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Physico-chemical and biological properties of the subunits of bovine colostral anti-DNP immunoglobulin M (IgM)
Irnmunochemistry, 1973. Vol. 10. pp. 477--479. Pergamon Press.
Printedin Great Britain
PHYSICO-CHEMICAL A N D BIOLOGICAL PROPERTIES OF THE S U B U N I T S OF BOVINE COLOSTRAL A N T I - D N P I M M U N O G L O B U L I N M (lgM) T. K. S. M U K K U R and U S H A J. T E W A R I Department of Biology, University of Windsor, Windsor, Ontario, Canada (First received 15 Janua~. 1973; in revised fi~rm 14 March 1973) Abstract-Bovine colostral anti-DNP IgM which possessed five measurable combining sites per molecule (K0 = 4.8 x 10~M-~) was reduced with/3-Mercaptoethylamine and alkylated with neutralized iodoacetic acid to form lgMs (Szo.w= 7.6S at 3-0 mg/ml). The lgMs was shown to possess only one combining site per molecule (K0 = 4.6 × 104M-~) and failed to cause hemagglutination of the DNPconjugated red blood cells thus indicating their functional univalency. The Fab subunits produced by papain digestion (Sz0,~ = 3.6S at 2.1 mg/ml) were shown to bind 0-5 mole of hapten/mole (K0 = 4.3 × 10~M- ~)and also did not cause hemagglutination of the DN P-conjugated red blood cells.
INTRODUCTION
Previous studies from this laboratory (Mukkur, 1972) h a v e shown that Bovine colostra] antidinitrophenyl ( D N P ) I m m u n o g l o b u l i n M (IgM) possessed five combining sites per molecule. Similar findings have also been reported from o t h e r laboratories (Frank and H u m p h r e y , 1968: Lindquist and Bauer, 1966; O n o u e et at., 1965; S c h r o h e n l o h e r and Barry, 1968; Voss et al., 1969). L a t e r on it was r e p o r t e d that if one used antibody I g M with an average binding constant (Ko) greater than 10SM -I, it was possible to detect two sets of sites ( O n o u e et al., 1968; K i s h i m o t o and O n o u e , 1971 ; Oriol et al., 1971 ), one half of the sites having a higher affinity constant than the second set. T h i s led to a suggestion that in case of antibody IgM with a Ko smaller than 106M -t, the second set of sites would not be measurable ( K i s h i m o t o and O n o u e , 1971 ; Mukkur, 1972). T h e present investigation was designed to study the binding properties of the reductive and proteolytic subunits, lgMs and F a b respectively o f a n t i - D N P IgM. In addition, s o m e biological properties of these subunits w e r e also studied by the use of hemagglutination technique. MATERIALS AND METHODS
Purification of bovine colostral anti-DNP IgM Anti-DNP lgM was purified using the same procedure as described previously (Mukkur, 1972). Briefly, IgM was isolated from the colostrum of cows immunized with dinitrophenylated porcine gamma globulin, using a procedure described previously (Mukkur and Froese, 1971). Antibodies from this preparation were removed using a DN P-immunosorbent according to Robbins et al. (1967). The elution of anti-DNP IgM from the latter was performed using 0.05M 2,4-dinitrophenol, pH 8.0 (DNP-OH) which was removed by passage of the eluted antibody through a column (40.0 cm x 2.5 cm) consisting of DEAE-
cellulose in the top half and Dowex (I-XS) in the bottom half, both the ion exchangers having been equilibrated with 0-32 MNaCI-0.01 MTris-HCI, pH 8.0 (0.32 ST). The recovery of anti-DNP IgM was found to be 2-4 rag/100 ml of colostrum. Percent activity of this antibody preparation was judged by immunosorption as described previously (Mukkur, 1972) and was found to be 90 per cent. Preparation of the subunits of anti-DNP IgM (A ) Reductive subunit (igM,). Reduction of lgM was carried out under N~ atmosphere at 37°C using fl-mercaptoethylamine (MEA) at a final concentration of 0-2M in 0.2MTris-HCI buffer (pH 8.0) for 30 min. followed by alkylation using 100 per cent excess of neutralized iodacetic acid. The subunits produced by this reduction sedimented with a S20.w of 7.6S at 3'0 mg per ml and had the same heavy and light chain composition as the native lgM. Bovine anti-DNP Immunoglobulin G isolated according to Froese (1971) was also reduced and alkylated for use as a control in hemagglutination. (B) Proteolytic subunit (Fab). The univalent Fab fragments were produced essentially according to the procedure of Goodman and lnman (1969). Briefly, the purified lgM antibody was digested with 3% papain in 0.1 M phosphate buffer (pH 7.0) containing 0.002M E D T A and 0.002MO-mercaptoethylamine (MEA) for 30 hr under N2 atmosphere at 37°C followed by alkylation with neutralized iodoacetic acid (pH 8.0), The digestion mixture was then dialyzed exhaustively against 0.32 and subjected to gel filtration on Bio-Gel P-150 equilibrated with the same buffer in order to remove papain. At the same time, a portion of Fab preparation was filtered through Bio-Gel P- 150 equilibriated with 5M GuanidineHCI (pH 8.0). Only a single peak was realized indicating that the disulfide bridge between Fd(/z) and the light chain was intact. On Ouchterlony analysis, it was shown to react with anti-bovine light chain anti sera (anti-bovine lgG was used as a source of anti-light chain antibodies) but not with specific anti-Fc0z) antisera. On analytical ultracentrifugation, the sedimentation coefficient was (S20,w) found to be 3.6S at 2. Img per ml. 477
478
T.K.S.
M U K K U R and U S H A J. TEWARI I
.~ole,'ular weights and e.~tinction coefficients E_,x. I'z~
Mentioned below are the tool. wt and the extinction coefficients used in calculations for the following preparations. mol. wt igM
30C
E~,~
1.03 X 1(~ (Mukkur, 1972)
12.6 (Mukkur, 1972)
IgM~
2.00 x 10~ (Mukkur, 1972)
12.6 (Determined)
Fab
6.0x 104 (Assumed to be the same as in human Fab (lgM), Goodman and lnman, 1969)
14.0 Determined)
% 20C
IOC
\. 25
Determination
o f the
average
intrinsic
50
association
t'onsl~lnl ( gql)
The determination of the average intrinsic association constant was carried out according to Stupp et al. (19691 with one modification. Cold Trichloracetic acid to a final concentration of 20% instead of (NH4),SO4 was used to precipitate the antibody-ligand mixture. Tritiated ~-(3H)DNP-L-Lysine (3.22Ci/MM, New England Nuclear) was used as a ligand. The concentrations of anti-DNP lgM. IgM~ and Fab in triplicate experiments were 6 x 10-6 M, 3'5 x 10-"M and 7-13 x 10-a M respectively. q'he association constants and the heterogeneity indices were calculated from the Scatchard plot (Scatchard, 1949) and Sip's plot (Sip's. 1948) respectively. Hernagglutination
The hemagglutination of the intact lgM and the subunits were measured using diazotized red blood cells employing substituted dinitrophenylated human serum albumin (30 DNP groups/mole of HSA) as an antigen, according to the procedure of Gordon et al. (19581 as reported previously (Mukkur. 1972).
Fig. I. Scatchard plot of the binding data obtained by the interaction of Bovine Anti-DNP IgM and ~-DNP-LLysine at 25°C. pH 8.0. two sets of sites (Mukkur, 1972; O n o u e et al., 1968; Oriol et al., 1971; Voss, 19691, the undetectability of one half of the sites could possibly be ascribed to a relatively low average association constant (less than 10a I/mole) of a steric hinderance in the antibody combining site despite cleavage to Fab. U n f o r t u n a t e l y there is no direct e v i d e n c e available in the literature to support the a b o v e assumptions. O n the other hand, A s h m a n and M e t z g e r (19691 have reported the presence of 10 combining sites of uniform al~nity per molecule
\
RESULTS AND DISCUSSION
Analysis of the binding data obtained by the interaction of a n t i - D N P - I g M with e - D N P - L Lysine revealed that the molecule possessed five binding sites as o b s e r v e d previously (Mukkur, 1972) with an average association constant ( K . ) o f 4.8 × 104M-' as calculated from the Scatchard plot (Fig. l). T h e heterogeneity index (a) was calculated to be 0.81 using Sip's plot. T h e binding constants for I g M s and F a b were found to be 4.6 × 104M -1 and 4.3 x 104M -~ respectively (Figs. 2 and 3). T h e heterogeneity indices as calculated from the Sip's plot were the same for both the subunits (c~ = (I.801. T h e n u m b e r of measurable combining sites on IgM.~ was found to be I while the Fab preparation was shown to possess 0.5 sites per molecule. This data thus suggests the possible existence of two sets of sites in the a n t i - D N P - I g M , one of which is not measurable. T h e lack of detectability of half of the total available sites (one set) could be due to several reasons. If one a s s u m e s the existence of
2O
I
O
05
0
Fig. 2. Scatchard plot of the binding data obtained by the interaction of Bovine Anti-DNP lgM~ and ~-DNP-t.l.ysine at 25~C. pH 8.0.
Subunits of Bovine Colostral anti-DNP immunoglobulin M (IgM)
479
Table 1. Hemagglutination titres of lgM and its subunits I
Fraction Igm IgMs (MEA) Fab lgG igG (reduced with MEA)
2.0
~t
Optical density Hemaggluper ml tination titre 2.0 2.0 1.0 2.0 2.0
512 0 0 64 128
cause hemagglutination after having undergone reduction and alkylation.
l0
Acknowledgements-This project was supported by a
National Research Council of Canada grant (1". K. S. Mukkur).
\ 05
Fig. 3. Scatchard plot of the binding data obtained by the interaction of Bovine Anti-DNP Fab and ~-DNP-LLysine at 25°C, pH 8.0. of a myeioma lgM which had a natural binding activity for the nitrophenyl group. T h e y have suggested that the previous observations of the existence of two sets of sites may be due to the heterogeneity of the antibody molecules. Our experiments do not exclude this possibility. A definitive answer to the question of heterogeneity and hence the presence or absence of two sets of sites in the lgM antibody molecules shall have to await the results of binding studies that would have to be done on homogeneous antigen-induced antibody preparations. T h e biological properties of the subunits which were investigated by the use of hemagglutination technique are presented in Table 1. Both the IgMs and Fab did not cause hemagglutination of the DNP-conjugated red blood cells. T h e lack of hemagglutination by IgMs thus points out to its functional univalency. The control a n t i - D N P IgG, on the other hand, did not lose its capability to
REFERENCES Ashman R. F. and Metzger H. (1969) J. biol. Chem. 244, 3405. Frank M. M. and Humphrey J. H. (1968) J. exp. Med. 127, 967. Goodman A. and Inman F. P. (1969) J. lmmun. 102, 1032. Gordon J., Rose B. and Sehon A. H. (1958) J. exp. Med. 108, 27. Kishimoto T. and Onoue K. ( 1971) J. Immun. 106, 341. Lindquist K. and Bauer D. C. ~1966) Immunochemistry 3, 373. Mukkur T. K. S. (1972) lmmunochemistry 9, 1049. Mukkur T. K. S. and Froese A. (1971) ImmunochemistO' 8, 257. Onoue K., Yagi Y., Grossberg A. L. and Pressman D. (1965) lmmunochemistry 2, 40 I. Onoue K., Grossberg A. L. and Pressman D. (1968) Science 162, 574. Oriol R., Binaghi R. and Coltorti E. (1971) J. immun. 104, 932. Robbins J. B., Haimovich J. and Sela M. (1967) Immunochemistry 4, 11.
Scathchard G. (1949) Ann. N. Y. ,4 cad. Sci. 51,660. Schrohenloher R. E. and Barry C. B. (1968) J. Immun. 100, 1006. Sips R. (1948) J. chem. Phys. 16, 490. Stupp Y., Yoshida T. and Paul W. E. (1969) J. Immun. 103, 625. Voss E. W., Warren J. R. and Sigel M. M. (1969) Biochemistry 8, 4866.
Printedin Great Britain
PHYSICO-CHEMICAL A N D BIOLOGICAL PROPERTIES OF THE S U B U N I T S OF BOVINE COLOSTRAL A N T I - D N P I M M U N O G L O B U L I N M (lgM) T. K. S. M U K K U R and U S H A J. T E W A R I Department of Biology, University of Windsor, Windsor, Ontario, Canada (First received 15 Janua~. 1973; in revised fi~rm 14 March 1973) Abstract-Bovine colostral anti-DNP IgM which possessed five measurable combining sites per molecule (K0 = 4.8 x 10~M-~) was reduced with/3-Mercaptoethylamine and alkylated with neutralized iodoacetic acid to form lgMs (Szo.w= 7.6S at 3-0 mg/ml). The lgMs was shown to possess only one combining site per molecule (K0 = 4.6 × 104M-~) and failed to cause hemagglutination of the DNPconjugated red blood cells thus indicating their functional univalency. The Fab subunits produced by papain digestion (Sz0,~ = 3.6S at 2.1 mg/ml) were shown to bind 0-5 mole of hapten/mole (K0 = 4.3 × 10~M- ~)and also did not cause hemagglutination of the DN P-conjugated red blood cells.
INTRODUCTION
Previous studies from this laboratory (Mukkur, 1972) h a v e shown that Bovine colostra] antidinitrophenyl ( D N P ) I m m u n o g l o b u l i n M (IgM) possessed five combining sites per molecule. Similar findings have also been reported from o t h e r laboratories (Frank and H u m p h r e y , 1968: Lindquist and Bauer, 1966; O n o u e et at., 1965; S c h r o h e n l o h e r and Barry, 1968; Voss et al., 1969). L a t e r on it was r e p o r t e d that if one used antibody I g M with an average binding constant (Ko) greater than 10SM -I, it was possible to detect two sets of sites ( O n o u e et al., 1968; K i s h i m o t o and O n o u e , 1971 ; Oriol et al., 1971 ), one half of the sites having a higher affinity constant than the second set. T h i s led to a suggestion that in case of antibody IgM with a Ko smaller than 106M -t, the second set of sites would not be measurable ( K i s h i m o t o and O n o u e , 1971 ; Mukkur, 1972). T h e present investigation was designed to study the binding properties of the reductive and proteolytic subunits, lgMs and F a b respectively o f a n t i - D N P IgM. In addition, s o m e biological properties of these subunits w e r e also studied by the use of hemagglutination technique. MATERIALS AND METHODS
Purification of bovine colostral anti-DNP IgM Anti-DNP lgM was purified using the same procedure as described previously (Mukkur, 1972). Briefly, IgM was isolated from the colostrum of cows immunized with dinitrophenylated porcine gamma globulin, using a procedure described previously (Mukkur and Froese, 1971). Antibodies from this preparation were removed using a DN P-immunosorbent according to Robbins et al. (1967). The elution of anti-DNP IgM from the latter was performed using 0.05M 2,4-dinitrophenol, pH 8.0 (DNP-OH) which was removed by passage of the eluted antibody through a column (40.0 cm x 2.5 cm) consisting of DEAE-
cellulose in the top half and Dowex (I-XS) in the bottom half, both the ion exchangers having been equilibrated with 0-32 MNaCI-0.01 MTris-HCI, pH 8.0 (0.32 ST). The recovery of anti-DNP IgM was found to be 2-4 rag/100 ml of colostrum. Percent activity of this antibody preparation was judged by immunosorption as described previously (Mukkur, 1972) and was found to be 90 per cent. Preparation of the subunits of anti-DNP IgM (A ) Reductive subunit (igM,). Reduction of lgM was carried out under N~ atmosphere at 37°C using fl-mercaptoethylamine (MEA) at a final concentration of 0-2M in 0.2MTris-HCI buffer (pH 8.0) for 30 min. followed by alkylation using 100 per cent excess of neutralized iodacetic acid. The subunits produced by this reduction sedimented with a S20.w of 7.6S at 3'0 mg per ml and had the same heavy and light chain composition as the native lgM. Bovine anti-DNP Immunoglobulin G isolated according to Froese (1971) was also reduced and alkylated for use as a control in hemagglutination. (B) Proteolytic subunit (Fab). The univalent Fab fragments were produced essentially according to the procedure of Goodman and lnman (1969). Briefly, the purified lgM antibody was digested with 3% papain in 0.1 M phosphate buffer (pH 7.0) containing 0.002M E D T A and 0.002MO-mercaptoethylamine (MEA) for 30 hr under N2 atmosphere at 37°C followed by alkylation with neutralized iodoacetic acid (pH 8.0), The digestion mixture was then dialyzed exhaustively against 0.32 and subjected to gel filtration on Bio-Gel P-150 equilibrated with the same buffer in order to remove papain. At the same time, a portion of Fab preparation was filtered through Bio-Gel P- 150 equilibriated with 5M GuanidineHCI (pH 8.0). Only a single peak was realized indicating that the disulfide bridge between Fd(/z) and the light chain was intact. On Ouchterlony analysis, it was shown to react with anti-bovine light chain anti sera (anti-bovine lgG was used as a source of anti-light chain antibodies) but not with specific anti-Fc0z) antisera. On analytical ultracentrifugation, the sedimentation coefficient was (S20,w) found to be 3.6S at 2. Img per ml. 477
478
T.K.S.
M U K K U R and U S H A J. TEWARI I
.~ole,'ular weights and e.~tinction coefficients E_,x. I'z~
Mentioned below are the tool. wt and the extinction coefficients used in calculations for the following preparations. mol. wt igM
30C
E~,~
1.03 X 1(~ (Mukkur, 1972)
12.6 (Mukkur, 1972)
IgM~
2.00 x 10~ (Mukkur, 1972)
12.6 (Determined)
Fab
6.0x 104 (Assumed to be the same as in human Fab (lgM), Goodman and lnman, 1969)
14.0 Determined)
% 20C
IOC
\. 25
Determination
o f the
average
intrinsic
50
association
t'onsl~lnl ( gql)
The determination of the average intrinsic association constant was carried out according to Stupp et al. (19691 with one modification. Cold Trichloracetic acid to a final concentration of 20% instead of (NH4),SO4 was used to precipitate the antibody-ligand mixture. Tritiated ~-(3H)DNP-L-Lysine (3.22Ci/MM, New England Nuclear) was used as a ligand. The concentrations of anti-DNP lgM. IgM~ and Fab in triplicate experiments were 6 x 10-6 M, 3'5 x 10-"M and 7-13 x 10-a M respectively. q'he association constants and the heterogeneity indices were calculated from the Scatchard plot (Scatchard, 1949) and Sip's plot (Sip's. 1948) respectively. Hernagglutination
The hemagglutination of the intact lgM and the subunits were measured using diazotized red blood cells employing substituted dinitrophenylated human serum albumin (30 DNP groups/mole of HSA) as an antigen, according to the procedure of Gordon et al. (19581 as reported previously (Mukkur. 1972).
Fig. I. Scatchard plot of the binding data obtained by the interaction of Bovine Anti-DNP IgM and ~-DNP-LLysine at 25°C. pH 8.0. two sets of sites (Mukkur, 1972; O n o u e et al., 1968; Oriol et al., 1971; Voss, 19691, the undetectability of one half of the sites could possibly be ascribed to a relatively low average association constant (less than 10a I/mole) of a steric hinderance in the antibody combining site despite cleavage to Fab. U n f o r t u n a t e l y there is no direct e v i d e n c e available in the literature to support the a b o v e assumptions. O n the other hand, A s h m a n and M e t z g e r (19691 have reported the presence of 10 combining sites of uniform al~nity per molecule
\
RESULTS AND DISCUSSION
Analysis of the binding data obtained by the interaction of a n t i - D N P - I g M with e - D N P - L Lysine revealed that the molecule possessed five binding sites as o b s e r v e d previously (Mukkur, 1972) with an average association constant ( K . ) o f 4.8 × 104M-' as calculated from the Scatchard plot (Fig. l). T h e heterogeneity index (a) was calculated to be 0.81 using Sip's plot. T h e binding constants for I g M s and F a b were found to be 4.6 × 104M -1 and 4.3 x 104M -~ respectively (Figs. 2 and 3). T h e heterogeneity indices as calculated from the Sip's plot were the same for both the subunits (c~ = (I.801. T h e n u m b e r of measurable combining sites on IgM.~ was found to be I while the Fab preparation was shown to possess 0.5 sites per molecule. This data thus suggests the possible existence of two sets of sites in the a n t i - D N P - I g M , one of which is not measurable. T h e lack of detectability of half of the total available sites (one set) could be due to several reasons. If one a s s u m e s the existence of
2O
I
O
05
0
Fig. 2. Scatchard plot of the binding data obtained by the interaction of Bovine Anti-DNP lgM~ and ~-DNP-t.l.ysine at 25~C. pH 8.0.
Subunits of Bovine Colostral anti-DNP immunoglobulin M (IgM)
479
Table 1. Hemagglutination titres of lgM and its subunits I
Fraction Igm IgMs (MEA) Fab lgG igG (reduced with MEA)
2.0
~t
Optical density Hemaggluper ml tination titre 2.0 2.0 1.0 2.0 2.0
512 0 0 64 128
cause hemagglutination after having undergone reduction and alkylation.
l0
Acknowledgements-This project was supported by a
National Research Council of Canada grant (1". K. S. Mukkur).
\ 05
Fig. 3. Scatchard plot of the binding data obtained by the interaction of Bovine Anti-DNP Fab and ~-DNP-LLysine at 25°C, pH 8.0. of a myeioma lgM which had a natural binding activity for the nitrophenyl group. T h e y have suggested that the previous observations of the existence of two sets of sites may be due to the heterogeneity of the antibody molecules. Our experiments do not exclude this possibility. A definitive answer to the question of heterogeneity and hence the presence or absence of two sets of sites in the lgM antibody molecules shall have to await the results of binding studies that would have to be done on homogeneous antigen-induced antibody preparations. T h e biological properties of the subunits which were investigated by the use of hemagglutination technique are presented in Table 1. Both the IgMs and Fab did not cause hemagglutination of the DNP-conjugated red blood cells. T h e lack of hemagglutination by IgMs thus points out to its functional univalency. The control a n t i - D N P IgG, on the other hand, did not lose its capability to
REFERENCES Ashman R. F. and Metzger H. (1969) J. biol. Chem. 244, 3405. Frank M. M. and Humphrey J. H. (1968) J. exp. Med. 127, 967. Goodman A. and Inman F. P. (1969) J. lmmun. 102, 1032. Gordon J., Rose B. and Sehon A. H. (1958) J. exp. Med. 108, 27. Kishimoto T. and Onoue K. ( 1971) J. Immun. 106, 341. Lindquist K. and Bauer D. C. ~1966) Immunochemistry 3, 373. Mukkur T. K. S. (1972) lmmunochemistry 9, 1049. Mukkur T. K. S. and Froese A. (1971) ImmunochemistO' 8, 257. Onoue K., Yagi Y., Grossberg A. L. and Pressman D. (1965) lmmunochemistry 2, 40 I. Onoue K., Grossberg A. L. and Pressman D. (1968) Science 162, 574. Oriol R., Binaghi R. and Coltorti E. (1971) J. immun. 104, 932. Robbins J. B., Haimovich J. and Sela M. (1967) Immunochemistry 4, 11.
Scathchard G. (1949) Ann. N. Y. ,4 cad. Sci. 51,660. Schrohenloher R. E. and Barry C. B. (1968) J. Immun. 100, 1006. Sips R. (1948) J. chem. Phys. 16, 490. Stupp Y., Yoshida T. and Paul W. E. (1969) J. Immun. 103, 625. Voss E. W., Warren J. R. and Sigel M. M. (1969) Biochemistry 8, 4866.