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Serologic reactions of bovine procarboxypeptidase A and carboxypeptidase A
362
BIOCHIMICA ET BIOPHYSICA A(;T.\
BBA 25 606 SEROLOGIC REACTIONS OF BOVINE PROCARBOXYPEPTIDASE A AND CARBOXYPEPTIDASE A H A R R Y N. BEATY*
Department of Biochemistry, Umversity of Washington, Seattle, Wash. (U.S.A .) (Received January 3rd, I966)
SUMMARY
Serologic reactions of bovine procarboxypeptidase A and carboxypeptidase A (peptidyl-L-amino-acid hydrolase, EC 3.4.2.1) are compared through immunological reactions in agar and studies of the inhibition of enzymic reactions by antibody. I. Rabbits immunized with carboxypeptidase Aa or procarboxypeptidase A-S~ form antibodies with physical and immunological properties of 7-S y-globulin. A procedure for the isolation of 7-S y-globulin from the serum of immunized rabbits is described and utilized for the evaluation of the serologic reactions of the antigens and related proteins in a system relatively free of non-specific protein. 2. Some of the antigenic relationships between carboxypeptidase A~, procarboxypeptidase A-S~, procarboxypeptidase A-S 5 and Fraction II of procarboxypeptidase A are demonstrated by immunologic reactions in agar. The effect of homologous and heterologous antibody on the enzymic activity of carboxypeptidase A and partially activated procarboxypeptidase A-S 6 are compared.
INTRODUCTION
Chemical and enzymic properties of bovine carboxypeptidase A (peptidyl-L amino-acid hydrolase, EC 3.4.2.1) and its zymogen procarboxypeptidase A have been studied in this and other laboratories. However, despite the recent trend in immunochemistry to use enzymes as antigens, few studies on the immunologic reactions of carboxypeptidase or procarboxypeptidase have been reported since SMrrH et al. 1 in x952 demonstrated that carboxypeptidase A, injected into rabbits, produced a heterogeneous population of antibodies capable of precipitating the enzyme and noncompetitively inhibiting its enzymic activity against carbobenzoxyglycyl+-tryptophan. Recently, BARRETT 2 a n d LEKRER AND VAN VUNAKISa have reported immunodiffusion and precipitin data derived from the use of these proteins as antigens. CINADER 4, in discussing developments in immunochemistry, has pointed out some of the advantages of studying a system of three components, e.g. antibody, enzyme, and substrate. With the isolation and purification of bovine procarboxypeptidase A by KELLER, COHEN" AND NEURATH 5,6, and the extensive study of the chemical properties and enzymic functions of this zymogen by BRowx and coAbbreviations: ATEE, acetyl-L-tyrosine ethyl ester; HPLA, hippuryl-DL-phenyllactic acid. * Present address: Department of Medicine, King County Hospital, Seattle, Wash., I!.S.A.
Diochim. 13iophys. Acta, 124 (i066) 362 373
363
SEROLOGIC REACTIONS OF CARBOXYPEPTIDASE
workers v-9 and YAMASAKI et al. TM, it is now possible to examine the serologic relationships of procarboxypeptidase A and carboxypeptidase A in such a system. Other zymogens and their enzymes have been studied in this mannern, 12 with the general observation that enzyme and precursor have antigenic determinants in common, presumably related by areas of similarity in tertiary structure. The immunochemistry of procarboxypeptidase A and carboxypeptidase A is of particular interest because of the unique quaternary structure of the zymogen which is characterized by the presence of three subunits. Subunit I has been identified as the zymogen of carboxypeptidase A, and Subunit II as the zymogen of an endopeptidase similar in specificity to chymotrypsin. No biological function has yet been demonstrated for Subunit III (see refs. 7-1o). Activation of procarboxypeptidase A by relatively high concentrations of trypsin at 37 ° proceeds slowly to produce carboxypeptidase A and proteolytic destruction of the remainder of the zymogen molecule. However, activation at o ° by much lower concentrations of trypsin is more suited to immunologic study in a three-component system since it progresses rapidly, yielding only endopeptidase activity without disaggregation or extensive destruction of the zymogen. Most of the investigation into the immunochemistry of enzymes has been done in serum, allowing for non-specific protein-protein interaction which could conceivably lead to difficulties in interpretation of results. In addition, complement fixation by the reaction of carboxypeptidase with its antibody has been demonstrated 3, introducing another variable to be considered in the analysis of data. For these reasons, the present studies were conducted in a system using 7-S 7-globulin isolated from the serum of immunized rabbits. MATERIALS AND METHODS
Procarboxypeptidase A-S s, procarboxypeptidase A-Ss*, carboxypeptidase Aa and purified Fraction II* were prepared as described by YAMASAKI et al. TM, BROWN, YAMASAKI AND N E U R A T H 13 and Cox et al. 14 respectively. Adult, female rabbits were immunized by four weekly subcutaneous injections containing 20 mg of procarboxypeptidase A-S s or carboxypeptidase Aa dissolved in I ml of o.15 M NaC1, o.o15 M Tris-HC1 (pH 7.5). Immediately prior to injection, each antigen solution was emulsified in an equal volume of Freund's adjuvant (Difco). Antiserum was obtained by bleeding the rabbits 7-1o days after the final injection. 7-Globulin was isolated from the antisera by precipitation with (NH4)~SO 4 and chromatography on TEAE-cellulose. TEAE-cellulose was a product of Bio-Rad Corp., with a stated capacity of 0.78 mequiv/g or 0.55 mequiv/g according to the lot used. Trypsin (twice crystallized and salt free) was obtained from Worthington Biochemical Corp., HPLA from Cyclo Chemical Corp. (as the sodium salt), N-carbonaphthoxy-DL-phenylalanine from K and K Laboratories, and ATEE from the California Corp. for Biochemical Research. All other chemicals were reagent grade. fi-Lactoglobulin was obtained from Dr. P. E. WILCOX. It had been isolated and purified as "Lot B " described by DAVIE, NEWMAN AND WILCOX17. Measurements of pH were made with a Radiometer pH meter 22 using a GK 202 C * K i n d l y p r o v i d e d b y J. H. FREISHEIM a n d W. D. BEHNKE Of this l a b o r a t o r y .
Biochim. Biophys. Acta, 124 (1966) 362-373
364
H . N . BEATY
combined glass electrode. During measurements of enzyme activity, temperature was controlled b y means of circulating water with a Haake thermostat. Protein concentrations were determined b y absorbance measurements at 28o m#, using values of E~ ~i~ = 13.5 for rabbit v-globulin (ref. I5), E~ ~,;, - - 19.o for procarboxypeptidase A (ref. 5) and E~ ~i',~= 18.8 for carboxypeptidase A (ref. 16).
Isolation of v-globulin 7-Globulin was isolated from immune sera a n d for control purposes, from the serum of non-immunized rabbits. After centrifugation, the sermn was adjusted to 4 ° % saturation of (NH4)2SO 4 b y the slow addition of the crystalline salt (242 g/1 serum) with constant stirring at room temperature for 2 h. The suspension was then allowed to stand without stirring for i 6 - 2 o h at 4 ° and further purification was carried out at this temperature. The resulting precipitate was removed b y centrifugation, dissolved in 20-40 ml of distilled water and dialyzed 24 h against four changes of 21 of 5 mM K 2 H P O 4 (pH 7.5). Fresh TEAE-cellulose was prepared for each chromat o g r a p h y according to the procedure of PETERSON AND SOBER18. The washed cellulose was packed under a pressure of 5 lb/inch2 into a 3.3 cm ~ 45 em column from a thick slurry in 5 mM K 2 H P O 4 (pH 7.5)- 2-3 1 of the same buffer were flushed t h r o u g h the column at 4 ° for equilibration. The dialyzed protein solution was p u m p e d onto the the column and followed b y a volume of equilibrating buffer sufficient to elute a faint red pigment b a n d which had relatively low absorbance at 280 m/x. At this point, a concave gradient of increasing phosphate concentration from 0.005 M to o.15 M was applied utilizing the c o n e - s p h e r e technique of SOBER AND PETERSON 19. Effluent was collected in I o - I 2 - m l fractions which were screened for absorbance at 28o m/x, yielding a c h r o m a t o g r a m as represented in Fig. IA. The fractions of the major protein peak were pooled as shown, dialyzed 18-24 h against three changes of distilled water, lyophilized, and stored at o ° as a white, fluffy powder. A solution containing IO mg of the protein per ml was dialyzed 24 h against a buffer composed of o.15 M NaC1, O.Ol 5 M Tris-HC1 (pH 7.5). Approx. 5 IO/xl of this solution were subjected to immunoelectrophoresis in 1 % agar using veronal buffer (pH 8.6, I o.I) at 4-5 V/cm for IOO rain. After electrophoresis a central trough cut in the agar was filled with goat serum containing antibodies against rabbit serum (Hyland Laboratories), and the materials allowed to diffuse for 20 h at 22-25 ° in a humid chamber. Unf)recipitated protein was removed b y immersing the nficroscope slide in a 1 % NaC1 solution for 24 h. The salt was removed b y rinsing in two changes of distilled water for an additional 24 h. After drying in air, the precipitin bands were stained with amido black. A solution of anticarboxypeptidase v-globulin containing 9.4 mg/ml of 0.05 M K i H P O 4 (pH 7.47) was subjected to ultracentrifugation in a Spinco Model-E ultracentrifuge.
Immunodiffusion reactions All immunodiffusion reactions were carried out in 1% agar (Difco) gel in o.15 M NaC1, O.Ol 5 M Tris-HC1 (pH 7.5) according to the double diffusion technique of OUGHTERLONY24. One central and six peripheral wells were cut in a I-ram layer of agar on a microscope slide using an L K B gel cutter, die No. 6866A. Each peripheral well was 5 m m from its neighbor and 5 m m from the central well. Approx. io ~,1 of
Biochim. Biophys. Acta, 124 (1966) 302 373
365
SEROLOGIC REACTIONS OF CARBOXYPEPTIDASE
15-20 FM solutions of carboxypeptidase A~, procarboxypeptidase A-S e, procarboxypeptidase A-S 5 and Fraction I I were arranged in the peripheral wells and allowed to diffuse in a humid chamber at 22-25 o against a 200 ~M solution of antibody y-globulin in the central well. After 20 h of diffusion, the slides were washed in 1 % NaC1, rinsed in distilled water, dried in room air and stained with amido black.
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Fig. I. A. C h r o m a t o g r a p h y on T E A E - c e l l u l o s e of dissolved, dialyzed (NH4)2SO 4 p r e c i p i t a t e f r o m r a b b i t a n t i s e r u m . A n t i s e r u m was a d j u s t e d to 4 o % s a t u r a t i o n of (NH4)zSO 4 a n d t h e r e s u l t i n g precipitate redissolved a n d dialyzed a g a n s t 0.005 M K 2 H P O 4 (pH 7.5) ; t h e s a m e buffer used to equilibrate t h e TEAE-cellulose. After t h e p r o t e i n was flushed t h r o u g h t h e c o l u m n , e l u t i n g a s m a l l a m o u n t of protein, a c o n c a v e g r a d i e n t of 0.005 M to o.15 M K 2 H P O 4 (pH 7.5) w a s applied at t h e p o i n t i n d i c a t e d b y t h e arrow. T h e b r a c k e t d e n o t e s t h e fractions pooled for c h a r a c t e r i z a t i o n . B. T h e i m m u n o e l e c t r o p h o r e s i s of r a b b i t v-globulin. A s a m p l e of pooled m a t e r i a l f r o m t h e a b o v e c h r o m a t o g r a m was placed in t h e u p p e r well a n d n o r m a l r a b b i t s e r u m in t h e lower well. A f t e r electrophoresis, goat s e r u m c o n t a i n i n g antibodies to r a b b i t s e r u m was placed in t h e central t r o u g h . T h e single precipitin b a n d f o r m e d w i t h t h e m a t e r i a l f r o m t h e c h r o m a t o g r a p h y c o r r e s p o n d s to t h e v-globulin b a n d of n o r m a l s e r u m . C. S e d i m e n t a t i o n p a t t e r n of isolated v-globulin. A solution of 9.4 m g / m l of p r o t e i n in o.o 5 M K ~ H P O 4 (pH 7.47) after 80 m i n of s e d i m e n t a t i o n a t 59778 r e v . / m i n in t h e Spinco Model-E u l t r a c e n t r i f u g e .
Enzyme characterization reactions were carried out on dried, unstained duplicates of immunodiffusion preparations following the procedure described b y URIEL2°, An agar solution containing the chromogenic substrate N-carbonaphthoxy-DL-phenylaianine in N,N-dimethylformamide and Blue BNS salt (Allied Chemical) was layered Biochim. Biophys. Acta, I24 (1966) 362-373
366
H . N . BEATY
over t h e slide, which was first i n c u b a t e d at 37 °, then washed in 2 o; acetic acid. W h e n active c a r b o x y p e p t i d a s e was present, the precipitin b a n d t o o k on a characteristic color. A s s a y procedures
C a r b o x y p e p t i d a s e a c t i v i t y was m e a s u r e d against the ester s u b s t r a t e H P L A a n d the p r o t e i n s u b s t r a t e fi-lactoglobulin. R o u t i n e analyses were carried out in o . o i M racemic H P L A as described 1)57 BARGETZI et al. 1~ using a R a d i o m e t e r T T T - I a u t o t i t r a t o r a n d Ole Dich recorder. Studies of the d e p e n d e n c e of the inhibition reactions on t h e c o n c e n t r a t i o n of s u b s t r a t e were carried out in more dilute H P L A solutions using t h e s p e c t r o p h o t o m e t r i c t e c h n i q u e of MCCLURE, NEURATH ANY) \~,'ALSH2l. F o r these studies a stock solution of I mM racemic H P L A in 5 mM Tris .HC1, o.I M NaC1 (pH 7-5) was d i l u t e d to the desired s u b s t r a t e c o n c e n t r a t i o n with t h e same buffer. The increase in a b s o r b a n c e at 254 m/, was followed in the P e r k i n - E l m e r Model-35o recording u l t r a v i o l e t s p e c t r o p h o t o m e t e r . I n i t i a l velocities were derived from the expression ~. -
AA eM"t
(i)
where v is the initial v e l o c i t y in rnoles/1.min, AA is the change in absorbance, and eM is t h e m o l a r e x t i n c t i o n coefficient of H P L A at 254 mtz (ref. 2x), a n d t is t i m e in minutes. Assays of a c t i v i t y using fl-lactoglobulin as s u b s t r a t e were b a s e d on the release of n i n h y d r i n - p o s i t i v e m a t e r i a l w i t h time, according to t h e t e c h n i q u e of DAVIE, NEWMAN AND WILCOX17 a n d McCLURE 22. These assays were p e r f o r m e d at 25 ° with solutions c o n t a i n i n g 5 m g / m l of fl-lactoglobulin in o . o i M veronal-HC1 (pH 7.5)- A t a p p r o p r i a t e intervals, aliquots of t h e reaction m i x t u r e were a d d e d to an equal v o l u m e of a solution c o n t a i n i n g 30 % trichloroacetic acid a n d 4 % p h o s p h o t u n g s t i c acid which p r e c i p i t a t e d t h e p r o t e i n a n d s t o p p e d t h e reaction. The acid solutions were centrifuged a n d aliquots of t h e s u p e r n a t a n t were a d d e d to I ml of n i n h y d r i n solution (see ref. 23), boiled 20 min, d i l u t e d to 5 ml a n d the a b s o r b a n e e r e a d at 57 ° m / , . Velocities, e x p r e s s e d in t e r m s of moles leucine released p e r 1 per min, were c a l c u l a t e d from Eqn. I. The eM at 57 ° m/~ for a leucine s t a n d a r d was d e t e r m i n e d to be 2.54" IO ~ 1/mole. cm. The progress of t h e a c t i v a t i o n of t h e e n d o p e p t i d a s e a c t i v i t y of p r o c a r b o x y p e p t i d a s e A-S 6 was followed using t h e ester A T E E as s u b s t r a t e . P r o c a r b o x y p e p t i d a s e was a c t i v a t e d at o ° b y a d d i t i o n of t r y p s i n (weight r a t i o of 500" I). A t a p p r o p r i a t e t i m e intervals, aliquots of t h e reaction m i x t u r e s were a s s a y e d in the p H s t a t with o . o i M solutions of A T E E in I mM Tris-HC1, o.I M KC1, at p H 8.o a n d 25 °. I n i t i a l velocities of t h e e n d o p e p t i d a s e a s s a y were expressed as ~ e q u i v O H - per m g of p l o c a r b o x y p e p t i d a s e p r o t e i n p e r rain. F i r s t - o r d e r r a t e c o n s t a n t s for t h e a c t i v a t i o n process were c a l c u l a t e d from t h e expression. k __ log a / a - - x 2.303 t
(2)
where a is t h e m a x i m u m e n d o p e p t i d a s e per m g of p r o t e i n after t o t a l a c t i v a t i o n a n d x is t h e a m o u n t of e n d o p e p t i d a s e f o r m e d per m g of p r o t e i n at a n y given time. Biochim. Biophys. Acta, 124 (1966) 362 373
SEROLOGIC REACTIONS OF CARBOXYPEPT]DASE
367
RESULTS
Fig. IB represents the immunoelectrophoretic pattern of material isolated from chromatography on TEAE-cellulose, showing a single precipitin band corresponding to the 7-S y-globulin of a normal rabbit serum control. The ultracentrifuge pattern of this material (Fig. IC) reveals a homogeneous peak with an s°20, w of 6.51 S. These y-globulin fractions were shown to contain antibody against the sensitizing antigens, but no estimates of relative potency were made.
Immunodiffusion reactions Fig. 2 represents an example of an immunodiffusion reaction used to demonstrate the serologic relationships of the various proteins under consideration. It can be seen that antibody directed against carboxypeptidase forms a single dense precipitin band with its homologous antigen, and a less dense single band with both procarboxypeptidase A-S 6 and S5. At the concentrations of antigen used in this preparation, this cross-reaction appears to be a reaction of identity. However, at higher concentrations, which produce less distinct bands, there is faint but definite spur formation at the junction of the enzyme-zymogen precipitin bands. Antibody against carboxypeptidase does not cross-react with Fraction II irrespective of the concentration tested.
Fig. 2. Immunodiffusion in agar showing antigenic relationships of c a r b o x y p e p t i d a s e and related proteins. The central well to the left contained a n t i c a r b o x y p e p t i d a s e A (A-CP), and t h e central well to the right contained a n t i p r o c a r b o x y p e p t i d a s e (A-S~). The peripheral wells contained carb o x y p e p t i d a s e A(CP), p r o c a r b o x y p e p t i d a s e A-S 6 ($6), p r o c a r b o x y p e p t i d a s e A-S 5 (S~) and Fraction I[ (II) as shown.
With antiprocarboxypeptidase A-S6 in the central well, the immunodiffusion results are more complicated. There appear to be at least three precipitin bands formed by the reaction between the procarboxypeptidase antigens and homologous antibody, although the two bands nearest the antigen well are almost superimposed, giving the appearance of a single dense band in some preparations. The intercept between the $6 and S5 zymogen forms shows a reaction of identity, but the precipitin bands formed by procarboxypeptidase A-S5 are usually more widely separated and smeared. Antiprocarboxypeptidase forms a single band on cross-reaction with carboxypeptidase and two bands on cross-reaction with Fraction II. Where the carboxypeptidase band fuses with the middle of the three zymogen bands, it exhibits the reaction of multideterminant antigens described by OUCHTERLONY2¢. One of the Fraction II bands shows a reaction of identity with the zymogen band nearest the central well. The Biochim. Biophys. Acta, 124 (1966) 362-373
368
H.N.
BEATY
second Fraction I I band is less distinct, but again demonstrates the relatively nonspecific reaction of multideterminant antigens with the outer zymogen band. One of the Fraction I I bands, probably the latter, m a y be due to cross-reaction of antiprocarboxypeptidase with a small amount of Fraction I I I which is difficult to exclude in the chromatographic purification of Fraction II (ref. 9)As URIEL~° has pointed out, the characterization reactions of e n z y m e - a n t i enzyme precipitates in agar aid in the study of the purity, homogeneity and native state of an enzyme preparation. Carbonaphthoxyphenylalanine seems to be a highly specific substrate for carboxypeptidase and when hydrolyzed in the presence of a diazonium salt yields a red-violet color. This reaction was carried out in duplicates of immunodiffusion preparations of the type shown in Fig. 2 with the exception that they were not stained with amido black. When anticarboxypeptidase was used in the central well, the single precipitin band formed by its reaction with carboxypeptidase took on the characteristic color described above after incubation with the chromogenic substrate. The precipitin bands formed by the cross-reaction of anticarboxypeptidase with the zymogen antigens were not colored. This indicates that cross-reaction is indeed due to antigenic similarity between the enzyme and its zymogen rather than to contamination of the zymogen preparation with small amounts of active enzyme. As expected, in preparations where the antigens were allowed to diffuse against antiprocarboxypeptidase, only the precipitin band resulting from the cross-reaction of this antibody with carboxypeptidase was colored.
Inhibition of enzymic activity All of the inhibition studies to be described were designed to compare either the progress of activation of procarboxypeptidase or the enzymic activity of carboxypeptidase in equimolar concentrations of y-globulin solutions containing homologous or heterologous antibody. The most meaningful control for these reactions was one containing an equal concentration of non-specific y-globulin since the mere presence of protein invariably resulted in rate constants for the activation of procarboxypeptidase which were lower than in saline controls and initial velocities for the hydrolysis of H P L A by carboxypeptidase which were greater than in saline controls. The inhibitory effect of non-specific protein on the activation of procarboxypet~tidase varied from 22 % at low v-globulin concentrations to about 45 % at higher concentrations. The apparent increase in activity of carboxypeptidase in a solution of nonspecific v-globulin averaged about 25 % over a wide range of concentration. It is desirable to study the enzymic activity of a soluble antigen ~antibody complex rather than activity remaining in solution after precipitation of antigen by antibody. For this reason, these studies were done at molar ratios of y-globulin to antigen which resulted in significant inhibition of enzymic activity without development of a demonstrable precipitate during the period of incubation and assay. This was assumed to be in the zone of antigen excess since an increase in antibody concentration resulted in prompt precipitate formation. Carboxvpeptidase A. Since the carboxypeptidase concentrations required to adequately measure the hydrolysis of H P L A and fl-lactoglobulin are so different, separate conditions were established for each assay. Solutions containing anticarEoxypeptidase and carboxypeptidase in a molar ratio of 20 : I, incubated at 25~', exhibited m a x i m u m inhibition of enzymic activity (5° %) within 15 min when subjected to Biochim. 13iophys. Acla, 124 (1966) 362 373
369
SEROLOGIC REACTIONS OF CARBOXYPEPTIDASE
the spectrophotometric assay of HPLA at enzyme concentrations of O.l-O.6 tzM. After 60 rain incubation, there was no visible precipitate, but the enzymic activity of the incubation mixture was found to decrease with centrifugation at 3000 rev./min for 5 min, indicating that the antigen-antibody complexes had formed fairly large aggregates. Therefore, studies of the effect of antibody on the enzymic activity of carboxypeptidase were performed on low concentrations of enzyme in 2o-fold molar excess of antibody solution after 30 min incubation at 25 °. At the higher enzyme concentrations (12-13 ~M), necessary for assays of the hydrolysis of ~-lactoglobulin, a 2o-fold molar excess of antibody solution resulted in visible precipitation within 30 min. When antibody was present in a 4-fold molar excess, incubation for 3 ° min at 25 ° resulted in significant inhibition of enzymic activity without evidence of precipitate formation. Because of the well established inhibition of carboxypeptidase activity by excess HPLA (ref. 2I), it was desirable to examine the relationship of substrate concentration to inhibition by antibody at low HPLA concentrations where a conventional dependence of the velocity of the enzyme reaction upon substrate concentration can be demonstrated. Fig. 3 represents a Lineweaver-Burk plot of data from a typical experiment which compared the rate of hydrolysis at four concentrations of HPLA in the presence of antibody. Both Km and V appear to be altered by homo logous antibody, indicating that the mechanism of inhibition is neither the classical competive nor non-competitive type. Inhibition by heterologous antibody is less marked than with the homologous antibody, but is associated with a clear-cut increase in Km. The effect of substrate size on the inhibition of carboxypeptidase activity by antibody was studied in a separate experiment. A 12-13 FM solution of carboxypeptidase was incubated for 3 ° min at 25 ° in a 4-fold molar excess of v-globulin containing homologous or heterologous antibody. The enzymic activity of the incubation 6,40
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I/Is] (x I0 ~) Fig. 3. L i n e w e a v e r - B u r k plot showing the dependence of antibody inhibition of carboxypeptidase activity on the concentration of substrate (HPLA), The Q points represent inhibition by y-globulin containing antibodies against carboxypeptidase, the • points represent inhibition by yglobulin containing antibody to procarboxypeptidase, and the • points represent the non-specific y-globulin control. The y-globulin:enzyme ratio in the incubation mixture was 2o: I. The enzyme concentration in the assay mixture was 1.64 m/zM.
Biochim. Biophys. Acta, 124 (1966) 362-373
37 °
H.N.
I-H,:.VI'Y
mixture was assayed against fl-lactoglobulin (mol. wt. 35 400) by the ninhvdrin technique previously described and H P L A (mol. wt. 349) according to the titration technique of BARC;ETZI et al. 16. Under these conditions, there was a 35 % reduction in the velocity of the hydrolysis of fi-lactoglobulin in the presence of h(mlologous antibody and a 25 % reduction of heterologous antibody. However, no inhibition of enzymic activity could be detected under these conditions when assayed with H PLA in the autotitrator. In other systems, data of this sort is cited as evidence that steric hindrance by antibody prevents the larger substrate molecules flora reaching the binding or catalytic site of the enzyme molecule °-:'. Procarbo:Evpeptidase A-S~i. Solutions of 0. 5 ffM t)rocarboxypeptidase A-S, in o.15 M NaC1, O.Ol5 M Tris-HCl (t)H 7.5) were incubated with antibody for 3o rain at 25 °, and rapidly cooled to o '~ heft)re activation with trypsin, The progress of activation was followed by assaying aliquots of the aetivated incubation mixture against ATEE at various time intervals after the addition of trypsin. y-Globulin solutions containing antiprocarboxypeptidasc in c(meentrations necessary to achieve molar ratios of 2o: z with this concentration of t)rocarboxyt)et> tidase resulted in almost immediate formation of a heavy precipitate. Consequently, subsequent inhibition studies were performed with molar ratios of 7-gh~bulin to procarboxypeptidase ranging from I : I to 8:~. At the lowest concentrations ()f antibody used, it was consistently found that anticaH)oxypeptidase was a re.re effectiv(~ inhibitor of procarboxypeptidase activation than antiprocarboxypeptidase. As the molar ratio of y-globulin to t)rocarb()xypeptidas~ ' was increased, the inhibitor\' ('apacity of homologous antibody more closely approximated that of heterologous antibody until the ratio of ~;:I was reached (Fig. 4). At that point, the antib(~tlv solutions were equally effective as inhibitors. At greater molar ratios, both h o m . logous and heterologous antibody formed a precipitate with pr()carboxyt)eptidase, although with homologous antibody, the precipitate appeared more promt)tly an(l in greater quantity. From these data, it was not clear whether the decrease in rate ('(instants observed during the activiation of proearl)oxypeptidase in the presence ~f antibody was due to inhibition of the actiwttion process itself or of the endopeptidase formed by activation. ]% resolve this question, procarboxypeptidase solutions were incubated 0.080.070060.05-
0.030.02O.01
Rgtio "~- globulin/procorboxypeptidase
Fig. 4. The effect of a n t i b o d y and non-specific y-globulin on the rate of activation of procarl)oxypeptidase A-S 6. Solutions of" p r o c a r b o x y p e p t i d a s e A-S, (6 ffM) were activated at o ° by addition of small a m o u n t s of trypsin. The first-order rate c o n s t a n t of activation (K1) was determined in increasing molar ratios of non-specific v-globulin, (o), },-globulin containing a n t i p r o c a r b o x y peptidase (O), and y-globulin containing a n t i c a r b o x y p e p t i d a s e (O).
Biockim. Hiopk),s..4cla, l~ 4 (~900) 362-37 ~,
SEROLOGIC REACTIONS OF CARBOXYPEPTIDASE
371
6o min at o ° with appropriate concentrations of trypsin to allow complete activation. Endopeptidase activity against ATEE was then determined b y the titration method previously described and compared with the activity measured in similarly activated solutions incubated 30 min at o ° with homologous antibody, heterologous antibody, and non-specific ~,-globulin (3:1 molar ratio of ~,-globulin to procarboxypeptidase) respectively. In saline controls, the non-specific ~,-globulin controls, and reaction mixtures containing homologous antibody, there was no significant difference in the level of enzymic activity before and after incubation with ~,-globulin. Again, heterologous antibody was found to have a greater inhibiting effect than the homologous antibody, since it produced a 12-14 % reduction in endopeptidase activity. These data indicate that antibodies capable of reacting with procarboxypeptidase interfere with the activation of the zymogen rather than with the enzymic activity of the product of activation, and that in low concentrations, anticarboxypeptidase is a more effective inhibitor than antiprocarboxypeptidase. DISCUSSION
The use of ~-globulin fractions isolated from immune serum for the study of immunologic reactions of enzymes appears to offer certain advantages, e.g. the elimination of most non-specific protein in serum, the removal of complement which is frequently involved in enzyme-antienzyme reactions, and the ability to overcome the limitation of weak antisera by increasing antibody concentrations. The procedure for the isolation of ~,-globulin described in this report was repeatedly demonstrated to yield preparations of 7-S ~-globulin which were free of contamination by other serum proteins or hemoglobin after a single chromatography. The immunodiffusion results reported here demonstrate some of the serologic relationships of carboxypeptidase A, procarboxypeptidase A and related proteins. Clearly, procarboxypeptidase A-S6, procarboxypeptidase A-S 5 and carboxypeptidase As have broad areas of antigenic similarity as previously reported by BARRETT2 and LEHRER AND VAN VUNAKIS3. Indeed, the vast majority of the antigenic determinant sites of carboxypeptidase seem to be available on the surface of the procarboxypeptidase molecules since spur formation at the iunction of the enzyme-zymogen precipitin bands is difficult to demonstrate. This concept is in agreement with the absorption data of LEHRER AND VAN VUNAKIS3 in which procarboxypeptidase was shown to precipitate with anticarboxypeptidase to iemove most but not all of the antibodies capable of complement fixation with carboxypeptidase. The complete lack of crossreaction between anticarboxypeptidase and Fraction II is consistent with the concept that carboxypeptidase A and Fraction II are different proteins which, combined with Fraction III, comprise the subunit structure of procarboxypeptidase A-S6. The reaction of carboxypeptidase with carbonaphthoxyphenylalanine in agar demonstrates that antibodies formed in response to immunization with carboxypeptidase react with native carboxypeptidase and that the antigen-antibody precipitate retains some enzymic activity. This is of importance since it has been observed in some systems that antibodies formed by immunization with denatured enzyme cross-react poorly with the native molecule ~6, and one might anticipate significant denaturation of a protein emulsified in Freund's adjuvant. Procarboxypeptidase A-S 6 and procarboxypeptidase A-S 5 are immunologically Biochim. Biophys. Acta, 124 (1966) 362-373
372
n . N . BEATY
indistinguishable in these studies. The demonstration of three precipitin bands in the reaction of antiprocarboxypeptidase with these zymogens is in agreement with the report of BARRETT2 who was able to separate these bands by electrophoresis in agar before diffusion against procarboxypeptidase. One of the precipitin bands formed b y the reaction of procarboxypeptidase and anticarboxypeptidase seems unrelated to either carboxypeptidase of Fraction II. It is possible that the antigenic determinants responsible for this band are located on Subunit I I I of procarboxypeptidase A-S6 and that the band is seen in the reaction of antibody with the $5 zymogen because of contamination by S s. These relationships cannot be resolved until each of the fractions of procarboxypeptidase A-Ss is available in pure form and is used to induce formation of specific antibodies which can be compared in immunodiffusion experiments. The mechanism responsible for the observed effect of non-specific y-globulin on the hydrolysis of H P L A and on the activation of procarboxypeptidase is unknown. Partial release of the substrate inhibition of H P L A m a y be an explanation for the former but seems unlikely since the effect is observed at low ranges of substrate concentration where inhibition by substrate is minimal 2~. The non-specific protein effect on the activation of procarboxypeptidase is probably related to competition between y-globulin and zymogen for available trypsin, since the endopeptidase activity of completely activated procarboxypeptidase is the same in saline as in solutions containing non-specific protein. It is virtually impossible to formulate principles of inhibition which can be applied to e n z y m e - a n t i e n z y m e systems in general. The degree of inhibition of enzymic activity by antibody depends upon several factors including the number of antibody molecules reacting with each enzyme molecule, the firmness of binding of enzyme by antibody, spatial relationships of a n t i b o d y - e n z y m e combining sites with the catalytic site, and the size of substrate molecule being hydrolyzed. These factors are in turn influenced by the complex antigenic structure of proteins, variability in the immune response, and the heterogeneity of any population of antibodies with respect to their binding affinities 27. For these reasons, classification of antibody inhibition as competitive or non-competitive must represent a gross oversimplification. Anticarboxypeptidase clearly inhibits enzymic activity of carboxypeptidase. In the hydrolysis of a large protein substrate this inhibition is at least partially accomplished by steric hindrance of the catalytic site by antibody, a mechanism previously demonstrated in other enzyme systems 2'~. In addition, the inhibiting capacity of the antibody is affected by substrate concentration when H P L A is used for the assay. Heterologous antibody has a similar, less potent, effect. Apparent discrepancies between the degree of inhibition of enzymic activity reported here and that previously reported b y LEHRER ANt)VAN VUNAKIS3 are probably related to differences in experimental procedure. In contrast to this study, LEHRER AND VAN VUNAKIS incubated enzyme and antiserum for 18 h at 2-4 ° before assaying for peptidase activity. These conditions m a y be associated with precipitate formation, particularly at the lower serum dilutions where the greatest inhibition of activity was observed. Activation of procarboxypeptidase with trypsin is inhibited by antibody, presumably through combination of antibody molecules with antigenic determinants at or near the point of attack by trypsin. Little is known about the location of the site of activation, but it is presumably on the surface of Subunit II (the endopeptidase fliochim. Biophys. dcta, 124 (1966) 302-373
373
SEROLOGIC REACTIONS OF CARBOXYPEPTIDASE
precursor). A clue to t h e m o r e precise l o c a l i z a t i o n of t h i s site m i g h t lie in t h e s u r p r i s i n g o b s e r v a t i o n s t h a t low c o n c e n t r a t i o n s of a n t i b o d i e s w h i c h r e a c t w i t h c a r b o x y p e p t i d a s e b u t n o t F r a c t i o n 21 are m o r e effective i n h i b i t o r s of t h e process of a c t i v a t i o n a n d t h e e n d o p e p t i d a s e a c t i v i t y of t h e f u l l y a c t i v a t e d z y m o g e n t h a n e q u i m o l a r c o n c e n t r a t i o n s of a n t i b o d y c a p a b l e of r e a c t i n g w i t h t h e e n t i r e z y m o g e n m o l e c u l e . T h e s e o b s e r v a t i o n s i n d i c a t e t h a t t h e site of.. a c t i v a t i o n a n d t h e c a t a l y t i c site of t h e e n d o p e p t i d a s e p o r t i o n of procarboxyi~ep.tidase are in a p o s i t i o n w h e r e t h e y m a y b e s t e r i c a l l y i n h i b i t e d b y a n t i b o d i e s c o m b i n i n g w i t h S u b u n i t I only. A t h i g h e r a n t i b o d y c o n c e n t r a t i o n s , a n t i p r o c a r b o x y p e p t t d a s e c o n t a i n s a g r e a t e r p r o p o r t i o n of m o l e c u l e s c a p a b l e of r e a c t i n g w i t h m o r e a n t i g e n i c d e t e r m i n a n t s on S u b u n i t s I I a n d 122 w h i c h m a y be n e a r t h e s e sites, i n c r e a s i n g its e f f e c t i v e n e s s as an i n h i b i t o r . A n e x t e n s i o n of t h e s e studies c o u l d offer v a l u a b l e clues to t h e s p a t i a l r e l a t i o n s h i p s of t h e s u b u n i t s o f procarboxypeptidase. ACKNOWLEDGEMENTS T h e a u t h o r wishes to t h a n k Dr. H . NEURATH for his s u p p o r t a n d e n c o u r a g e m e n t d u r i n g t h e course of t h i s w o r k , a n d Dr. K. A. WALSH for m a n y h e l p f u l discussions. T h e i n v a l u a b l e a s s i s t a n c e of Dr. W . O . McCLURE, Dr. P . E . GUIRE, a n d Mr. R . D . WADE is also a c k n o w l e d g e d . T h i s i n v e s t i g a t i o n was s u p p o r t e ~ i n p a r t b y U.S. P u b l i c H e a l t h S e r v i c e f e l l o w s h i p in i F 2 A I - I 9 , 8 8 0 - 0 I f r o m t h e N a t i o n a l I n s t i t u t e of A l l e r g y a n d I n f e c t i o u s D i s e a s e . REFERENCES
I 2 3 4 5 6 7 8 9 IO II
12 13 14 15 16 17 18
19 20 21 22 23 24 25
26 27
.
.
.
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E. L. SMITH, B. V. JAGER, R. LUMRY AND R. R. GLANTZ,J. Biol. Chem., 199 (i952) 789 . J. T. BARRETT, Immunology, 8 (I965) 129. H. I. LEHRER AND H. VAN VUNAKIS, Immunochemistry, 2 (1965) 255. B. CINADER,Ann. N . Y . Acad. Sci., lO 3 (1963) 495P. J- KELLER, E. COHEN AND H. NEURATH, J. Biol. Chem., 223 (1956) 457. P. J. KELLER, E. COHEN AND H. NEURATH, J. Biol. Chem., 230 (1958) 905. J' R. BROWN, D. J. Cox, R. N. GREENSHIELDS, K. A. WALSH, M. YAMASAKIAND H. NEURATH, Proc. Natl. Acad. Sci., 47 (1961) 1554. J. R. BROWN, R.N. GREENSHIELDS,M.YAMASAKIAND H. NEURATH, Biochemistry, 2 (1963) 867. J. R. BROWN, Ph.D. Thesis, University of Washington, Seattle, Wash., 1963. M. YAMASAKI, J. R. BROWN, D. J. Cox, R. N. GREENSHIELDS, R. D. WADE AND H. NEURATH, Biochemistry, 2 (1963) 859. ]~I. VAN VUNAKIS AND L. LEVINE, Ann. N . Y . Acad. Sci., lO3 (1963) 735. J. T. BARRETT AND L. D. THOMPSON, Immunology, 8 (1965) 136. J. R. BROWN, M. YAMASAKIAND H. NEURATH, Biochemistry, 2 (1963) 877. D. J. Cox, F. C. BOVARD, J-P. BAROETZI, K. A. WALSH AND H. NEURATH, Biochemistry~ 3 (1963) 44. M. J. CRUMPTONAND J. M. WILKENSON, Biochem. J., 88 (1963) 228. J-P. BARGETZI, K. S. V. S. KUMAR, D. J. Cox,=K. A. WALSH AND H. NEURATH, Biochemistry, 2 (1963) 1468. E. W. DAVIE, C. R. NEWMAN AND P. E. WILCOX, J. Biol. Chem., 234 (1959) 2635. E, A. PETERSON AND H. A. SOBER, in S. P. COLOWICK AND N. O. KAPLAN, Methods in E n zymology, Vol. 5, Academic Press, New York, 1962, p. 3. H. A. SOBER AND E. A. PETERSON, Federation Proc., 17 (1958) 1116. J. URIEL, Ann. N . Y . Acad. Sci., lO3 (1963) 956. W. O. MCCLURE, H. NEURATH AND K. A. WALSH, Biochemistry, 3 (1964) 1897. W. O. MCCLURE, unpublished experiments. S. MOORE AND W. H. STEIN, J. Biol. Chem., 211 (1954) 907. 0. OUCHTERLONY,in J. F. ACKROYD, Immunological Methods, Davis, Philadelphia, 1964, p. 55B. CINADER AND K. J. LAFFERTY, Ann. N . Y . Acad. Sci., lO3 (1963) 653. M. R. POLLOCK, Ann. N. Y. Acad. Sci., lO3 (1963) 989. A. H. SEHON, Ann. N . Y . Acad. Sci., lO3 (1963) 626. Biochim. Biophys. Acta, 124 (1966) 362-373
BIOCHIMICA ET BIOPHYSICA A(;T.\
BBA 25 606 SEROLOGIC REACTIONS OF BOVINE PROCARBOXYPEPTIDASE A AND CARBOXYPEPTIDASE A H A R R Y N. BEATY*
Department of Biochemistry, Umversity of Washington, Seattle, Wash. (U.S.A .) (Received January 3rd, I966)
SUMMARY
Serologic reactions of bovine procarboxypeptidase A and carboxypeptidase A (peptidyl-L-amino-acid hydrolase, EC 3.4.2.1) are compared through immunological reactions in agar and studies of the inhibition of enzymic reactions by antibody. I. Rabbits immunized with carboxypeptidase Aa or procarboxypeptidase A-S~ form antibodies with physical and immunological properties of 7-S y-globulin. A procedure for the isolation of 7-S y-globulin from the serum of immunized rabbits is described and utilized for the evaluation of the serologic reactions of the antigens and related proteins in a system relatively free of non-specific protein. 2. Some of the antigenic relationships between carboxypeptidase A~, procarboxypeptidase A-S~, procarboxypeptidase A-S 5 and Fraction II of procarboxypeptidase A are demonstrated by immunologic reactions in agar. The effect of homologous and heterologous antibody on the enzymic activity of carboxypeptidase A and partially activated procarboxypeptidase A-S 6 are compared.
INTRODUCTION
Chemical and enzymic properties of bovine carboxypeptidase A (peptidyl-L amino-acid hydrolase, EC 3.4.2.1) and its zymogen procarboxypeptidase A have been studied in this and other laboratories. However, despite the recent trend in immunochemistry to use enzymes as antigens, few studies on the immunologic reactions of carboxypeptidase or procarboxypeptidase have been reported since SMrrH et al. 1 in x952 demonstrated that carboxypeptidase A, injected into rabbits, produced a heterogeneous population of antibodies capable of precipitating the enzyme and noncompetitively inhibiting its enzymic activity against carbobenzoxyglycyl+-tryptophan. Recently, BARRETT 2 a n d LEKRER AND VAN VUNAKISa have reported immunodiffusion and precipitin data derived from the use of these proteins as antigens. CINADER 4, in discussing developments in immunochemistry, has pointed out some of the advantages of studying a system of three components, e.g. antibody, enzyme, and substrate. With the isolation and purification of bovine procarboxypeptidase A by KELLER, COHEN" AND NEURATH 5,6, and the extensive study of the chemical properties and enzymic functions of this zymogen by BRowx and coAbbreviations: ATEE, acetyl-L-tyrosine ethyl ester; HPLA, hippuryl-DL-phenyllactic acid. * Present address: Department of Medicine, King County Hospital, Seattle, Wash., I!.S.A.
Diochim. 13iophys. Acta, 124 (i066) 362 373
363
SEROLOGIC REACTIONS OF CARBOXYPEPTIDASE
workers v-9 and YAMASAKI et al. TM, it is now possible to examine the serologic relationships of procarboxypeptidase A and carboxypeptidase A in such a system. Other zymogens and their enzymes have been studied in this mannern, 12 with the general observation that enzyme and precursor have antigenic determinants in common, presumably related by areas of similarity in tertiary structure. The immunochemistry of procarboxypeptidase A and carboxypeptidase A is of particular interest because of the unique quaternary structure of the zymogen which is characterized by the presence of three subunits. Subunit I has been identified as the zymogen of carboxypeptidase A, and Subunit II as the zymogen of an endopeptidase similar in specificity to chymotrypsin. No biological function has yet been demonstrated for Subunit III (see refs. 7-1o). Activation of procarboxypeptidase A by relatively high concentrations of trypsin at 37 ° proceeds slowly to produce carboxypeptidase A and proteolytic destruction of the remainder of the zymogen molecule. However, activation at o ° by much lower concentrations of trypsin is more suited to immunologic study in a three-component system since it progresses rapidly, yielding only endopeptidase activity without disaggregation or extensive destruction of the zymogen. Most of the investigation into the immunochemistry of enzymes has been done in serum, allowing for non-specific protein-protein interaction which could conceivably lead to difficulties in interpretation of results. In addition, complement fixation by the reaction of carboxypeptidase with its antibody has been demonstrated 3, introducing another variable to be considered in the analysis of data. For these reasons, the present studies were conducted in a system using 7-S 7-globulin isolated from the serum of immunized rabbits. MATERIALS AND METHODS
Procarboxypeptidase A-S s, procarboxypeptidase A-Ss*, carboxypeptidase Aa and purified Fraction II* were prepared as described by YAMASAKI et al. TM, BROWN, YAMASAKI AND N E U R A T H 13 and Cox et al. 14 respectively. Adult, female rabbits were immunized by four weekly subcutaneous injections containing 20 mg of procarboxypeptidase A-S s or carboxypeptidase Aa dissolved in I ml of o.15 M NaC1, o.o15 M Tris-HC1 (pH 7.5). Immediately prior to injection, each antigen solution was emulsified in an equal volume of Freund's adjuvant (Difco). Antiserum was obtained by bleeding the rabbits 7-1o days after the final injection. 7-Globulin was isolated from the antisera by precipitation with (NH4)~SO 4 and chromatography on TEAE-cellulose. TEAE-cellulose was a product of Bio-Rad Corp., with a stated capacity of 0.78 mequiv/g or 0.55 mequiv/g according to the lot used. Trypsin (twice crystallized and salt free) was obtained from Worthington Biochemical Corp., HPLA from Cyclo Chemical Corp. (as the sodium salt), N-carbonaphthoxy-DL-phenylalanine from K and K Laboratories, and ATEE from the California Corp. for Biochemical Research. All other chemicals were reagent grade. fi-Lactoglobulin was obtained from Dr. P. E. WILCOX. It had been isolated and purified as "Lot B " described by DAVIE, NEWMAN AND WILCOX17. Measurements of pH were made with a Radiometer pH meter 22 using a GK 202 C * K i n d l y p r o v i d e d b y J. H. FREISHEIM a n d W. D. BEHNKE Of this l a b o r a t o r y .
Biochim. Biophys. Acta, 124 (1966) 362-373
364
H . N . BEATY
combined glass electrode. During measurements of enzyme activity, temperature was controlled b y means of circulating water with a Haake thermostat. Protein concentrations were determined b y absorbance measurements at 28o m#, using values of E~ ~i~ = 13.5 for rabbit v-globulin (ref. I5), E~ ~,;, - - 19.o for procarboxypeptidase A (ref. 5) and E~ ~i',~= 18.8 for carboxypeptidase A (ref. 16).
Isolation of v-globulin 7-Globulin was isolated from immune sera a n d for control purposes, from the serum of non-immunized rabbits. After centrifugation, the sermn was adjusted to 4 ° % saturation of (NH4)2SO 4 b y the slow addition of the crystalline salt (242 g/1 serum) with constant stirring at room temperature for 2 h. The suspension was then allowed to stand without stirring for i 6 - 2 o h at 4 ° and further purification was carried out at this temperature. The resulting precipitate was removed b y centrifugation, dissolved in 20-40 ml of distilled water and dialyzed 24 h against four changes of 21 of 5 mM K 2 H P O 4 (pH 7.5). Fresh TEAE-cellulose was prepared for each chromat o g r a p h y according to the procedure of PETERSON AND SOBER18. The washed cellulose was packed under a pressure of 5 lb/inch2 into a 3.3 cm ~ 45 em column from a thick slurry in 5 mM K 2 H P O 4 (pH 7.5)- 2-3 1 of the same buffer were flushed t h r o u g h the column at 4 ° for equilibration. The dialyzed protein solution was p u m p e d onto the the column and followed b y a volume of equilibrating buffer sufficient to elute a faint red pigment b a n d which had relatively low absorbance at 280 m/x. At this point, a concave gradient of increasing phosphate concentration from 0.005 M to o.15 M was applied utilizing the c o n e - s p h e r e technique of SOBER AND PETERSON 19. Effluent was collected in I o - I 2 - m l fractions which were screened for absorbance at 28o m/x, yielding a c h r o m a t o g r a m as represented in Fig. IA. The fractions of the major protein peak were pooled as shown, dialyzed 18-24 h against three changes of distilled water, lyophilized, and stored at o ° as a white, fluffy powder. A solution containing IO mg of the protein per ml was dialyzed 24 h against a buffer composed of o.15 M NaC1, O.Ol 5 M Tris-HC1 (pH 7.5). Approx. 5 IO/xl of this solution were subjected to immunoelectrophoresis in 1 % agar using veronal buffer (pH 8.6, I o.I) at 4-5 V/cm for IOO rain. After electrophoresis a central trough cut in the agar was filled with goat serum containing antibodies against rabbit serum (Hyland Laboratories), and the materials allowed to diffuse for 20 h at 22-25 ° in a humid chamber. Unf)recipitated protein was removed b y immersing the nficroscope slide in a 1 % NaC1 solution for 24 h. The salt was removed b y rinsing in two changes of distilled water for an additional 24 h. After drying in air, the precipitin bands were stained with amido black. A solution of anticarboxypeptidase v-globulin containing 9.4 mg/ml of 0.05 M K i H P O 4 (pH 7.47) was subjected to ultracentrifugation in a Spinco Model-E ultracentrifuge.
Immunodiffusion reactions All immunodiffusion reactions were carried out in 1% agar (Difco) gel in o.15 M NaC1, O.Ol 5 M Tris-HC1 (pH 7.5) according to the double diffusion technique of OUGHTERLONY24. One central and six peripheral wells were cut in a I-ram layer of agar on a microscope slide using an L K B gel cutter, die No. 6866A. Each peripheral well was 5 m m from its neighbor and 5 m m from the central well. Approx. io ~,1 of
Biochim. Biophys. Acta, 124 (1966) 302 373
365
SEROLOGIC REACTIONS OF CARBOXYPEPTIDASE
15-20 FM solutions of carboxypeptidase A~, procarboxypeptidase A-S e, procarboxypeptidase A-S 5 and Fraction I I were arranged in the peripheral wells and allowed to diffuse in a humid chamber at 22-25 o against a 200 ~M solution of antibody y-globulin in the central well. After 20 h of diffusion, the slides were washed in 1 % NaC1, rinsed in distilled water, dried in room air and stained with amido black.
[
I
7.06.0-
~b 5.0e~
4.0-
.8.~
3.0-
-~
Z.O1.0-
/-.
j
~'o 2'0 3'o 4'0 5'0 6~
7'0 8'o 9'0
Froclion number
Fig. I. A. C h r o m a t o g r a p h y on T E A E - c e l l u l o s e of dissolved, dialyzed (NH4)2SO 4 p r e c i p i t a t e f r o m r a b b i t a n t i s e r u m . A n t i s e r u m was a d j u s t e d to 4 o % s a t u r a t i o n of (NH4)zSO 4 a n d t h e r e s u l t i n g precipitate redissolved a n d dialyzed a g a n s t 0.005 M K 2 H P O 4 (pH 7.5) ; t h e s a m e buffer used to equilibrate t h e TEAE-cellulose. After t h e p r o t e i n was flushed t h r o u g h t h e c o l u m n , e l u t i n g a s m a l l a m o u n t of protein, a c o n c a v e g r a d i e n t of 0.005 M to o.15 M K 2 H P O 4 (pH 7.5) w a s applied at t h e p o i n t i n d i c a t e d b y t h e arrow. T h e b r a c k e t d e n o t e s t h e fractions pooled for c h a r a c t e r i z a t i o n . B. T h e i m m u n o e l e c t r o p h o r e s i s of r a b b i t v-globulin. A s a m p l e of pooled m a t e r i a l f r o m t h e a b o v e c h r o m a t o g r a m was placed in t h e u p p e r well a n d n o r m a l r a b b i t s e r u m in t h e lower well. A f t e r electrophoresis, goat s e r u m c o n t a i n i n g antibodies to r a b b i t s e r u m was placed in t h e central t r o u g h . T h e single precipitin b a n d f o r m e d w i t h t h e m a t e r i a l f r o m t h e c h r o m a t o g r a p h y c o r r e s p o n d s to t h e v-globulin b a n d of n o r m a l s e r u m . C. S e d i m e n t a t i o n p a t t e r n of isolated v-globulin. A solution of 9.4 m g / m l of p r o t e i n in o.o 5 M K ~ H P O 4 (pH 7.47) after 80 m i n of s e d i m e n t a t i o n a t 59778 r e v . / m i n in t h e Spinco Model-E u l t r a c e n t r i f u g e .
Enzyme characterization reactions were carried out on dried, unstained duplicates of immunodiffusion preparations following the procedure described b y URIEL2°, An agar solution containing the chromogenic substrate N-carbonaphthoxy-DL-phenylaianine in N,N-dimethylformamide and Blue BNS salt (Allied Chemical) was layered Biochim. Biophys. Acta, I24 (1966) 362-373
366
H . N . BEATY
over t h e slide, which was first i n c u b a t e d at 37 °, then washed in 2 o; acetic acid. W h e n active c a r b o x y p e p t i d a s e was present, the precipitin b a n d t o o k on a characteristic color. A s s a y procedures
C a r b o x y p e p t i d a s e a c t i v i t y was m e a s u r e d against the ester s u b s t r a t e H P L A a n d the p r o t e i n s u b s t r a t e fi-lactoglobulin. R o u t i n e analyses were carried out in o . o i M racemic H P L A as described 1)57 BARGETZI et al. 1~ using a R a d i o m e t e r T T T - I a u t o t i t r a t o r a n d Ole Dich recorder. Studies of the d e p e n d e n c e of the inhibition reactions on t h e c o n c e n t r a t i o n of s u b s t r a t e were carried out in more dilute H P L A solutions using t h e s p e c t r o p h o t o m e t r i c t e c h n i q u e of MCCLURE, NEURATH ANY) \~,'ALSH2l. F o r these studies a stock solution of I mM racemic H P L A in 5 mM Tris .HC1, o.I M NaC1 (pH 7-5) was d i l u t e d to the desired s u b s t r a t e c o n c e n t r a t i o n with t h e same buffer. The increase in a b s o r b a n c e at 254 m/, was followed in the P e r k i n - E l m e r Model-35o recording u l t r a v i o l e t s p e c t r o p h o t o m e t e r . I n i t i a l velocities were derived from the expression ~. -
AA eM"t
(i)
where v is the initial v e l o c i t y in rnoles/1.min, AA is the change in absorbance, and eM is t h e m o l a r e x t i n c t i o n coefficient of H P L A at 254 mtz (ref. 2x), a n d t is t i m e in minutes. Assays of a c t i v i t y using fl-lactoglobulin as s u b s t r a t e were b a s e d on the release of n i n h y d r i n - p o s i t i v e m a t e r i a l w i t h time, according to t h e t e c h n i q u e of DAVIE, NEWMAN AND WILCOX17 a n d McCLURE 22. These assays were p e r f o r m e d at 25 ° with solutions c o n t a i n i n g 5 m g / m l of fl-lactoglobulin in o . o i M veronal-HC1 (pH 7.5)- A t a p p r o p r i a t e intervals, aliquots of t h e reaction m i x t u r e were a d d e d to an equal v o l u m e of a solution c o n t a i n i n g 30 % trichloroacetic acid a n d 4 % p h o s p h o t u n g s t i c acid which p r e c i p i t a t e d t h e p r o t e i n a n d s t o p p e d t h e reaction. The acid solutions were centrifuged a n d aliquots of t h e s u p e r n a t a n t were a d d e d to I ml of n i n h y d r i n solution (see ref. 23), boiled 20 min, d i l u t e d to 5 ml a n d the a b s o r b a n e e r e a d at 57 ° m / , . Velocities, e x p r e s s e d in t e r m s of moles leucine released p e r 1 per min, were c a l c u l a t e d from Eqn. I. The eM at 57 ° m/~ for a leucine s t a n d a r d was d e t e r m i n e d to be 2.54" IO ~ 1/mole. cm. The progress of t h e a c t i v a t i o n of t h e e n d o p e p t i d a s e a c t i v i t y of p r o c a r b o x y p e p t i d a s e A-S 6 was followed using t h e ester A T E E as s u b s t r a t e . P r o c a r b o x y p e p t i d a s e was a c t i v a t e d at o ° b y a d d i t i o n of t r y p s i n (weight r a t i o of 500" I). A t a p p r o p r i a t e t i m e intervals, aliquots of t h e reaction m i x t u r e s were a s s a y e d in the p H s t a t with o . o i M solutions of A T E E in I mM Tris-HC1, o.I M KC1, at p H 8.o a n d 25 °. I n i t i a l velocities of t h e e n d o p e p t i d a s e a s s a y were expressed as ~ e q u i v O H - per m g of p l o c a r b o x y p e p t i d a s e p r o t e i n p e r rain. F i r s t - o r d e r r a t e c o n s t a n t s for t h e a c t i v a t i o n process were c a l c u l a t e d from t h e expression. k __ log a / a - - x 2.303 t
(2)
where a is t h e m a x i m u m e n d o p e p t i d a s e per m g of p r o t e i n after t o t a l a c t i v a t i o n a n d x is t h e a m o u n t of e n d o p e p t i d a s e f o r m e d per m g of p r o t e i n at a n y given time. Biochim. Biophys. Acta, 124 (1966) 362 373
SEROLOGIC REACTIONS OF CARBOXYPEPT]DASE
367
RESULTS
Fig. IB represents the immunoelectrophoretic pattern of material isolated from chromatography on TEAE-cellulose, showing a single precipitin band corresponding to the 7-S y-globulin of a normal rabbit serum control. The ultracentrifuge pattern of this material (Fig. IC) reveals a homogeneous peak with an s°20, w of 6.51 S. These y-globulin fractions were shown to contain antibody against the sensitizing antigens, but no estimates of relative potency were made.
Immunodiffusion reactions Fig. 2 represents an example of an immunodiffusion reaction used to demonstrate the serologic relationships of the various proteins under consideration. It can be seen that antibody directed against carboxypeptidase forms a single dense precipitin band with its homologous antigen, and a less dense single band with both procarboxypeptidase A-S 6 and S5. At the concentrations of antigen used in this preparation, this cross-reaction appears to be a reaction of identity. However, at higher concentrations, which produce less distinct bands, there is faint but definite spur formation at the junction of the enzyme-zymogen precipitin bands. Antibody against carboxypeptidase does not cross-react with Fraction II irrespective of the concentration tested.
Fig. 2. Immunodiffusion in agar showing antigenic relationships of c a r b o x y p e p t i d a s e and related proteins. The central well to the left contained a n t i c a r b o x y p e p t i d a s e A (A-CP), and t h e central well to the right contained a n t i p r o c a r b o x y p e p t i d a s e (A-S~). The peripheral wells contained carb o x y p e p t i d a s e A(CP), p r o c a r b o x y p e p t i d a s e A-S 6 ($6), p r o c a r b o x y p e p t i d a s e A-S 5 (S~) and Fraction I[ (II) as shown.
With antiprocarboxypeptidase A-S6 in the central well, the immunodiffusion results are more complicated. There appear to be at least three precipitin bands formed by the reaction between the procarboxypeptidase antigens and homologous antibody, although the two bands nearest the antigen well are almost superimposed, giving the appearance of a single dense band in some preparations. The intercept between the $6 and S5 zymogen forms shows a reaction of identity, but the precipitin bands formed by procarboxypeptidase A-S5 are usually more widely separated and smeared. Antiprocarboxypeptidase forms a single band on cross-reaction with carboxypeptidase and two bands on cross-reaction with Fraction II. Where the carboxypeptidase band fuses with the middle of the three zymogen bands, it exhibits the reaction of multideterminant antigens described by OUCHTERLONY2¢. One of the Fraction II bands shows a reaction of identity with the zymogen band nearest the central well. The Biochim. Biophys. Acta, 124 (1966) 362-373
368
H.N.
BEATY
second Fraction I I band is less distinct, but again demonstrates the relatively nonspecific reaction of multideterminant antigens with the outer zymogen band. One of the Fraction I I bands, probably the latter, m a y be due to cross-reaction of antiprocarboxypeptidase with a small amount of Fraction I I I which is difficult to exclude in the chromatographic purification of Fraction II (ref. 9)As URIEL~° has pointed out, the characterization reactions of e n z y m e - a n t i enzyme precipitates in agar aid in the study of the purity, homogeneity and native state of an enzyme preparation. Carbonaphthoxyphenylalanine seems to be a highly specific substrate for carboxypeptidase and when hydrolyzed in the presence of a diazonium salt yields a red-violet color. This reaction was carried out in duplicates of immunodiffusion preparations of the type shown in Fig. 2 with the exception that they were not stained with amido black. When anticarboxypeptidase was used in the central well, the single precipitin band formed by its reaction with carboxypeptidase took on the characteristic color described above after incubation with the chromogenic substrate. The precipitin bands formed by the cross-reaction of anticarboxypeptidase with the zymogen antigens were not colored. This indicates that cross-reaction is indeed due to antigenic similarity between the enzyme and its zymogen rather than to contamination of the zymogen preparation with small amounts of active enzyme. As expected, in preparations where the antigens were allowed to diffuse against antiprocarboxypeptidase, only the precipitin band resulting from the cross-reaction of this antibody with carboxypeptidase was colored.
Inhibition of enzymic activity All of the inhibition studies to be described were designed to compare either the progress of activation of procarboxypeptidase or the enzymic activity of carboxypeptidase in equimolar concentrations of y-globulin solutions containing homologous or heterologous antibody. The most meaningful control for these reactions was one containing an equal concentration of non-specific y-globulin since the mere presence of protein invariably resulted in rate constants for the activation of procarboxypeptidase which were lower than in saline controls and initial velocities for the hydrolysis of H P L A by carboxypeptidase which were greater than in saline controls. The inhibitory effect of non-specific protein on the activation of procarboxypet~tidase varied from 22 % at low v-globulin concentrations to about 45 % at higher concentrations. The apparent increase in activity of carboxypeptidase in a solution of nonspecific v-globulin averaged about 25 % over a wide range of concentration. It is desirable to study the enzymic activity of a soluble antigen ~antibody complex rather than activity remaining in solution after precipitation of antigen by antibody. For this reason, these studies were done at molar ratios of y-globulin to antigen which resulted in significant inhibition of enzymic activity without development of a demonstrable precipitate during the period of incubation and assay. This was assumed to be in the zone of antigen excess since an increase in antibody concentration resulted in prompt precipitate formation. Carboxvpeptidase A. Since the carboxypeptidase concentrations required to adequately measure the hydrolysis of H P L A and fl-lactoglobulin are so different, separate conditions were established for each assay. Solutions containing anticarEoxypeptidase and carboxypeptidase in a molar ratio of 20 : I, incubated at 25~', exhibited m a x i m u m inhibition of enzymic activity (5° %) within 15 min when subjected to Biochim. 13iophys. Acla, 124 (1966) 362 373
369
SEROLOGIC REACTIONS OF CARBOXYPEPTIDASE
the spectrophotometric assay of HPLA at enzyme concentrations of O.l-O.6 tzM. After 60 rain incubation, there was no visible precipitate, but the enzymic activity of the incubation mixture was found to decrease with centrifugation at 3000 rev./min for 5 min, indicating that the antigen-antibody complexes had formed fairly large aggregates. Therefore, studies of the effect of antibody on the enzymic activity of carboxypeptidase were performed on low concentrations of enzyme in 2o-fold molar excess of antibody solution after 30 min incubation at 25 °. At the higher enzyme concentrations (12-13 ~M), necessary for assays of the hydrolysis of ~-lactoglobulin, a 2o-fold molar excess of antibody solution resulted in visible precipitation within 30 min. When antibody was present in a 4-fold molar excess, incubation for 3 ° min at 25 ° resulted in significant inhibition of enzymic activity without evidence of precipitate formation. Because of the well established inhibition of carboxypeptidase activity by excess HPLA (ref. 2I), it was desirable to examine the relationship of substrate concentration to inhibition by antibody at low HPLA concentrations where a conventional dependence of the velocity of the enzyme reaction upon substrate concentration can be demonstrated. Fig. 3 represents a Lineweaver-Burk plot of data from a typical experiment which compared the rate of hydrolysis at four concentrations of HPLA in the presence of antibody. Both Km and V appear to be altered by homo logous antibody, indicating that the mechanism of inhibition is neither the classical competive nor non-competitive type. Inhibition by heterologous antibody is less marked than with the homologous antibody, but is associated with a clear-cut increase in Km. The effect of substrate size on the inhibition of carboxypeptidase activity by antibody was studied in a separate experiment. A 12-13 FM solution of carboxypeptidase was incubated for 3 ° min at 25 ° in a 4-fold molar excess of v-globulin containing homologous or heterologous antibody. The enzymic activity of the incubation 6,40
-
G005.60 52048O
-
4404OO
-
4" 360% 3204 280-.~ 2 . 4 0 2.001,60h200.80-
0.40-5
-4
-3
-2
-I
0
i I
i 2
i 3
i 4
i 5
I/Is] (x I0 ~) Fig. 3. L i n e w e a v e r - B u r k plot showing the dependence of antibody inhibition of carboxypeptidase activity on the concentration of substrate (HPLA), The Q points represent inhibition by y-globulin containing antibodies against carboxypeptidase, the • points represent inhibition by yglobulin containing antibody to procarboxypeptidase, and the • points represent the non-specific y-globulin control. The y-globulin:enzyme ratio in the incubation mixture was 2o: I. The enzyme concentration in the assay mixture was 1.64 m/zM.
Biochim. Biophys. Acta, 124 (1966) 362-373
37 °
H.N.
I-H,:.VI'Y
mixture was assayed against fl-lactoglobulin (mol. wt. 35 400) by the ninhvdrin technique previously described and H P L A (mol. wt. 349) according to the titration technique of BARC;ETZI et al. 16. Under these conditions, there was a 35 % reduction in the velocity of the hydrolysis of fi-lactoglobulin in the presence of h(mlologous antibody and a 25 % reduction of heterologous antibody. However, no inhibition of enzymic activity could be detected under these conditions when assayed with H PLA in the autotitrator. In other systems, data of this sort is cited as evidence that steric hindrance by antibody prevents the larger substrate molecules flora reaching the binding or catalytic site of the enzyme molecule °-:'. Procarbo:Evpeptidase A-S~i. Solutions of 0. 5 ffM t)rocarboxypeptidase A-S, in o.15 M NaC1, O.Ol5 M Tris-HCl (t)H 7.5) were incubated with antibody for 3o rain at 25 °, and rapidly cooled to o '~ heft)re activation with trypsin, The progress of activation was followed by assaying aliquots of the aetivated incubation mixture against ATEE at various time intervals after the addition of trypsin. y-Globulin solutions containing antiprocarboxypeptidasc in c(meentrations necessary to achieve molar ratios of 2o: z with this concentration of t)rocarboxyt)et> tidase resulted in almost immediate formation of a heavy precipitate. Consequently, subsequent inhibition studies were performed with molar ratios of 7-gh~bulin to procarboxypeptidase ranging from I : I to 8:~. At the lowest concentrations ()f antibody used, it was consistently found that anticaH)oxypeptidase was a re.re effectiv(~ inhibitor of procarboxypeptidase activation than antiprocarboxypeptidase. As the molar ratio of y-globulin to t)rocarb()xypeptidas~ ' was increased, the inhibitor\' ('apacity of homologous antibody more closely approximated that of heterologous antibody until the ratio of ~;:I was reached (Fig. 4). At that point, the antib(~tlv solutions were equally effective as inhibitors. At greater molar ratios, both h o m . logous and heterologous antibody formed a precipitate with pr()carboxyt)eptidase, although with homologous antibody, the precipitate appeared more promt)tly an(l in greater quantity. From these data, it was not clear whether the decrease in rate ('(instants observed during the activiation of proearl)oxypeptidase in the presence ~f antibody was due to inhibition of the actiwttion process itself or of the endopeptidase formed by activation. ]% resolve this question, procarboxypeptidase solutions were incubated 0.080.070060.05-
0.030.02O.01
Rgtio "~- globulin/procorboxypeptidase
Fig. 4. The effect of a n t i b o d y and non-specific y-globulin on the rate of activation of procarl)oxypeptidase A-S 6. Solutions of" p r o c a r b o x y p e p t i d a s e A-S, (6 ffM) were activated at o ° by addition of small a m o u n t s of trypsin. The first-order rate c o n s t a n t of activation (K1) was determined in increasing molar ratios of non-specific v-globulin, (o), },-globulin containing a n t i p r o c a r b o x y peptidase (O), and y-globulin containing a n t i c a r b o x y p e p t i d a s e (O).
Biockim. Hiopk),s..4cla, l~ 4 (~900) 362-37 ~,
SEROLOGIC REACTIONS OF CARBOXYPEPTIDASE
371
6o min at o ° with appropriate concentrations of trypsin to allow complete activation. Endopeptidase activity against ATEE was then determined b y the titration method previously described and compared with the activity measured in similarly activated solutions incubated 30 min at o ° with homologous antibody, heterologous antibody, and non-specific ~,-globulin (3:1 molar ratio of ~,-globulin to procarboxypeptidase) respectively. In saline controls, the non-specific ~,-globulin controls, and reaction mixtures containing homologous antibody, there was no significant difference in the level of enzymic activity before and after incubation with ~,-globulin. Again, heterologous antibody was found to have a greater inhibiting effect than the homologous antibody, since it produced a 12-14 % reduction in endopeptidase activity. These data indicate that antibodies capable of reacting with procarboxypeptidase interfere with the activation of the zymogen rather than with the enzymic activity of the product of activation, and that in low concentrations, anticarboxypeptidase is a more effective inhibitor than antiprocarboxypeptidase. DISCUSSION
The use of ~-globulin fractions isolated from immune serum for the study of immunologic reactions of enzymes appears to offer certain advantages, e.g. the elimination of most non-specific protein in serum, the removal of complement which is frequently involved in enzyme-antienzyme reactions, and the ability to overcome the limitation of weak antisera by increasing antibody concentrations. The procedure for the isolation of ~,-globulin described in this report was repeatedly demonstrated to yield preparations of 7-S ~-globulin which were free of contamination by other serum proteins or hemoglobin after a single chromatography. The immunodiffusion results reported here demonstrate some of the serologic relationships of carboxypeptidase A, procarboxypeptidase A and related proteins. Clearly, procarboxypeptidase A-S6, procarboxypeptidase A-S 5 and carboxypeptidase As have broad areas of antigenic similarity as previously reported by BARRETT2 and LEHRER AND VAN VUNAKIS3. Indeed, the vast majority of the antigenic determinant sites of carboxypeptidase seem to be available on the surface of the procarboxypeptidase molecules since spur formation at the iunction of the enzyme-zymogen precipitin bands is difficult to demonstrate. This concept is in agreement with the absorption data of LEHRER AND VAN VUNAKIS3 in which procarboxypeptidase was shown to precipitate with anticarboxypeptidase to iemove most but not all of the antibodies capable of complement fixation with carboxypeptidase. The complete lack of crossreaction between anticarboxypeptidase and Fraction II is consistent with the concept that carboxypeptidase A and Fraction II are different proteins which, combined with Fraction III, comprise the subunit structure of procarboxypeptidase A-S6. The reaction of carboxypeptidase with carbonaphthoxyphenylalanine in agar demonstrates that antibodies formed in response to immunization with carboxypeptidase react with native carboxypeptidase and that the antigen-antibody precipitate retains some enzymic activity. This is of importance since it has been observed in some systems that antibodies formed by immunization with denatured enzyme cross-react poorly with the native molecule ~6, and one might anticipate significant denaturation of a protein emulsified in Freund's adjuvant. Procarboxypeptidase A-S 6 and procarboxypeptidase A-S 5 are immunologically Biochim. Biophys. Acta, 124 (1966) 362-373
372
n . N . BEATY
indistinguishable in these studies. The demonstration of three precipitin bands in the reaction of antiprocarboxypeptidase with these zymogens is in agreement with the report of BARRETT2 who was able to separate these bands by electrophoresis in agar before diffusion against procarboxypeptidase. One of the precipitin bands formed b y the reaction of procarboxypeptidase and anticarboxypeptidase seems unrelated to either carboxypeptidase of Fraction II. It is possible that the antigenic determinants responsible for this band are located on Subunit I I I of procarboxypeptidase A-S6 and that the band is seen in the reaction of antibody with the $5 zymogen because of contamination by S s. These relationships cannot be resolved until each of the fractions of procarboxypeptidase A-Ss is available in pure form and is used to induce formation of specific antibodies which can be compared in immunodiffusion experiments. The mechanism responsible for the observed effect of non-specific y-globulin on the hydrolysis of H P L A and on the activation of procarboxypeptidase is unknown. Partial release of the substrate inhibition of H P L A m a y be an explanation for the former but seems unlikely since the effect is observed at low ranges of substrate concentration where inhibition by substrate is minimal 2~. The non-specific protein effect on the activation of procarboxypeptidase is probably related to competition between y-globulin and zymogen for available trypsin, since the endopeptidase activity of completely activated procarboxypeptidase is the same in saline as in solutions containing non-specific protein. It is virtually impossible to formulate principles of inhibition which can be applied to e n z y m e - a n t i e n z y m e systems in general. The degree of inhibition of enzymic activity by antibody depends upon several factors including the number of antibody molecules reacting with each enzyme molecule, the firmness of binding of enzyme by antibody, spatial relationships of a n t i b o d y - e n z y m e combining sites with the catalytic site, and the size of substrate molecule being hydrolyzed. These factors are in turn influenced by the complex antigenic structure of proteins, variability in the immune response, and the heterogeneity of any population of antibodies with respect to their binding affinities 27. For these reasons, classification of antibody inhibition as competitive or non-competitive must represent a gross oversimplification. Anticarboxypeptidase clearly inhibits enzymic activity of carboxypeptidase. In the hydrolysis of a large protein substrate this inhibition is at least partially accomplished by steric hindrance of the catalytic site by antibody, a mechanism previously demonstrated in other enzyme systems 2'~. In addition, the inhibiting capacity of the antibody is affected by substrate concentration when H P L A is used for the assay. Heterologous antibody has a similar, less potent, effect. Apparent discrepancies between the degree of inhibition of enzymic activity reported here and that previously reported b y LEHRER ANt)VAN VUNAKIS3 are probably related to differences in experimental procedure. In contrast to this study, LEHRER AND VAN VUNAKIS incubated enzyme and antiserum for 18 h at 2-4 ° before assaying for peptidase activity. These conditions m a y be associated with precipitate formation, particularly at the lower serum dilutions where the greatest inhibition of activity was observed. Activation of procarboxypeptidase with trypsin is inhibited by antibody, presumably through combination of antibody molecules with antigenic determinants at or near the point of attack by trypsin. Little is known about the location of the site of activation, but it is presumably on the surface of Subunit II (the endopeptidase fliochim. Biophys. dcta, 124 (1966) 302-373
373
SEROLOGIC REACTIONS OF CARBOXYPEPTIDASE
precursor). A clue to t h e m o r e precise l o c a l i z a t i o n of t h i s site m i g h t lie in t h e s u r p r i s i n g o b s e r v a t i o n s t h a t low c o n c e n t r a t i o n s of a n t i b o d i e s w h i c h r e a c t w i t h c a r b o x y p e p t i d a s e b u t n o t F r a c t i o n 21 are m o r e effective i n h i b i t o r s of t h e process of a c t i v a t i o n a n d t h e e n d o p e p t i d a s e a c t i v i t y of t h e f u l l y a c t i v a t e d z y m o g e n t h a n e q u i m o l a r c o n c e n t r a t i o n s of a n t i b o d y c a p a b l e of r e a c t i n g w i t h t h e e n t i r e z y m o g e n m o l e c u l e . T h e s e o b s e r v a t i o n s i n d i c a t e t h a t t h e site of.. a c t i v a t i o n a n d t h e c a t a l y t i c site of t h e e n d o p e p t i d a s e p o r t i o n of procarboxyi~ep.tidase are in a p o s i t i o n w h e r e t h e y m a y b e s t e r i c a l l y i n h i b i t e d b y a n t i b o d i e s c o m b i n i n g w i t h S u b u n i t I only. A t h i g h e r a n t i b o d y c o n c e n t r a t i o n s , a n t i p r o c a r b o x y p e p t t d a s e c o n t a i n s a g r e a t e r p r o p o r t i o n of m o l e c u l e s c a p a b l e of r e a c t i n g w i t h m o r e a n t i g e n i c d e t e r m i n a n t s on S u b u n i t s I I a n d 122 w h i c h m a y be n e a r t h e s e sites, i n c r e a s i n g its e f f e c t i v e n e s s as an i n h i b i t o r . A n e x t e n s i o n of t h e s e studies c o u l d offer v a l u a b l e clues to t h e s p a t i a l r e l a t i o n s h i p s of t h e s u b u n i t s o f procarboxypeptidase. ACKNOWLEDGEMENTS T h e a u t h o r wishes to t h a n k Dr. H . NEURATH for his s u p p o r t a n d e n c o u r a g e m e n t d u r i n g t h e course of t h i s w o r k , a n d Dr. K. A. WALSH for m a n y h e l p f u l discussions. T h e i n v a l u a b l e a s s i s t a n c e of Dr. W . O . McCLURE, Dr. P . E . GUIRE, a n d Mr. R . D . WADE is also a c k n o w l e d g e d . T h i s i n v e s t i g a t i o n was s u p p o r t e ~ i n p a r t b y U.S. P u b l i c H e a l t h S e r v i c e f e l l o w s h i p in i F 2 A I - I 9 , 8 8 0 - 0 I f r o m t h e N a t i o n a l I n s t i t u t e of A l l e r g y a n d I n f e c t i o u s D i s e a s e . REFERENCES
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12 13 14 15 16 17 18
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26 27
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