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Inavtivation of trypsin by antibodies of high affinity
Immunochemlstrv, 1974, Vol 11, pp. 4 1 4 5
Pergamon Press
Printed in Great Britain
INACTIVATION OF TRYPSIN BY ANTIBODIES OF HIGH AFFINITY R O B E R T P. E R I C K S O N Division of Experimental Biology, National Institute of Medical Research, Mill Hill, London, N.W.7, England and the Department of Pediatrics, University of California-San Francisco, San Francisco, California 94122, U.S.A.*
(First received 19 June 1973 ; in revised form 17 August 1973) Abstract--Antibodies were raised to unmodified trypsin and to trypsin stabilized by soybean trypsin inhibitor or glutaraldehyde cross-linking. The antibody molecules were studied for affinity by measuring the slope of drop-off in binding as a function of antibody dilution in a Farr test. They were also assayed for inactivation of trypsin activity as assayed with a small mol. wt substrate. A positive correlation of affinity and effectiveness of inactivation was found for antibodies prepared against nonstabilized trypsin (presented in Freund's complete adjuvant). Little or no inhibition was found when the antibodies were raised against conformationally-stabilized trypsin. The results suggest that changes in affinity of antitrypsin antibodies are major determinants of the inhibitory capacity of such antibodies. INTRODUCTION
(2)Low-dose trypsin. 1 mg/ml trypsin in pH 6, 0.05 M Trismaleate was homogenized with Freund's complete adjuvant, 40 #g was given i.m. followed by 100 ,ag i.m. at 7, 21 and 28 days. 10/~g of the trypsin solution was given intravenously (i.v.) at 42 days and rabbits were bled 7 days before and 7 days after the last injection. (3) Trypsin-soybean trypsin inhibitor, 10 mg/mi trypsin and :7 mg/ml soybean trypsin inhibitor in Tris-Ca were homogenized with equal volumes of Freund's complete adjuvant and the rabbit immunized as in (l). (4) glutaraldehyde trypsin. 1 mg/ml trypsin in phosphatebuffered saline (PBS) was added to equal volumes of 0'2~o glutaraldehyde in PBS and left at 4°C for about 20 hr. The resulting (40 per cent of the initial tryptic activity remained) precipitate was washed three times in PBS and adjusted to 2.5 mg (initial weight) per ml in PBS. 5 mg was injected i.m. three times at about 10-day intervals followed by 100 #g i.v. at the lower concentration of 0.5 mg/ml in PBS. The rabbit was bled 10 days before and 10 days after the last injection.
It is a common observation that the inactivating capacity of antibody to enzymes increases during the course of immunization (Cinader, 1967; Shapira and Arnon, 1968; Fuller and Marucci, 1971). It is also a well established observation that antibody to haptens (where affinity may be directly measured) shows an average increase in affinity with time after immunization (Eisen, 1966; Siskind and Benacerraf, 1969) although a later decrease in affinity was found in one study of anti-protein antibodies (Urbain et al., 1972). The studies on relative affinity and inhibitory capacity of anti-trypsin antibodies reported in this paper suggest that the increase in affinity is responsible for the increased inhibitory capacity. MATERIALS AND METHODS
Chemicals Twice crystallized, dialyzed and lyophilized bovine trypsin; Type 1-S soybean trypsin inhibitor; and ~-N-benzoyloL-arginine-p-nitroanilide hydrochloride were purchased from Sigma Chemical Company, St. Louis, Missouri, U.S.A. Glutaraldehyde (25~o solution in water) was purchased from J. T. Baker Chem. Co., Phillipsburg, N.J. Freund's complete adjuvant was purchased from Difco Laboratories, Detroit, Michigan.
Partial purification of antisera The 0-40~o saturation, ammonium sulfate precipitate of the individual bleeds of antisera was dissolved and dialyzed against Trts-Ca. Following this, the 0-33~o saturatton ammonium sulfate precipitate of this was taken and handled in the same way. The resultant solution was adjusted to the original volume of sera. Trypsin inhibition determinations Tryptic activity was assayed on benzoyl-oL-arginine-p-nitroanilide (BANA) in Tris-Ca according to Erlanger et al. (1961). Increasing volumes of the purified antisera were added to 20 #g trypsin in a constant volume and the residual activity measured. The results are expressed relative to the degree of inhibition found with the similarly purified, preimmunization sera obtained from each rabbit.
Immunizations (1) High-dose trypsin. 10 mg/ml trypsin in pH 8, 0'05 M Tris-HC1, 0.01 M CaC12 (Tris-Ca) was homogenized with equal volumes of Freund's complete adjuvant and 10 mg trypsin equivalent was given to multiple intradermal sites followed by 10 nag intramuscularly (i.m.); twice at 20-day intervals. The rabbit was bled at 10-day intervals starting after the last injection.
Affinity and equivalence point measurements Trypsin was radio-iodinated with ~25I by the chloramine T method (Freeman, 1967). The percentage of antigen preci-
* Current address. 41
42
ROBERT P. ERICKSON
pltated with serial dilutmns of purified antibody was measured by the Farr test (Minden and Farr, 1967). RESULTS
The fractionation of the antisera before inhibition testing was required to partially separate immunoglobulins from proteolytic activity and trypsin inhibitors (e.g. ~-anti-trypsin) normally found in serum. The persistence of small amounts of these materials and the substrate competition (of the added protein for BANA) required control inhibition curves with pre-immunization sera. Fractionated normal sera inhibited tryptic hydrolysis of BANA less than 20 per cent at a 40-fold ratio of proteins and, based on these normal sera controls, inhibition curves such as those found in Fig. 1 were obtained. Here a significantly more rapid inhibition is found for the later sera (from a series of bleeds) 100q
8(] % OF INHIBITION
6(]
BY CONTROL
o 4¢
2c
0 " ' ~ ,,,.,,. II I 51015 25
,oso
,,
0
,oo
/~1 PURIFIED ANTI-TRYPSIN
Fig. 1. Inhibition of trypsin by purified anti-trypsin antibodies expressed as the percent of inhibition by purified preimmunization sera: --O--- high dose anti-trypsin, third bleed, 79.2 pg 125I-trypsin/ml is the 33 per cent antigenbinding capacity; - - ~ - - high-dose anti-trypsin, fourth bleed, ABC-33 = 42.2 #g 12sI-trypsin/ml. 10G O 8C % OF ANTIGEN PRECIPITATED
6C 4(
LOG 2 OF RECIPROCAL OF DILUTION
Fig. 2. Binding of 125I-trypsin by two, representative purified antisera in the Farr test. 0.25 ml of the indicated dilution of antisera and 0.1 ,ug of 125i_trypsin were incubated for 30 min at 20°C and then quickly brought to 50~o saturation with saturated ammonium sulfate. The reaction was then incubated for 18 hr at 4°C and spun at 1400 g for 30 min. Supernatants of these and 5~ trichloracetic acid controls were carefully transferred and counted: - - A - - anti-trypsin soybean trypsin inhibitor, fourth bleed ABC-33 at 0.1 pg -0.53 gg 125I-trypsin bound/ml serum; slope -- - 0 . 2 7 ; - - O - low dose anti-trypsin, first bleed ABC-33 at 0.1 #g = 3.2/2g 25I-trypsin bound/ml sera, slope = - 0.19.
even though there is only about half as much antibody per ml (as defined by the 33 per cent antigen-binding capacity). The amount and relative affinities of each of the partially purified antisera was determined by the Farr test utilizing 0'1/~g of 1-~sI-trypsin in the reaction mixture. As seen in Fig. 2, the data provided the antigen-binding capacity (ABC) which is the calculated amount of antigen bound per ml of undiluted sera. (ABC-33 indicates the point where 33 per cent of antigen is precipitated; end-points with more than 33 per cent precipitation are not generally used because of spontaneous precipitation.) The data also provided the relative affinity, as determined by the slope of drop-off in binding with successive dilutions in serum. A slope of - 1 would represent a 50 per cent decrease in binding with a 50 per cent decrease in sera concentration, i.e. an affinity so great that there is no free antibody and the binding would drop-off with antibody dilution. A lower negative slope represents a lower affinity with free antibody present, then as dilution occurs the higher antigen/antibody ratio results in more binding. The percentage inhibition of trypsin by the individual antisera at the ratio of antibody to trypsin of the ABC-33 was determined. The use of this relatively low ratio of antibody/trypsin was required in order to use the Farr test data as an antibody-counting determination. For the higher avidity antibodies, the ABC-33 would approach 1 divalent antibody molecule/molecule trypsin while, at the 200-fold higher concentration of trypsin in the enzyme inhibition test, tri-molecular reactions (2 trypsin:l antibody) would be expected. These inhibition points could then be related to affinities as in Fig. 3. Here the relative correlation in affinity and inhibitory capacity for anti-trypsin antibodies is shown during the course of immunization. However, when the animals were immunized with the soybean trypsin inhibitor complex which has an association constant of about 1 0 - 1 ° M , although moderate high affinities develop, there is no inhibition of the~tryptic activity for the small tool. wt substrate (BANAl. The effect of soybean trypsin inhibitor on the lack of inhibition of anti-trypsin antibodies might be due to blockage of haptenic sites (sites for which antibody would be made late in the course of immunization) which have to be exposed for antibody inhibition ('anti-active site inhibition') or to a stabilization of trypsin's conformation during the immunization process so that even high affinity antibody is not inactivating. Immunizations with glutaraldehyde trypsin (another way of stabilizing enzyme conformation during immunization) and attempts to raise very high affinity antibodies (low-dose immunizations) were performed to further explore the alternatives. As seen in Fig. 4, there was a good correlation of the inhibiting capacity of antibodies to untreated trypsin with their affinity (when judged by the slope of drop-off in the Farr test; r = 0"73, P < 0"05). On the other hand, antibodies raised against glutaraldehyde cross-linked trypsin are similar to those to trypsin-soybean trypsin in-
Affinity and Trypsin Inactivation
43 --I .0
I00
80 % INHIBITION
SLOPE OF
60 --0.5
AT EQUIVALENCE
40 2(
g.,.....o.~ ~ 10m9 ~" COMPL AOJ
I
lO
lorae
f
coMPt ADJ
I
2o
I
3o
DROP-OFF IN FARR TEST
~,,
t lore e7 COMPL I 40
ADJ
b---x--50 60
x___~ 70 80
DAYS
Fig. 3. Development of inhibitory capacity and affimty m anti-trypsin antibodies during the course of immunization; arrows indicate the time of injection of 10 mg of trypsin in Freund's complete adjuvant: circles: anti-trypsin immunization; triangles; anti-trypsin-soybean trypsin inhibitor complex immunization: --(3-- inhibition of trypsin by anti-trypsin antibodies; - - A - - inhibition of trypsin by antitrypsin-soybean trypsin inhibitor complex antibodxes; - - O - - affinity of anti-trypsin antibodies; - - A - affinity of anti-trypsin-soybean trypsin inhibitor complex antibodies.
,oo
8(3 /~
•
% INHIBITION
6C
AT EQUIVALENCE
4C
20 /
n I
0.2
0.4
0.6
0.8
AFFINITY (SLOPE OF DROP-OFF IN FARR TEST x --1)
Fig. 4. Relationship of inhibition of trypsin to affinity of anti-trypsin antibodies for antibodies raised against untreated trypsin (circles) compared to anti-trypsin antibodies to conformationally-stabilized trypsin (squares): • highdose anti-trypsin antibodies; O low-dose anti-trypsin; • antibodies to trypsin-soybean trypsin inhibitor; [] antibodies to glutaraldehyde cross-linked trypsin, r = 0.73, P < 0'05. hibitor complex in that they do not inactivate the enzymatic activity even when of relatively high affinity. Ouchterlony double diffusion analysis was performed to look for changing specificities. Double diffusion of the antisera with trypsin showed a single arc of precipitation with no spur of late anti-nonstabilized trypsin over early anti-nonstabilized trypsin or of late anti-nonstabilized trypsin over anti-trypsin-soybean trypsin inhibitor. DISCUSSION
It is always possible that the correlation of avidity and efficacy of inactivation of the anti-trypsin antibodies was fortuitous. A lack of significant inhibition despite similar avidities when conformationally-stabi-
lized trypsin was the antigen argues that the correlation is not fortuitous. What is the evidence that the soybean trypsin inhibitor-reacted trypsin and glutaraldehyde cross-linked trypsin are conformationally stabilized? The trypsin-soybean trypsin inhibitor complex is formed with the cleavage of a peptide bond in the inhibitor and spectral measurements (circular dichroic and fluorescent) suggest little change in conformation (Ishida et al., 1970). The glutaraldehyde crosslinked trypsin retained 40 per cent of its specific activity which suggests probably full catalytic activity for substrate accessible sites--many having become inaccessible because of the aggregated condition seen visibly as a coarse precipitate. Similarly, Quiocho and Richards (1964) used glutaraldehyde-stabilized ribonuclease for X-ray crystallographic studies. It is clear that the activity of many enzymes on macromolecular substrates is readily inhibited by the steric interference of antibodies to the enzyme (Cinader, 1967). For a single (divalent) antibody molecule and a single enzyme molecule, this would mean that the antibody-enzyme reaction has a higher affinity constant than does the substrate enzyme reaction. However, lattice formation with many antibody and enzyme molecules creates a substrate impermeable complex of high stability (if not a precipitate, which will remove the enzyme from the aqueous phase). For many years, most of the data on the inhibition by antibodies of enzyme reactions involving small mol. wt substrates was also interpreted in terms of steric interference by antibody directed at 'active-site-haptens' (Cinader, 1967), e.g. portions of the enzyme molecule so close to the active site that the combining antibody would prevent substrate approach or product release. A variety of observations have indicated that the effect of the antibody molecules on the conformation of the enzyme may be more important: (1) Mutant enzymes could be activated by antibodies directed against the native enzyme. Penicillinases were the first example of this (Pollock et al., 1966); bacterial fl-galactosidase (Rotman and Celada, 1968) and mammalian catalase
44
ROBERT P. ERICKSON
(Feinstein et al., 1971) are more recently studied examples; (2) The enzymatic activity of some enzymes can be increased by some antibodies to the enzymes. Ribonuclease is the best studied enzyme in this regard; originally one activating sera was found (Cinader, 1967) then 5 of 16 antisera tested were found to have activating components (Suzuki et al., 1969), these components seem to be ones with higher affinity for ribonuclease than are the inhibiting components (Pelichova et al., 1970) and the activating effect also occurs with a two polypeptide-chain derivative of ribonuclease (Cinader et al., 1971). Amylase (Okada et al., 1963), L-amino acid oxidase (Zimmerman et al., 1971) and phospho-fructokinase (Donnicke et al., 1972) are further examples of antibody-activated enzymes; (3) Many enzymes may be protected from antibody-inactivation by prior addition of substrate or by addition of coenzyme. Cinader (1967) has reviewed most of the examples of this. In all these examples, the stabilizing effect of substrate or cofactors is superseded by antibodies with affinities greater than those between the enzyme and effectors. These three groups of observations are most easily explained by a model recognizing the plurality of conformations for enzymes in solution. The equilibria between such states has been studied in the case of staphylococcal nuclease by preparing antibodies to fragment 99-149 of nativ~ nuclease (isolated with the insoluble fragment) and to fragment 99-126 which is in a random configuration (Sachs et al., 1972). Sometimes antibodies can shift the equilibria between states, Antiapomyglobin precipitates metamyoglobin, releasing the haem (Crumpton, 1966) while new haptens are exposed on ferritin when it interacts with antibody (data in Henney and Stanworth, 1966). A well studied system for such equilibria shifts utilized polymers of Try-Ala-Glu with and without an inserted hapten (Conway-Jacobs et al., 1970; Schechter et al., 1971), Celada and Strom (1972) have reviewed the topic. Since solubilization of an enzyme in Freund's adjuvant or the phase transition as it is released from this depot, proteolytic digestion during antigen processing, or other aspects of the immunization process may result in conformation alterations in the antigen, antibodies may result that are directed against a less active (partially unfolded) configuration. Such antibodies should be inhibitory if their affinity constants are sufficiently large. The present work presents evidence that antibodies to trypsin are more inhibitory when of higher affinity except when attempts were made to maintain the native conformation during immunization. A variety of methods have been used to measure the affinity of anti-protein antibodies. The rate of association and dissociation of I*-bovine serum albumin (BSA) antibody complexes was initially used to study affinity with this antigen (Talmage, 1960; Grey, 1964). The ratio of antibody binding capacity at two different concentrations of antigen has also been used with I*BSA (Farr 1958; Minden and Farr, 1967); this is essentially the method used here. The 'dilutional' measure-
ment has been confirmed with fluorescein labelled BSA (Hudson, 1968) and anti-IgG co-precipitation (Steward and Petty, 1972) and made more sensitive by plotting the log of the fraction of antibody bound against the log of the antibody/antigen ratio for a series of concentrations of antigen (Celada et al., 1969). Urbain et al. (1972) in the work cited earlier have used the competition of the experimental 13XI-labelled antibody with a standard l~5I-labelled antibody to measure the affinity of anti-tobacco mosaic virus antibodies. These methods are all affected by antibody heterogeneity; the antibody dilutional method (measuring the slope of drop-offin binding)proved to be sufficiently reproducible to provide good data for comparing the antibodies made in this study. Trypsin has been a popular enzyme for immunochemical research. Of the many studies (Arnon and Schechter, 1966; Arnon and Neurath, 1969, 1970; Pfleiderer et al., 1970), few have studied the inactivation of trypsin by its specific antibody. Arnon and Schechter (1966) concluded that steric factors were most important because the action of trypsin on larger substrates was much more effectively inhibited. However, the role of affinity was not pursued. There are many observations in the literature on the inactivation of proteolytic enzymes by their specific antibodies ' which might be best explained by the high-affinity hypothesis. Trop et al. (1972) found that specific antibody can displace the insoluble egg white trypsin inhibitor derivative from pronase trypsin. This was interpreted in terms of a steric interaction but could well represent conformational alteration of the enzyme by high affinity antibody. Similarly, the inactivation of papain by antibodies adsorbed by chymopapain (Arnon and Shapira, 1967) might well reflect selection of antibodies with the highest affinity rather than selection of ones directed at a particular hapten present on the cross-reacting antigens which is of special significance for inactivation. Acknowledgements--A portion of this work was done during the tenure of N.I.H. Special Fellowship IF03HD 43252. I thank Dr. N. A. Mitchison and the Division of Experimental Biology for their warm hospitality and Ms. Janet Cowan for her skillful technical assistance. A portion of the work was supported by the Population Council, New York. REFERENCES
Arnon R. and Neurath H. (1969) Proc. natn. Acad. Sci. U.S.A. 64, 1323. Arnon R. and Neurath H. (1970) lmmunochemistry 7, 241. Arnon R. and Schechter B. (1966) Immunochemistry 3, 451. Arnon R. and Shapira E. (1967)Biochemistry 6, 3942. Celada F. and Strom R. (1972) Q. Rev. Biophys. 5, 395. Celada F., Schmidt D. and Strom R. (1969) Immunology 17, 189. Cinader B. (1967) Antibodies to Biologically Active Molecules (Edited by Cinader B.), p. 85. Pergamon Press, Oxford. Cinader B., Suzuki T. and Pelichova T. (1971) J. Immun. 106, 1381.
Affinity and Trypsin Inactivation Conway-Jacobs A., Schechter B. and Sela M. (19701 Biochemistry 9, 4870. Crumpton M. J. (1966) Antibo&es to Biologically Active Molecules (Edited by Cinader B.), p. 61. Pergamon Press, Oxford. Donnicke M., Hofer H. W. and Pette D. (1972) FEBS Letters 20, 184. Eisen H. N. (1966) Harvey Lect. 60, 1. Erlanger B. F,, Kokowsky N. and Cohen W. 11961) Archs Btochem. 96, 271. Farr R. S. (1958) J. Infect. Dis. 103, 239. Feinsteln R. N., Jaroslow B. N., Howard J. B. and Faulhaber J. T. (1971) J lmmun. 106, 1316. Freeman T. (1967) Handbook of Experimental Immunology (Edited by Weir D. M.), p. 597. Blackwell, Oxford. Fuller T. C. and Maruccl A. (1971) J. lmmun. 106, 110. Grey H. M. (1964) Immunology 7, 82. Henney C. S. and Stanworth D. R. (1966) Nature, Lond. 210, 1071. Hudson B. W. (1968) Immunochemistry 5, 87. Ishida M., Hamaguchi K. and Ikenaka T. (1970) J. Biochem 67, 363. Minden P. and Farr R. S. (1967) Handbook of Expertmental Immunology (Edited by Weir D. M.), p. 463. Blackwell, Oxford. Okada Y., Ikenaka T., Yagura T. and Yamamura Y. (1963) J. Biochem. (Tokyo) 54, 101. Pelichova H., Suzuki T. and Cinader B. (1970) J. lmmun. 104, 195.
45
Ptteiderer G., Linke R. and Reinhardt A. (1970) Comp. Biochem. Phystol. 33, 955. Pollock M. R., Fleming J. and Petrie S. (1967) Anttbodies to Biologically Actwe Molecules (Edited by Cmader B.), p. 139. Pergamon Press, Oxford. Quiocho F. A. and Richards F. M. (1964) Proc. hath. Acad. : Sci. U.S.A. 52, 833. ;Rotman M. B. and Celada F. (1968) Proc. natn. Acad. Sci. U.S.A. 60, 660. Sachs D. H., Schechter A. N., Eastlake A. and Anfinsen C. B. (1972) Proc natn. Acad. Scl. U.S.A. 69, 3790. Schechter B., Conway-Jacobs A. and Sela M. (1971) Eur. J. Bioehem. 20, 321. Shapira E. and Arnon R. (1968) Immunochemtstry 5, 501. Siskind, G. W. and Benacerraf B. (1969) Adv. hnmunol. 10, 1. Steward M. W. and Petty R. E. Immunology 22, 747, Suzuki T., Pelichova H, and Cinader B. (1969) J. Immun. 103, 1366. Talmage D. W, (1960) J. Infect. Dis. 107, 115. Trop M., Pinsky A. and Avtalion R. (1972) Immunology 22, 531. Urbain J., Van Acker A., De Vos-Cloetens C. and UrbainVansanten G. (1972) hnmunochemistry 9, 121. Zlmmerman S. E., Brown R K., Curtl B. and Massley V. (1971) Biochim. biophys. Acta 229, 260.
Pergamon Press
Printed in Great Britain
INACTIVATION OF TRYPSIN BY ANTIBODIES OF HIGH AFFINITY R O B E R T P. E R I C K S O N Division of Experimental Biology, National Institute of Medical Research, Mill Hill, London, N.W.7, England and the Department of Pediatrics, University of California-San Francisco, San Francisco, California 94122, U.S.A.*
(First received 19 June 1973 ; in revised form 17 August 1973) Abstract--Antibodies were raised to unmodified trypsin and to trypsin stabilized by soybean trypsin inhibitor or glutaraldehyde cross-linking. The antibody molecules were studied for affinity by measuring the slope of drop-off in binding as a function of antibody dilution in a Farr test. They were also assayed for inactivation of trypsin activity as assayed with a small mol. wt substrate. A positive correlation of affinity and effectiveness of inactivation was found for antibodies prepared against nonstabilized trypsin (presented in Freund's complete adjuvant). Little or no inhibition was found when the antibodies were raised against conformationally-stabilized trypsin. The results suggest that changes in affinity of antitrypsin antibodies are major determinants of the inhibitory capacity of such antibodies. INTRODUCTION
(2)Low-dose trypsin. 1 mg/ml trypsin in pH 6, 0.05 M Trismaleate was homogenized with Freund's complete adjuvant, 40 #g was given i.m. followed by 100 ,ag i.m. at 7, 21 and 28 days. 10/~g of the trypsin solution was given intravenously (i.v.) at 42 days and rabbits were bled 7 days before and 7 days after the last injection. (3) Trypsin-soybean trypsin inhibitor, 10 mg/mi trypsin and :7 mg/ml soybean trypsin inhibitor in Tris-Ca were homogenized with equal volumes of Freund's complete adjuvant and the rabbit immunized as in (l). (4) glutaraldehyde trypsin. 1 mg/ml trypsin in phosphatebuffered saline (PBS) was added to equal volumes of 0'2~o glutaraldehyde in PBS and left at 4°C for about 20 hr. The resulting (40 per cent of the initial tryptic activity remained) precipitate was washed three times in PBS and adjusted to 2.5 mg (initial weight) per ml in PBS. 5 mg was injected i.m. three times at about 10-day intervals followed by 100 #g i.v. at the lower concentration of 0.5 mg/ml in PBS. The rabbit was bled 10 days before and 10 days after the last injection.
It is a common observation that the inactivating capacity of antibody to enzymes increases during the course of immunization (Cinader, 1967; Shapira and Arnon, 1968; Fuller and Marucci, 1971). It is also a well established observation that antibody to haptens (where affinity may be directly measured) shows an average increase in affinity with time after immunization (Eisen, 1966; Siskind and Benacerraf, 1969) although a later decrease in affinity was found in one study of anti-protein antibodies (Urbain et al., 1972). The studies on relative affinity and inhibitory capacity of anti-trypsin antibodies reported in this paper suggest that the increase in affinity is responsible for the increased inhibitory capacity. MATERIALS AND METHODS
Chemicals Twice crystallized, dialyzed and lyophilized bovine trypsin; Type 1-S soybean trypsin inhibitor; and ~-N-benzoyloL-arginine-p-nitroanilide hydrochloride were purchased from Sigma Chemical Company, St. Louis, Missouri, U.S.A. Glutaraldehyde (25~o solution in water) was purchased from J. T. Baker Chem. Co., Phillipsburg, N.J. Freund's complete adjuvant was purchased from Difco Laboratories, Detroit, Michigan.
Partial purification of antisera The 0-40~o saturation, ammonium sulfate precipitate of the individual bleeds of antisera was dissolved and dialyzed against Trts-Ca. Following this, the 0-33~o saturatton ammonium sulfate precipitate of this was taken and handled in the same way. The resultant solution was adjusted to the original volume of sera. Trypsin inhibition determinations Tryptic activity was assayed on benzoyl-oL-arginine-p-nitroanilide (BANA) in Tris-Ca according to Erlanger et al. (1961). Increasing volumes of the purified antisera were added to 20 #g trypsin in a constant volume and the residual activity measured. The results are expressed relative to the degree of inhibition found with the similarly purified, preimmunization sera obtained from each rabbit.
Immunizations (1) High-dose trypsin. 10 mg/ml trypsin in pH 8, 0'05 M Tris-HC1, 0.01 M CaC12 (Tris-Ca) was homogenized with equal volumes of Freund's complete adjuvant and 10 mg trypsin equivalent was given to multiple intradermal sites followed by 10 nag intramuscularly (i.m.); twice at 20-day intervals. The rabbit was bled at 10-day intervals starting after the last injection.
Affinity and equivalence point measurements Trypsin was radio-iodinated with ~25I by the chloramine T method (Freeman, 1967). The percentage of antigen preci-
* Current address. 41
42
ROBERT P. ERICKSON
pltated with serial dilutmns of purified antibody was measured by the Farr test (Minden and Farr, 1967). RESULTS
The fractionation of the antisera before inhibition testing was required to partially separate immunoglobulins from proteolytic activity and trypsin inhibitors (e.g. ~-anti-trypsin) normally found in serum. The persistence of small amounts of these materials and the substrate competition (of the added protein for BANA) required control inhibition curves with pre-immunization sera. Fractionated normal sera inhibited tryptic hydrolysis of BANA less than 20 per cent at a 40-fold ratio of proteins and, based on these normal sera controls, inhibition curves such as those found in Fig. 1 were obtained. Here a significantly more rapid inhibition is found for the later sera (from a series of bleeds) 100q
8(] % OF INHIBITION
6(]
BY CONTROL
o 4¢
2c
0 " ' ~ ,,,.,,. II I 51015 25
,oso
,,
0
,oo
/~1 PURIFIED ANTI-TRYPSIN
Fig. 1. Inhibition of trypsin by purified anti-trypsin antibodies expressed as the percent of inhibition by purified preimmunization sera: --O--- high dose anti-trypsin, third bleed, 79.2 pg 125I-trypsin/ml is the 33 per cent antigenbinding capacity; - - ~ - - high-dose anti-trypsin, fourth bleed, ABC-33 = 42.2 #g 12sI-trypsin/ml. 10G O 8C % OF ANTIGEN PRECIPITATED
6C 4(
LOG 2 OF RECIPROCAL OF DILUTION
Fig. 2. Binding of 125I-trypsin by two, representative purified antisera in the Farr test. 0.25 ml of the indicated dilution of antisera and 0.1 ,ug of 125i_trypsin were incubated for 30 min at 20°C and then quickly brought to 50~o saturation with saturated ammonium sulfate. The reaction was then incubated for 18 hr at 4°C and spun at 1400 g for 30 min. Supernatants of these and 5~ trichloracetic acid controls were carefully transferred and counted: - - A - - anti-trypsin soybean trypsin inhibitor, fourth bleed ABC-33 at 0.1 pg -0.53 gg 125I-trypsin bound/ml serum; slope -- - 0 . 2 7 ; - - O - low dose anti-trypsin, first bleed ABC-33 at 0.1 #g = 3.2/2g 25I-trypsin bound/ml sera, slope = - 0.19.
even though there is only about half as much antibody per ml (as defined by the 33 per cent antigen-binding capacity). The amount and relative affinities of each of the partially purified antisera was determined by the Farr test utilizing 0'1/~g of 1-~sI-trypsin in the reaction mixture. As seen in Fig. 2, the data provided the antigen-binding capacity (ABC) which is the calculated amount of antigen bound per ml of undiluted sera. (ABC-33 indicates the point where 33 per cent of antigen is precipitated; end-points with more than 33 per cent precipitation are not generally used because of spontaneous precipitation.) The data also provided the relative affinity, as determined by the slope of drop-off in binding with successive dilutions in serum. A slope of - 1 would represent a 50 per cent decrease in binding with a 50 per cent decrease in sera concentration, i.e. an affinity so great that there is no free antibody and the binding would drop-off with antibody dilution. A lower negative slope represents a lower affinity with free antibody present, then as dilution occurs the higher antigen/antibody ratio results in more binding. The percentage inhibition of trypsin by the individual antisera at the ratio of antibody to trypsin of the ABC-33 was determined. The use of this relatively low ratio of antibody/trypsin was required in order to use the Farr test data as an antibody-counting determination. For the higher avidity antibodies, the ABC-33 would approach 1 divalent antibody molecule/molecule trypsin while, at the 200-fold higher concentration of trypsin in the enzyme inhibition test, tri-molecular reactions (2 trypsin:l antibody) would be expected. These inhibition points could then be related to affinities as in Fig. 3. Here the relative correlation in affinity and inhibitory capacity for anti-trypsin antibodies is shown during the course of immunization. However, when the animals were immunized with the soybean trypsin inhibitor complex which has an association constant of about 1 0 - 1 ° M , although moderate high affinities develop, there is no inhibition of the~tryptic activity for the small tool. wt substrate (BANAl. The effect of soybean trypsin inhibitor on the lack of inhibition of anti-trypsin antibodies might be due to blockage of haptenic sites (sites for which antibody would be made late in the course of immunization) which have to be exposed for antibody inhibition ('anti-active site inhibition') or to a stabilization of trypsin's conformation during the immunization process so that even high affinity antibody is not inactivating. Immunizations with glutaraldehyde trypsin (another way of stabilizing enzyme conformation during immunization) and attempts to raise very high affinity antibodies (low-dose immunizations) were performed to further explore the alternatives. As seen in Fig. 4, there was a good correlation of the inhibiting capacity of antibodies to untreated trypsin with their affinity (when judged by the slope of drop-off in the Farr test; r = 0"73, P < 0"05). On the other hand, antibodies raised against glutaraldehyde cross-linked trypsin are similar to those to trypsin-soybean trypsin in-
Affinity and Trypsin Inactivation
43 --I .0
I00
80 % INHIBITION
SLOPE OF
60 --0.5
AT EQUIVALENCE
40 2(
g.,.....o.~ ~ 10m9 ~" COMPL AOJ
I
lO
lorae
f
coMPt ADJ
I
2o
I
3o
DROP-OFF IN FARR TEST
~,,
t lore e7 COMPL I 40
ADJ
b---x--50 60
x___~ 70 80
DAYS
Fig. 3. Development of inhibitory capacity and affimty m anti-trypsin antibodies during the course of immunization; arrows indicate the time of injection of 10 mg of trypsin in Freund's complete adjuvant: circles: anti-trypsin immunization; triangles; anti-trypsin-soybean trypsin inhibitor complex immunization: --(3-- inhibition of trypsin by anti-trypsin antibodies; - - A - - inhibition of trypsin by antitrypsin-soybean trypsin inhibitor complex antibodxes; - - O - - affinity of anti-trypsin antibodies; - - A - affinity of anti-trypsin-soybean trypsin inhibitor complex antibodies.
,oo
8(3 /~
•
% INHIBITION
6C
AT EQUIVALENCE
4C
20 /
n I
0.2
0.4
0.6
0.8
AFFINITY (SLOPE OF DROP-OFF IN FARR TEST x --1)
Fig. 4. Relationship of inhibition of trypsin to affinity of anti-trypsin antibodies for antibodies raised against untreated trypsin (circles) compared to anti-trypsin antibodies to conformationally-stabilized trypsin (squares): • highdose anti-trypsin antibodies; O low-dose anti-trypsin; • antibodies to trypsin-soybean trypsin inhibitor; [] antibodies to glutaraldehyde cross-linked trypsin, r = 0.73, P < 0'05. hibitor complex in that they do not inactivate the enzymatic activity even when of relatively high affinity. Ouchterlony double diffusion analysis was performed to look for changing specificities. Double diffusion of the antisera with trypsin showed a single arc of precipitation with no spur of late anti-nonstabilized trypsin over early anti-nonstabilized trypsin or of late anti-nonstabilized trypsin over anti-trypsin-soybean trypsin inhibitor. DISCUSSION
It is always possible that the correlation of avidity and efficacy of inactivation of the anti-trypsin antibodies was fortuitous. A lack of significant inhibition despite similar avidities when conformationally-stabi-
lized trypsin was the antigen argues that the correlation is not fortuitous. What is the evidence that the soybean trypsin inhibitor-reacted trypsin and glutaraldehyde cross-linked trypsin are conformationally stabilized? The trypsin-soybean trypsin inhibitor complex is formed with the cleavage of a peptide bond in the inhibitor and spectral measurements (circular dichroic and fluorescent) suggest little change in conformation (Ishida et al., 1970). The glutaraldehyde crosslinked trypsin retained 40 per cent of its specific activity which suggests probably full catalytic activity for substrate accessible sites--many having become inaccessible because of the aggregated condition seen visibly as a coarse precipitate. Similarly, Quiocho and Richards (1964) used glutaraldehyde-stabilized ribonuclease for X-ray crystallographic studies. It is clear that the activity of many enzymes on macromolecular substrates is readily inhibited by the steric interference of antibodies to the enzyme (Cinader, 1967). For a single (divalent) antibody molecule and a single enzyme molecule, this would mean that the antibody-enzyme reaction has a higher affinity constant than does the substrate enzyme reaction. However, lattice formation with many antibody and enzyme molecules creates a substrate impermeable complex of high stability (if not a precipitate, which will remove the enzyme from the aqueous phase). For many years, most of the data on the inhibition by antibodies of enzyme reactions involving small mol. wt substrates was also interpreted in terms of steric interference by antibody directed at 'active-site-haptens' (Cinader, 1967), e.g. portions of the enzyme molecule so close to the active site that the combining antibody would prevent substrate approach or product release. A variety of observations have indicated that the effect of the antibody molecules on the conformation of the enzyme may be more important: (1) Mutant enzymes could be activated by antibodies directed against the native enzyme. Penicillinases were the first example of this (Pollock et al., 1966); bacterial fl-galactosidase (Rotman and Celada, 1968) and mammalian catalase
44
ROBERT P. ERICKSON
(Feinstein et al., 1971) are more recently studied examples; (2) The enzymatic activity of some enzymes can be increased by some antibodies to the enzymes. Ribonuclease is the best studied enzyme in this regard; originally one activating sera was found (Cinader, 1967) then 5 of 16 antisera tested were found to have activating components (Suzuki et al., 1969), these components seem to be ones with higher affinity for ribonuclease than are the inhibiting components (Pelichova et al., 1970) and the activating effect also occurs with a two polypeptide-chain derivative of ribonuclease (Cinader et al., 1971). Amylase (Okada et al., 1963), L-amino acid oxidase (Zimmerman et al., 1971) and phospho-fructokinase (Donnicke et al., 1972) are further examples of antibody-activated enzymes; (3) Many enzymes may be protected from antibody-inactivation by prior addition of substrate or by addition of coenzyme. Cinader (1967) has reviewed most of the examples of this. In all these examples, the stabilizing effect of substrate or cofactors is superseded by antibodies with affinities greater than those between the enzyme and effectors. These three groups of observations are most easily explained by a model recognizing the plurality of conformations for enzymes in solution. The equilibria between such states has been studied in the case of staphylococcal nuclease by preparing antibodies to fragment 99-149 of nativ~ nuclease (isolated with the insoluble fragment) and to fragment 99-126 which is in a random configuration (Sachs et al., 1972). Sometimes antibodies can shift the equilibria between states, Antiapomyglobin precipitates metamyoglobin, releasing the haem (Crumpton, 1966) while new haptens are exposed on ferritin when it interacts with antibody (data in Henney and Stanworth, 1966). A well studied system for such equilibria shifts utilized polymers of Try-Ala-Glu with and without an inserted hapten (Conway-Jacobs et al., 1970; Schechter et al., 1971), Celada and Strom (1972) have reviewed the topic. Since solubilization of an enzyme in Freund's adjuvant or the phase transition as it is released from this depot, proteolytic digestion during antigen processing, or other aspects of the immunization process may result in conformation alterations in the antigen, antibodies may result that are directed against a less active (partially unfolded) configuration. Such antibodies should be inhibitory if their affinity constants are sufficiently large. The present work presents evidence that antibodies to trypsin are more inhibitory when of higher affinity except when attempts were made to maintain the native conformation during immunization. A variety of methods have been used to measure the affinity of anti-protein antibodies. The rate of association and dissociation of I*-bovine serum albumin (BSA) antibody complexes was initially used to study affinity with this antigen (Talmage, 1960; Grey, 1964). The ratio of antibody binding capacity at two different concentrations of antigen has also been used with I*BSA (Farr 1958; Minden and Farr, 1967); this is essentially the method used here. The 'dilutional' measure-
ment has been confirmed with fluorescein labelled BSA (Hudson, 1968) and anti-IgG co-precipitation (Steward and Petty, 1972) and made more sensitive by plotting the log of the fraction of antibody bound against the log of the antibody/antigen ratio for a series of concentrations of antigen (Celada et al., 1969). Urbain et al. (1972) in the work cited earlier have used the competition of the experimental 13XI-labelled antibody with a standard l~5I-labelled antibody to measure the affinity of anti-tobacco mosaic virus antibodies. These methods are all affected by antibody heterogeneity; the antibody dilutional method (measuring the slope of drop-offin binding)proved to be sufficiently reproducible to provide good data for comparing the antibodies made in this study. Trypsin has been a popular enzyme for immunochemical research. Of the many studies (Arnon and Schechter, 1966; Arnon and Neurath, 1969, 1970; Pfleiderer et al., 1970), few have studied the inactivation of trypsin by its specific antibody. Arnon and Schechter (1966) concluded that steric factors were most important because the action of trypsin on larger substrates was much more effectively inhibited. However, the role of affinity was not pursued. There are many observations in the literature on the inactivation of proteolytic enzymes by their specific antibodies ' which might be best explained by the high-affinity hypothesis. Trop et al. (1972) found that specific antibody can displace the insoluble egg white trypsin inhibitor derivative from pronase trypsin. This was interpreted in terms of a steric interaction but could well represent conformational alteration of the enzyme by high affinity antibody. Similarly, the inactivation of papain by antibodies adsorbed by chymopapain (Arnon and Shapira, 1967) might well reflect selection of antibodies with the highest affinity rather than selection of ones directed at a particular hapten present on the cross-reacting antigens which is of special significance for inactivation. Acknowledgements--A portion of this work was done during the tenure of N.I.H. Special Fellowship IF03HD 43252. I thank Dr. N. A. Mitchison and the Division of Experimental Biology for their warm hospitality and Ms. Janet Cowan for her skillful technical assistance. A portion of the work was supported by the Population Council, New York. REFERENCES
Arnon R. and Neurath H. (1969) Proc. natn. Acad. Sci. U.S.A. 64, 1323. Arnon R. and Neurath H. (1970) lmmunochemistry 7, 241. Arnon R. and Schechter B. (1966) Immunochemistry 3, 451. Arnon R. and Shapira E. (1967)Biochemistry 6, 3942. Celada F. and Strom R. (1972) Q. Rev. Biophys. 5, 395. Celada F., Schmidt D. and Strom R. (1969) Immunology 17, 189. Cinader B. (1967) Antibodies to Biologically Active Molecules (Edited by Cinader B.), p. 85. Pergamon Press, Oxford. Cinader B., Suzuki T. and Pelichova T. (1971) J. Immun. 106, 1381.
Affinity and Trypsin Inactivation Conway-Jacobs A., Schechter B. and Sela M. (19701 Biochemistry 9, 4870. Crumpton M. J. (1966) Antibo&es to Biologically Active Molecules (Edited by Cinader B.), p. 61. Pergamon Press, Oxford. Donnicke M., Hofer H. W. and Pette D. (1972) FEBS Letters 20, 184. Eisen H. N. (1966) Harvey Lect. 60, 1. Erlanger B. F,, Kokowsky N. and Cohen W. 11961) Archs Btochem. 96, 271. Farr R. S. (1958) J. Infect. Dis. 103, 239. Feinsteln R. N., Jaroslow B. N., Howard J. B. and Faulhaber J. T. (1971) J lmmun. 106, 1316. Freeman T. (1967) Handbook of Experimental Immunology (Edited by Weir D. M.), p. 597. Blackwell, Oxford. Fuller T. C. and Maruccl A. (1971) J. lmmun. 106, 110. Grey H. M. (1964) Immunology 7, 82. Henney C. S. and Stanworth D. R. (1966) Nature, Lond. 210, 1071. Hudson B. W. (1968) Immunochemistry 5, 87. Ishida M., Hamaguchi K. and Ikenaka T. (1970) J. Biochem 67, 363. Minden P. and Farr R. S. (1967) Handbook of Expertmental Immunology (Edited by Weir D. M.), p. 463. Blackwell, Oxford. Okada Y., Ikenaka T., Yagura T. and Yamamura Y. (1963) J. Biochem. (Tokyo) 54, 101. Pelichova H., Suzuki T. and Cinader B. (1970) J. lmmun. 104, 195.
45
Ptteiderer G., Linke R. and Reinhardt A. (1970) Comp. Biochem. Phystol. 33, 955. Pollock M. R., Fleming J. and Petrie S. (1967) Anttbodies to Biologically Actwe Molecules (Edited by Cmader B.), p. 139. Pergamon Press, Oxford. Quiocho F. A. and Richards F. M. (1964) Proc. hath. Acad. : Sci. U.S.A. 52, 833. ;Rotman M. B. and Celada F. (1968) Proc. natn. Acad. Sci. U.S.A. 60, 660. Sachs D. H., Schechter A. N., Eastlake A. and Anfinsen C. B. (1972) Proc natn. Acad. Scl. U.S.A. 69, 3790. Schechter B., Conway-Jacobs A. and Sela M. (1971) Eur. J. Bioehem. 20, 321. Shapira E. and Arnon R. (1968) Immunochemtstry 5, 501. Siskind, G. W. and Benacerraf B. (1969) Adv. hnmunol. 10, 1. Steward M. W. and Petty R. E. Immunology 22, 747, Suzuki T., Pelichova H, and Cinader B. (1969) J. Immun. 103, 1366. Talmage D. W, (1960) J. Infect. Dis. 107, 115. Trop M., Pinsky A. and Avtalion R. (1972) Immunology 22, 531. Urbain J., Van Acker A., De Vos-Cloetens C. and UrbainVansanten G. (1972) hnmunochemistry 9, 121. Zlmmerman S. E., Brown R K., Curtl B. and Massley V. (1971) Biochim. biophys. Acta 229, 260.