- Email: [email protected]
Ratios of the Fab fragments I and II from rabbit antibodies and non-specific γ-globulins
BIOCHIMICA ET BIOPHYSICA ACTA
BBA
333
35013
RATIOS OF T H E Fab FRAGMENTS I AND I I FROM R A B B I T A N T I B O D I E S AND NON-SPECIFIC v - G L O B U L I N S K. L. KNIGHT*, M. J. ROELOFS AND F. HAUROWITZ** Department of Chemistry, Indiana University, Bloomington, Ind. (U.S.A,)
(Received July I9th, 1966)
SUMMARY The ratio (R) of fragment I/fragment I I was determined in hapten-specific antibodies directed against Ars- and R4N+, and in non-specificv-globulins of rabbits. The R values showed great individual differences, but were always higher in antiR4N+ than in anti-Ars- antibodies of rabbits injected with the doubly labelled Ars--R4N+-bovine serum albumin antigen. The R values of the non-specific ~-globulin were always greater than unity, indicating predominance of the more acidic fragment I over the less acidic fragment II. The R values showed no relation to the age of the rabbits, to repeated bleeding, nor to the repeated injection of Ars--bovine serum albumin.
INTRODUCTION
PORTER1 found that rabbit antibodies or v-globulins on digestion b y papain were degraded to three fragments which could be separated from each other b y chromatography on CM-cellulose. Fragment Fc (III), the least acidic of the three fragments, crystallized easily and was identical in all rabbit antibodies and v-globulins. The more acidic fragments I and II, as shown later by PALMER et al. ~ are split products of two different types of antibody of the composition (I)2III and (II)2III. SELA et al. 3 succeeded in separating the two types of antibody by means of D E A E Sephadex. Later, SELA AND MOZES4 found that antibodies of the type (I)~III are formed predominantly in response to the administration of basic antigens whereas acidic antigens induce principally the formation of antibodies of the type (II)~III. The present investigation was undertaken to determine the ratio of fragment I to fragment I I in hapten-specific antibodies directed against Ars- and R4N+. We also investigated whether the ratio of I / I I in the non-specific v-globulins depended on: (I) the age of the rabbits, (2) the route of injection, or (3) the length of sensitization. Abbreviations: Ars% p-azophenylarsonate anion; R4N+, p-azophenyl-N-trimethylammonium cation. * Predoctoral fellow of the United States Public Health Service. ** To whom requests for reprints should be directed. Biochim. Biophys. Acta, 133 (1967) 333-337
334
K.L.
KNIGHT,
M. J
R O E L O F S , F. H A U R O W I T Z
EXPERIMENTAL
To determine the ratio of fragments I to I I in the hapten-specific antibodies, New Zealand white rabbits were immunized with either Ars--bovine serum albumin, R4N+-bovine serum albumin or the doubly labelled antigen, Ars--R4N+-bovine serum albumin, as described previouslyS, ~. The hapten-specific antibody from the serum of the animals injected with Ars--bovine serum albumin or R4N+-bovine serum albumin was isolated by precipitation with antigen and acid dissociation 5. The hapten-specific antibody from animals injected with doubly labelled antigen was isolated by i mmunosorbents:. Papain digestion of the antibodies and separation of fragments I and I I was carried out according to PORTER1. Fragment I I I was crystallized out and removed before placing samples on the CM-cellulose column. The eluate was collected in io-ml fractions and the absorbance determined at 28o m#. The ratio of fragment I to fragment I I was determined by: (I) adding up the corrected absorbance values for each peal(, and (2) dividing that value of fragment I by the value for fragment II. The value for fragment I was then divided by that found for fragment I I to give the ratio R (Table I). TABLE
[
RATIOS (R) OF THE CONCENTRATION OF I~RAGMENT I TO FRAGMENT II IN THE HAPTEN-SPECIFIC ANTIBODIES FROM R kBBIT SERA
Serum No.
Haplen group of antigen injected
R values in Anti-Ars-
Anti-R~N +
728 716
R4N + R~N +
-
t.53 2.33
718 (6/63) 718 (7/63) 718 (9/63)
Ars Ars Ars-
0.63 1.25 0.06
-
8 0 3 (7/65) 8 0 3 (12/65) 813 835
Ars--R4N+ Ars--R4N+ Ars -R4N+ Ars -RAN+
0.34 0.59 o.41 o.I 5
2.oo 1.o2 0.59 o.55
Similar analyses were performed on non-specific y-globulin obtained under various conditions from twelve rabbits during a two-month period. Rabbits weighing from 3.25 to 3.75 lb were designated as young, and those weighing from 4-75 to 7.25 lb were designated as old rabbits (Table II). The animals injected intravenously received 2 injections of 18 mg of Ars--bovine serum albumin per week for 3 weeks; they were then reinjected every two weeks with 3o mg of Ars--bovine serum albumin, given in two I5-mg doses, 3 days apart. Animals injected intramuscularly received one injection of 18 mg Ars--bovine serum albumin per week for three weeks. Following this series of injections an additional injection of 15 mg antigen was given every two weeks. The rabbits were bled each week for approx. 2o ml. The 7-globulin fraction from a two-week pool of serum from each rabbit was isolated by Na2SO 4 precipitation 8. The Ars -specific antibody was removed by passing the y-globulin through an Ars-Biochim. Biophys. Acta, 133 (3967) 333 3 3 7
ANTIBODY FRAGMENTS
I
AND
II
335
TABLE II RATIOS
Rabbit
(R) OF
F R A G M E N T I TO F R A G M E N T I1 IN N O N - S P E C I F I C RXI3BIT ~ - G L O B U L I N
Age
Injection route *
iNTo.
R values f r o m samples collected within five two-week periods c~fter the initial injection Periods :
First
Second
Third
Fourth
2.24 ---
Fifth
746 747
Old Old
Uninjected Uninjected
2.17 I.O3
3-94 8.23
7-25 9.35
7 !8 749 75 ° 751 752
Young Young Young Young Young
Intravenous Intravenous Intramuscular Intramuscular Intramuscular
4.84 8.87 1.43 4.31 6.25
6-83 -9.68 6.12 ---
5-99 4.5 ° 4.38 5.88 8.58
3,05
3 -lo
2,I2
6.8 5
2.55 1.99 4.27
17.85 4.31 5.98
753 754 755 756 757
Old O!d Old Old Old
Intravenous Intravenous Intramuscular Intramuscular Intramuscular
5 -8o -50.0 3-45 6.03
5-53 7-oo 14.29 o.oo 2.69
5 .68 3 .12 4.31 3.7 ° 4.05
8-23 5-99 2.66 5.35 6.37
4-84 6.88 lO.19 6.66 3.37
6-42 20.00
* The first injection was given to all animals on the same day.
specific immunosorbent 6 and the remainder of the y-globulin was treated with mercuri-papain. The separation of fragments I and I I was similar to that described above for the hapten-specific antibodies. RESULTS AND DISCUSSION
Fig. I shows a representative separation of fragments I and I I of the Arsand R4N+-specific antibodies from rabbit 803. The R ratios of the fragments I/II
<
°'a~! I
o.,!
0
g
I00
200
Eluate (mr) lqg. ~. F r a c t i o n a t i o n of f r a g m e n t s I and I I of a n t i - A r s - (solid line) and anti-R4N + (broken line) antibodies on CSI-cellulose. The concentration of f r a g m e n t I I was calculated from the absorbances of samples between i i o (or 12o) and 16o ml of the eluate.
Biochim. Biophys, Acta, 133 (1967) 333-337
336
K.L. KNIGHT, M. J. ROELOFS, F. HAUROWITZ
for the two hapten-specific antibodies in this rabbit and in other rabbits are presented in Table I. The table shows a wide range of R values; however, in all experiments on rabbits injected with the doubly labelled Ars -R~N+-bovine serum albumin antigen the ratio R is greater in the anti-R4N+ than in the anti-Ars- antibodies. This result agrees with the results obtained b y SELA et al. 4, who used acidic and basic polyamino acids as antigens and found that antibodies formed in response to acidic antigens were adsorbed to DEAE-Sephadex less firmly than antibodies directed against basic antigens. Our results also agree with the amino acid analyses of KOSHLAND AND ENGLBERGER9 who found a higher content of arginine in antibodies against/5-azophenylarsonate than in those directed against the fl-azophenyl-N-trimethylammonium groups. All three investigations, those of SELA et al. 4, those of KOSI~LAI~DAND ENGLBERGER9, and the present one, demonstrate clearly that the antibodies directed against anionic determinants differ from those directed against a cationic determinant by a higher positive or lower negative charge. It is not yet clear whether this is a rule of general validity since KOSHLAND et al. TM have not been able to detect an analogous change in the amino acid content of antibodies directed against the p-azophenylsulfonate anion. The considerable scatter of the R values recorded in Table I indicates that these ratios depend also on other factors which m a y vary from one animal to another. A similar conclusion can be drawn from the experiments recorded in Table II. We found in the non-specific v-globulins of all of the 12 rabbits, consistently more fragment I than fragment II, regardless of the age of the rabbits, the route of injection of Ars -bovine serum albumin, and the time of sensitization up to two months. Very high values for R were occasionally found in the animals 747 (not injected), 75o and 755 (both injected). Although we did not observe any pathological symptoms, we cannot exclude the possibility that these rabbits suffered from an inflammatory or another pathological process. The Fab fragnlents I and I I consist of the Fd portion of the heavy H chains and the entire L chains. Although our hapten-specific antibodies were serologically pure and free from antibodies directed against the protein carrier, Table I shows that they contain a mixture of fragments I and II. This is in accordance with our earlier finding 7 that the reduced and alkylated H chains of anti-Ars- and anti-R4N + on starch-gel electrophoresis at pH 8.2 migrate cathodically in two wide, overlapping bands. Papain digestion of these m a y yield the Fd portions of the Fab fragments I and II. Our observation that the heavy chains of anti-Ars- migrate faster cathodically than those of anti-R~N + (Figs. 2 and 4 in ref. 7) is in agreement with their higher content of the more basic fragment I I and the lower ratio I / I I in anti-Ars- as compared with anti-R4N + antibodies. ACKNOWLEDGEMENTS The work described in this paper has been supported by grants from the National Institute of Health (GM o1852) and the National Science Foundation (GB 2519), and by contracts of Indiana University with the Office of Naval Research (Nonr 31o4-oo ) and the U.S. Atomic Energy Commission (AT 11-1)209. This work was carried out with the technical assistance of Miss M. C. Tay, which we gratefully acknowledge. Biochim. Biophys. Acta, 133 (3967) 333-337
ANTIBODY FRAGMENTS I AND I I
337
REFERENCES I 2 3 4 5 6 7 8 9 IO
R. R. PORTER, Biochem. J., 73 (1959) 119. ]. L. PALMER, \~r. j. MANDY AND A. NISONOFF, Prom Natl. Acact. Sci. U.S., 48 (1962) 49M. SELA, D. GIVOL AND E. MOZES, Bioehim. Biophys. Acta, 78 (1963) 649. M. SELA AND E. MOZES, Proc. Natl. Acad. Sci. U.S., 55 (1966) 445J. L. GROFF AND F. HAUROWlTZ, Immunochemistry, i (1964) 31. E. F. GOLD, K. L. KNIGHT AND F. HAOROW~TZ, Biochem. Biophys. Res. Commun., 18 (1965) 76. K. L. B:N~GHT, M. A. LOPEZ AND F. HAUROWlTZ, J. Biol. Chem., 24t (1966) 2286. R. A. KEKWICK, Biochem. dr., 34 (194 o) 1248. M. E. KOSHLAND AND F. M. ENGLBERGER, Proc. Natl. Acad. Sci. U.S., 5 ° (1963) 61. M. E. KOSHLAND, V. ENGLBERGER AND I~. SHAPANKA, Federation Proc. Abstr., No. 1122, (1965) 332 .
Biochim. Biophys. Acta, I33 (1967) 333-337
BBA
333
35013
RATIOS OF T H E Fab FRAGMENTS I AND I I FROM R A B B I T A N T I B O D I E S AND NON-SPECIFIC v - G L O B U L I N S K. L. KNIGHT*, M. J. ROELOFS AND F. HAUROWITZ** Department of Chemistry, Indiana University, Bloomington, Ind. (U.S.A,)
(Received July I9th, 1966)
SUMMARY The ratio (R) of fragment I/fragment I I was determined in hapten-specific antibodies directed against Ars- and R4N+, and in non-specificv-globulins of rabbits. The R values showed great individual differences, but were always higher in antiR4N+ than in anti-Ars- antibodies of rabbits injected with the doubly labelled Ars--R4N+-bovine serum albumin antigen. The R values of the non-specific ~-globulin were always greater than unity, indicating predominance of the more acidic fragment I over the less acidic fragment II. The R values showed no relation to the age of the rabbits, to repeated bleeding, nor to the repeated injection of Ars--bovine serum albumin.
INTRODUCTION
PORTER1 found that rabbit antibodies or v-globulins on digestion b y papain were degraded to three fragments which could be separated from each other b y chromatography on CM-cellulose. Fragment Fc (III), the least acidic of the three fragments, crystallized easily and was identical in all rabbit antibodies and v-globulins. The more acidic fragments I and II, as shown later by PALMER et al. ~ are split products of two different types of antibody of the composition (I)2III and (II)2III. SELA et al. 3 succeeded in separating the two types of antibody by means of D E A E Sephadex. Later, SELA AND MOZES4 found that antibodies of the type (I)~III are formed predominantly in response to the administration of basic antigens whereas acidic antigens induce principally the formation of antibodies of the type (II)~III. The present investigation was undertaken to determine the ratio of fragment I to fragment I I in hapten-specific antibodies directed against Ars- and R4N+. We also investigated whether the ratio of I / I I in the non-specific v-globulins depended on: (I) the age of the rabbits, (2) the route of injection, or (3) the length of sensitization. Abbreviations: Ars% p-azophenylarsonate anion; R4N+, p-azophenyl-N-trimethylammonium cation. * Predoctoral fellow of the United States Public Health Service. ** To whom requests for reprints should be directed. Biochim. Biophys. Acta, 133 (1967) 333-337
334
K.L.
KNIGHT,
M. J
R O E L O F S , F. H A U R O W I T Z
EXPERIMENTAL
To determine the ratio of fragments I to I I in the hapten-specific antibodies, New Zealand white rabbits were immunized with either Ars--bovine serum albumin, R4N+-bovine serum albumin or the doubly labelled antigen, Ars--R4N+-bovine serum albumin, as described previouslyS, ~. The hapten-specific antibody from the serum of the animals injected with Ars--bovine serum albumin or R4N+-bovine serum albumin was isolated by precipitation with antigen and acid dissociation 5. The hapten-specific antibody from animals injected with doubly labelled antigen was isolated by i mmunosorbents:. Papain digestion of the antibodies and separation of fragments I and I I was carried out according to PORTER1. Fragment I I I was crystallized out and removed before placing samples on the CM-cellulose column. The eluate was collected in io-ml fractions and the absorbance determined at 28o m#. The ratio of fragment I to fragment I I was determined by: (I) adding up the corrected absorbance values for each peal(, and (2) dividing that value of fragment I by the value for fragment II. The value for fragment I was then divided by that found for fragment I I to give the ratio R (Table I). TABLE
[
RATIOS (R) OF THE CONCENTRATION OF I~RAGMENT I TO FRAGMENT II IN THE HAPTEN-SPECIFIC ANTIBODIES FROM R kBBIT SERA
Serum No.
Haplen group of antigen injected
R values in Anti-Ars-
Anti-R~N +
728 716
R4N + R~N +
-
t.53 2.33
718 (6/63) 718 (7/63) 718 (9/63)
Ars Ars Ars-
0.63 1.25 0.06
-
8 0 3 (7/65) 8 0 3 (12/65) 813 835
Ars--R4N+ Ars--R4N+ Ars -R4N+ Ars -RAN+
0.34 0.59 o.41 o.I 5
2.oo 1.o2 0.59 o.55
Similar analyses were performed on non-specific y-globulin obtained under various conditions from twelve rabbits during a two-month period. Rabbits weighing from 3.25 to 3.75 lb were designated as young, and those weighing from 4-75 to 7.25 lb were designated as old rabbits (Table II). The animals injected intravenously received 2 injections of 18 mg of Ars--bovine serum albumin per week for 3 weeks; they were then reinjected every two weeks with 3o mg of Ars--bovine serum albumin, given in two I5-mg doses, 3 days apart. Animals injected intramuscularly received one injection of 18 mg Ars--bovine serum albumin per week for three weeks. Following this series of injections an additional injection of 15 mg antigen was given every two weeks. The rabbits were bled each week for approx. 2o ml. The 7-globulin fraction from a two-week pool of serum from each rabbit was isolated by Na2SO 4 precipitation 8. The Ars -specific antibody was removed by passing the y-globulin through an Ars-Biochim. Biophys. Acta, 133 (3967) 333 3 3 7
ANTIBODY FRAGMENTS
I
AND
II
335
TABLE II RATIOS
Rabbit
(R) OF
F R A G M E N T I TO F R A G M E N T I1 IN N O N - S P E C I F I C RXI3BIT ~ - G L O B U L I N
Age
Injection route *
iNTo.
R values f r o m samples collected within five two-week periods c~fter the initial injection Periods :
First
Second
Third
Fourth
2.24 ---
Fifth
746 747
Old Old
Uninjected Uninjected
2.17 I.O3
3-94 8.23
7-25 9.35
7 !8 749 75 ° 751 752
Young Young Young Young Young
Intravenous Intravenous Intramuscular Intramuscular Intramuscular
4.84 8.87 1.43 4.31 6.25
6-83 -9.68 6.12 ---
5-99 4.5 ° 4.38 5.88 8.58
3,05
3 -lo
2,I2
6.8 5
2.55 1.99 4.27
17.85 4.31 5.98
753 754 755 756 757
Old O!d Old Old Old
Intravenous Intravenous Intramuscular Intramuscular Intramuscular
5 -8o -50.0 3-45 6.03
5-53 7-oo 14.29 o.oo 2.69
5 .68 3 .12 4.31 3.7 ° 4.05
8-23 5-99 2.66 5.35 6.37
4-84 6.88 lO.19 6.66 3.37
6-42 20.00
* The first injection was given to all animals on the same day.
specific immunosorbent 6 and the remainder of the y-globulin was treated with mercuri-papain. The separation of fragments I and I I was similar to that described above for the hapten-specific antibodies. RESULTS AND DISCUSSION
Fig. I shows a representative separation of fragments I and I I of the Arsand R4N+-specific antibodies from rabbit 803. The R ratios of the fragments I/II
<
°'a~! I
o.,!
0
g
I00
200
Eluate (mr) lqg. ~. F r a c t i o n a t i o n of f r a g m e n t s I and I I of a n t i - A r s - (solid line) and anti-R4N + (broken line) antibodies on CSI-cellulose. The concentration of f r a g m e n t I I was calculated from the absorbances of samples between i i o (or 12o) and 16o ml of the eluate.
Biochim. Biophys, Acta, 133 (1967) 333-337
336
K.L. KNIGHT, M. J. ROELOFS, F. HAUROWITZ
for the two hapten-specific antibodies in this rabbit and in other rabbits are presented in Table I. The table shows a wide range of R values; however, in all experiments on rabbits injected with the doubly labelled Ars -R~N+-bovine serum albumin antigen the ratio R is greater in the anti-R4N+ than in the anti-Ars- antibodies. This result agrees with the results obtained b y SELA et al. 4, who used acidic and basic polyamino acids as antigens and found that antibodies formed in response to acidic antigens were adsorbed to DEAE-Sephadex less firmly than antibodies directed against basic antigens. Our results also agree with the amino acid analyses of KOSHLAND AND ENGLBERGER9 who found a higher content of arginine in antibodies against/5-azophenylarsonate than in those directed against the fl-azophenyl-N-trimethylammonium groups. All three investigations, those of SELA et al. 4, those of KOSI~LAI~DAND ENGLBERGER9, and the present one, demonstrate clearly that the antibodies directed against anionic determinants differ from those directed against a cationic determinant by a higher positive or lower negative charge. It is not yet clear whether this is a rule of general validity since KOSHLAND et al. TM have not been able to detect an analogous change in the amino acid content of antibodies directed against the p-azophenylsulfonate anion. The considerable scatter of the R values recorded in Table I indicates that these ratios depend also on other factors which m a y vary from one animal to another. A similar conclusion can be drawn from the experiments recorded in Table II. We found in the non-specific v-globulins of all of the 12 rabbits, consistently more fragment I than fragment II, regardless of the age of the rabbits, the route of injection of Ars -bovine serum albumin, and the time of sensitization up to two months. Very high values for R were occasionally found in the animals 747 (not injected), 75o and 755 (both injected). Although we did not observe any pathological symptoms, we cannot exclude the possibility that these rabbits suffered from an inflammatory or another pathological process. The Fab fragnlents I and I I consist of the Fd portion of the heavy H chains and the entire L chains. Although our hapten-specific antibodies were serologically pure and free from antibodies directed against the protein carrier, Table I shows that they contain a mixture of fragments I and II. This is in accordance with our earlier finding 7 that the reduced and alkylated H chains of anti-Ars- and anti-R4N + on starch-gel electrophoresis at pH 8.2 migrate cathodically in two wide, overlapping bands. Papain digestion of these m a y yield the Fd portions of the Fab fragments I and II. Our observation that the heavy chains of anti-Ars- migrate faster cathodically than those of anti-R~N + (Figs. 2 and 4 in ref. 7) is in agreement with their higher content of the more basic fragment I I and the lower ratio I / I I in anti-Ars- as compared with anti-R4N + antibodies. ACKNOWLEDGEMENTS The work described in this paper has been supported by grants from the National Institute of Health (GM o1852) and the National Science Foundation (GB 2519), and by contracts of Indiana University with the Office of Naval Research (Nonr 31o4-oo ) and the U.S. Atomic Energy Commission (AT 11-1)209. This work was carried out with the technical assistance of Miss M. C. Tay, which we gratefully acknowledge. Biochim. Biophys. Acta, 133 (3967) 333-337
ANTIBODY FRAGMENTS I AND I I
337
REFERENCES I 2 3 4 5 6 7 8 9 IO
R. R. PORTER, Biochem. J., 73 (1959) 119. ]. L. PALMER, \~r. j. MANDY AND A. NISONOFF, Prom Natl. Acact. Sci. U.S., 48 (1962) 49M. SELA, D. GIVOL AND E. MOZES, Bioehim. Biophys. Acta, 78 (1963) 649. M. SELA AND E. MOZES, Proc. Natl. Acad. Sci. U.S., 55 (1966) 445J. L. GROFF AND F. HAUROWlTZ, Immunochemistry, i (1964) 31. E. F. GOLD, K. L. KNIGHT AND F. HAOROW~TZ, Biochem. Biophys. Res. Commun., 18 (1965) 76. K. L. B:N~GHT, M. A. LOPEZ AND F. HAUROWlTZ, J. Biol. Chem., 24t (1966) 2286. R. A. KEKWICK, Biochem. dr., 34 (194 o) 1248. M. E. KOSHLAND AND F. M. ENGLBERGER, Proc. Natl. Acad. Sci. U.S., 5 ° (1963) 61. M. E. KOSHLAND, V. ENGLBERGER AND I~. SHAPANKA, Federation Proc. Abstr., No. 1122, (1965) 332 .
Biochim. Biophys. Acta, I33 (1967) 333-337