of Allergy and Infectious Diseases, DHEW, and by grant No. P30-CA17609, awarded by the National Cancer Institute, DHEW. ~"A unit of the New York State Department of Health. :~Abbreviations used: MIT, monoiodotyrosine; DIT, diiodotyrosine; PTH, phenylthiohydantoin; Xp, p-azobenzoate. 269
In the heavy chain V-region there appear to be four hv regions, in the vicinities of positions 34, 58, 87 and 105, on the basis of human sequences (Capra & Kehoe, 1974). Again, as has been noted (Margolies et al., 1975), the third hv region, that around residue 87, is not in the binding site based on examination of crystallographic models (Poljak et al., 1973; Segal et al., 1974). Chemical evidence that it is the hv regions that constitute the binding site has been obtained by a number of site-labeling procedures using raised antibody or ligand-binding myeloma proteins followed by isolation and characterization of a peptide containing the labeled residue and localizing it within the heavy or light chain sequences (Takeda et al., 1973; Appella et al., 1973; Friedenson et al., 1976; Friedenson et al., 1972; Grossberg et al., 1976; Franek, 1971 ; Goetzl & Metzger, 1970; Haimovich et al., 1972; Koo & Cebra, 1973). All of the labeled residues reported and localized as site residues have been in hv regions. Thus, chemical methods as well as crystallographic analyses have contributed substantial evidence that antibody specificity is derived from the hv regions. However, no pattern between structure and specificity has developed and to determine a pattern would appear to require the continued accumulation of VL and VH sequences and information on the ligandbinding role of hv region residues. In our laboratory several studies on the isolation and characterization of tyrosyl peptides from the combining sites of rabbit anti-hapten antibodies using the paired-iodination technique have been reported. The locations of these tyrosyl residues were at position 102 in the fourth hv region of the heavy chain of an anti-p-azophenyltrimethylammonium antibody (Takeda et al., 1973), at position 96 in the third hv region of the light chain of an anti-p-azobenzoate antibody (Appella et al., 1973), and at position 30 in the first hv region of the light chain sequence of an anti-p-azobenzoate antibody (Friedenson et al., 1976). In one case (Friedenson et al., 1972), the labeled tyrosyl was between heavy chain residues 85 and 120 of a rabbit anti-p-azopyridine antibody.
270
Y. LEE, O. A. ROHOLT, E. APPELLA and D. PRESSMAN
In this communication we report the isolation and location of chymotryptic peptides containing a (monoido)tyrosyl residue from the site of a rabbit anti-p-azobenzoate antibody preparation of restricted heterogeneity (No. 5690) and the sequence of the first 38 residues from the N-terminus of the light chain. The tyrosyl residue in this peptide has been shown to be at position 30 of the light chain, in the first hv region. MATERIALS AND METHODS Preparation of antibody Rabbit No. 5690 was injected with 1 mg of a conjugate of diazotized-p-aminobenzoic acid and bovine-~-globulin intravenously three times a week for four weeks. Serum was then collected at weekly bleedings for 35 weeks and pooled to give 460 ml. One milligram of the conjugate was injected intravenously after each bleeding. The anti-p-azobenzoate (Xp) antibody was specifically purified by means of a solid absorbent consisting of diazotized p-aminobenzoic acid coupled to a polymer of rabbit serum albumin (Onoue et al., 1965). The yield was 385 mg. Pairecl-iodination of antibody Two equal portions of the antibody were each iodinated to a level of 21 iodine atoms/mole (Roholt & Pressman, 1972). One portion of antibody (20 mg) was iodinated with 13q-labeled hypoiodite in the presence of hapten (0.05 M p-nitrobenzoate) and the other portion (20 rag) was iodinated with x25I-labeled hypoiodite in the absence of hapten. The two labeled portions were mixed for the next experiments.
Amino-terminal amino acid sequences of light chain These were determined by sequential Edman degradation (Edman, 1950) with a Beckman model 890B protein sequencer. The resulting phenylthiohydantoin derivatives were identified by gas chromatography (Pisano & Brozert, 1969) and high pressure liquid chromatography (Zimmerman et al., 1976), by HI hydrolysis followed by amino acid analysis (Smithies et al., 1971), and by mass spectrometry (Fairwell & Brewer, 1973). Measurement of association constant of antibody by equilibrium dialysis Equilibrium dialysis was carried out using 12~I-labeled p-iodobenzoate as the hapten (Grossberg & Pressman, 1960). RESULTS Isoelectric focusing In Fig. 1 is shown the electrofocusing pattern of the specifically purified anti-Xp antibody, visualized by protein staining. Four equi-spaced major bands and the presence of only a few minor bands indicate that the antibody preparation was of very restricted heterogeneity.
Separation of heavy and light chains The pair-iodinated anti-Xp antibody was reduced and alkylated (Birshtein et al., 1971) using iodoacetamide as an alkylating agent, and the heavy and light chains were isolated by gel filtration through Sephadex G-100 in 1 M propionic acid at 5°C. Amino acid analyses These were carried out according to Spackman et al. (1958). Two microliters of 0.2 M phenol was added to prevent the destruction of tyrosine. Amino acid analyses were performed on Beckman model 120 C automatic amino acid analyzer. Subtractive micro-Edman degradation This was performed on 5 nmole of sample using N-ethylmorpholine-pyridine acetate buffer, pH 8.6, for the coupling reaction. A Durrum model D-500 automatic amino acid analyzer was used to identify the amino acid removed at each cycle in the degradation. COOH-terminus analysis For carboxypeptidase A digestion, the peptide (3 nmole) was dissolved in 0.2M N-ethylmorpholine, pH 8.3, and enzyme added to give an enzyme to substrate ratio of 1:250; the mixture was incubated at 37°C for 3hr. Two drops of 30% acetic acid were added to stop the reaction and the digest was lyophilized. The released amino acid residues were identified on the Durrum model D-500 amino acid analyzer. E dman-dans y lat ion This was performed using the conditions described for small peptides (Gray & Smith, 1970). The dansyl-amino acid was identified by thin-layer chromatography (Morse & Horecker, 1966).
Fig. 1. Isoelectric focusing pattern of anti-p-azobenzoate antibody of rabbit No. 5690 according to the procedure of Williamson (1971), as modified by Keck et al. (1973). A 0.8 mm layer of 5% acrylamide gel containing 3 M urea and 2% ampholine was photopolymerized. The gel area was 15 cm wide x 20 cm long. The sample size was 200 #g. A constant current of 2 mA/plate at 250 V was applied for 20hr at 5°C. The protein bands were fixed in 18% Na2SO4 containing 0.1% (v/v) glutaraldehyde and stained with 0.2% bromphenol blue. The pH gradient is from pH 3 at the top to pH 10 at the bottom.
Tyrosyl Residue in the Site of a Rabbit Antibody
,.of
high-ratio spots were identified in the whole antibody map (Fig. 3a). One of these, found on the H-chain map (Fig. 3b), is designated as C-l-H, and the other two, found on the L-chain map (Fig. 3c), are designated C-2-L and C-3-L. The values of the ratios were 3.5, 6.7 and 12.0, respectively. In order to obtain the high-ratio peptides, C-I-H, C-2-L, and C-3-L, in sufficient quantities for sequence analysis, larger amounts of chymotryptic digests of heavy and light chains separated from the pairiodinated antibody were fractionated by the same two-dimensional system as described in the legend of Fig. 3. The peptides eluted from three high-ratio spots were further purified by two-dimensional analysis using a different HVPE and descending chromatography system as described in the legend of Fig. 4.
! ! ! ! !
I
I
it
!
I
I
i
I I/"
I I
//
I
iI
/
/ I
/
!
i/ j~
/
271
/
Characterization of peptide C-1-H /
0
I
t
2
3
4
5
6
?
8
b x 10 6 M
Fig. 2. Plot of bindingofp-iodobenzoate by specificallypurified anti-Xp-antibody (No. 5690) measured by equilibrium dialysis (curve III). Curves I and II represent a resolution of curve III into the binding by two populations, one of high affinity (I) and the other of lower affinity (II). For population I, K = 3 × 106 M -1 with a site concentration of 5.8 x l 0 - 6 M and for population lI, K =0.6 x 10SM -1 with a site concentration of 1.6 x 10-6 M. The concentration of antibody was 0.6 mg per ml, equivalent to an antibody site concentration of 7.5 x 10-6M (taking the mol. wt of antibody as 160,000). Each dotted line has the slope, l/c, where c is the free hapten concentration at which the binding indicated by the points falling on a particular dotted line was measured. The resolution is such that the sum of the lengths of the segments marked off on a particular line 1/c by curve I and curve II is equal to the length of the segment marked off by curve III on that line 1/c.
Equilibrium dialysis The extent of binding results obtained by equilibrium dialysis are shown in Fig. 2 on a Scatchard plot. The binding curve can be resolved into two lines as described for another antibody preparation (Roholt et al., 1972). One line represents a high binding population of about 80% of the sites having a K of 3 x 106 and the other line represents a population of about 20~o of the sites having a K of 0.6 x 105. These data are consistent with the apparent restricted heterogeneity of the antibody preparation as indicated by the isoelectric focusing data above.
Identification of tyrosyl peptide of the antibody site Samples of the pair-labeled whole antibody, and the heavy chains and light chains derived therefrom were digested with ~-chymotrypsin and the digest subjected to mapping followed by radioautography (Fig. 3). The radioactive spots on the maps were assayed for the ratio of 1251/131I using a dual channel y-ray spectrometer. The 1251/131I ratio for each spot on the maps was normalized to the ratio in the unfractionated digest of whole antibody. Three distinct
This peptide was derived from the heavy chain of the pair-iodinated antibody and the amino acid analysis gave aspartic acid, serine, tyrosine, and three glycines (Table 1). According to the electrophoretic pattern at pH 6.5 (see Fig. 4), peptide C-1-H is acidic, indicating that the aspartic acid is present as Asp in the peptide. Electrophoresis of a pronase-pancreatic digest of this peptide (Dube et al., 1972) and the radioactive counts of a known quantity of peptide showed that the peptide contained monoidotyrosyl as the sole radioactive residue. For the sequence study of the peptide we carried out the Edman-dansylation technique on 12.5 nmole of the sample which was divided into five portions. One portion was used for each step. Thus the peptide was analyzed through the 5th cycle and its sequence was found to be Gly-GlyGly-Asp-Ser-MIT. The MIT was placed at the C-terminal end since this peptide was released by chymotryptic hydrolysis and also 65% of the radioactivity still remained in the water phase after the 5th cycle of Edman degradations. In an attempt to locate the position of this peptide in the heavy chain sequence, its sequence was compared with published sequences of rabbit heavy chains (Dayhoff, 1972). No similar sequence was found in either framework or hypervariable regions. Thus, the peptide appears to come from one of the hv regions of the heavy chain but the exact position remains unknown.
Characterization of peptide C-2-L Upon amino acid analysis this peptide was found to yield threonine, two serines, and one each of glutamic acid, valine, and tyrosine (Table 1). Since the electrophoretic pattern of this peptide at pH 6.5 showed the peptide to be neutral, this peptide must contain glutamine and not glutamic acid (Fig. 4). The radioactive amino acid of this peptide was identified as DIT by electrophoresis of a pronase-pancreatin digest of the peptide (Dube et al., 1964). The sequence analysis of this peptide was carried out following the procedures of substractive microEdman degradation through the 5th position. Because of the small quantity of this peptide, it was difficult for us to estimate very precisely the exact amount of amino acid removed after each cycle of degradation. However, the analysis clearly revealed the
m
rig. 3. Radioautograms of chymotryptic peptide maps of whole anti-p-azo~enzoate antibody from rabbit No. 5690 (a), separated heavy chains (b), and ight chains (c). Chymotryptic digestion of the anti-Xp-antibody, the heavy :hains, and the light chains was carried out at pH 8.0 at 37°C for 20 hr with ubstrate to enzyme weight ratio of 25. Two-dimensional analyses of the digests vere performed by high-voltage paper electrophoresis on Whatman 3MM )aper at 2 kV for 2.5 hr in 1 M formic acid. pH 1.8. followed by descending :hromatography in butanol-acetic acid water (4:1:5) for 10hr. The amount tpplied at the origin (X) was 1 mg, 0.75 mg and 0.25 mg, respectively, for digests ff the antibody, the heavy chain and the light chain. Radioautographs of the peptide maps were prepared with Kodak No-screen Medical X-ray film.
z
7~
...]
.
