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Immunochemical studies of angiotensin
Immunochemistry. Pergamon Press 1966. Vol. 3, pp. 223-231. Printed in Great Britain IMMUNOCHEMICAL
STUDIES
OF
ANGIOTENSIN*
THEODORE GOODFRIEND,~fGERALDFASMAN,:~DANIEL KEMP § and LAWRENCELEVlNE 11 Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts
(Received 14 October 1965) Abstract--Water-soluble carbodiimides and a benzisoxazolium salt were used to synthesize conjugates containing angiotensin coupled to albumin or poly-L-lysine. Antibodies to angiotensin were induced with a conjugate containing the hormone bound to rabbit serum albumin..The antigenic determinants of angiotensin were investigated by inhibition of complement fixation by analogues of the polypeptide. Among the structural features of angiotensin tested, those which had greatest importance for serologic activity were conformation as determined by an optical isomer in the middle of the hormone molecule; the presence of the two amino acids at the N-terminus ; and the single phenolic hydroxyl. The structural features shown to be important for serologic activity were those previously shown to be important for biological activity. INTRODUCTION THE SMALL p o l y p e p t i d e h o r m o n e s w h i c h affect s m o o t h muscles and blood vessels have b e e n studied extensively f r o m the point of view of their biological activity. Ill I m m u n o l o g i c a l investigations have b e e n h a m p e r e d b y the small size, widespread occurrence, and biological lability of these molecules. Successful i n d u c t i o n of antibodies to angiotensin, 12-~1 bradykinin, ~3~ and vasopressin 151 have been reported. W e have used conjugates containing angiotensin c o u p l e d to a l b u m i n to induce antibodies to angiotensin in rabbits, and have m e a s u r e d inhibition of these antibodies b y analogues of the natural polypeptide to s t u d y the antigenic determinants of angiotensin. Previous investigations, b y others, of the biological properties of the analogues enabled us to c o m p a r e biological and serologic properties of this polypepfide h o r m o n e . * Presented in part at the American Federation for Clinical Investigation, Atlantic City, N.J., May 1964. Publication No. 407 from the Graduate Department of Biochemistry, Brandeis University. ~f Work performed during tenure of a fellowship from the Helen Hay Whitney Foundation. Present address: Departments of Medicine and Pharmacology, School of Medicine, University of Wisconsin, Madison, Wis. ~: This work was performed during the tenure of an Established Investigatorship of the American Heart Association, and was supported in part by research grants from the National Institutes of Health (AM-05852) and the National Science Foundation (GB-428). § Department of Chemistry, Massachusetts Insitute of Technology, Cambridge, Mass. II American Cancer Society, Professor of Biochemistry (PRP-21). This work was supported in part by a research grant from the National Institutes of Health (AI-01940). Abbreviations used: RSA = rabbit serum albumin; Ethyl C D I = 1-Ethyl-3-(3-dimethylaminopropyl)ocarbodiimide hydrochloride; Morpho CDI = 1-Cyclohexyl-3-(2-morpholinyl (4) ethyl)-carbodiimide metho-p-toluene sulfonate; EBIZ = N-ethyl-benzisoxazole fluoborate; angio = 1-asparagine, 5-valine angiotensin; asp = aspartic acid; arg = arginine ; val = valine ; tyr = tyrosine ; his = histidine ; pro = proline ; phe = phenylalanine; asp-NH2 = asparagine; ileu = isoleucine; Des 1,2 = omitting the first two amino acids; polylysine = poly-L-lysine. The conjugates are abbreviated to include the coupling reagent used in their synthesis, e.g. RSA-angio (Morpho CDI) = the conjugate containing albumin and angiotensin, synthesized with Morpho CDI. 223
224
TH~ODOR~GOODFRIEND,GERALDFASMAN,DANII~LKEMP and LAWRENC~LEVINE
M A T E R I A L S AND M E T H O D S
Synthesis of the immunizing antigen: rabbit serum albumin-angiotensin (RSt1anglo). Immunogenic compounds of angiotensin and rabbit serum albumin (RSA) were synthesized with the water soluble carbodiimide 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (Ethyl CDI). The proportions of reagents were: 1-asparagine, 5-valine-angiotensin (angio) 10 mg (10/~moles); RSA 5 mg (0.1/~moles); Ethyl CDI 150 mg (1 m-mole) in 0.2 ml water. The reaction mixtttre was allowed to stand at room temperature for 1-12 hr. Appearance of a cloudy or colloidal precipitate was taken as the end-point of the reaction. (With some syntheses the precipitation end-point did not occur, and an overnight incubation was arbitrarily chosen.) Upon appearance of precipitate, or at the end of incubation, the reaction mixture was dialysed against water for 48 hr. The reaction yielded roughly 5 mg of prodtlct using the above proportions. Test antigens. Two 'test antigens' were synthesized. (1) The first was produced by a reaction similar to that used for the immunizing antigen, but a different carbodiimide was used, 1-cyclohexyl-3-(2-morpholinyl (4) ethyl)-c~rbodiimide metho-p-toluene sulfonate (Morpho CDI). The second test antigen contained angiotensin coupled to polylysine. This was synthesized by the use of an anlogue of 'Woodward's reagent K', the fluoborate salt of N-ethyl-benzisoxazole (EBIZ).t6~ The reaction was performed in two stages as follows: 10 mg of angiotensin was dissolved in 0.1 ml water at pH 5.0. To this solution was added 20 mg EBIZ, and the mixture warmed slightly to dissolve the reagent. Polylysine 5 mg, and 0.15 ml of dimethylformamide were added immediately, and the pH adjusted to 8.5 with concentrated sodium hydroxide. The mixture was allowed to stand at room temperature for 2 days, after which, water (1.0 ml) was added, and the pH brought to 2.0 with concentrated hydrochloric acid. Benzene, 2 ml, was added and the mixture shaken to extract the side-products of the EBIZ reagent. The aqueous layer was dialysed to purify the antigen. Five milligrams of soluble conjugate were produced by this procedure from the above reagents. The composition of the antigens was determined by amino acid analysis, using the method of Spackman, Stein, and Moore. tT~ All complexes synthesized with the carbodiimides and albumin contained carbodiimide derivatives (substituted ureas), linked via side-chain carboxyl groups of the albumin. Therefore all such complexes were in fact doubly-substituted albumin molecules:, angiox-RSA(CDI-urea)y. The quantity of carbodiimide thus bound was not determined. Immunization. One third of the product RSA-angio (Ethyl CDI), whether precipitated or soluble, roughly 2 mg of protein, was suspended in water, 0.5 ml, with an equal volume of complete Freund's adjuvant, emulsified, and injected into the toe pads and leg muscles of albino New Zealand rabbits. Three weeks later, a booster of the same amount was mixed with Freund's adjuvant and injected intraperitioneally. Ten to fourteen days later the animals were bled and boosted again as indicated. Two rabbits received 1LSA-angio, and both produced detectable antibodies after the first booster injection. The single antiserum used in the studies reported here was obtained after a total immunization schedule of 8 weeks, including two booster injections.
Immunochemistryof Angiotensin
225
Serologic procedures. Antibodies were detected by semi-micro-complement fixation, as described by Wasserman and Levine. ~s~ This method uses a final reaction volume of 7.0 ml. Hapten inhibition of complement fixation was also performed in the semimicro system. Varying amounts of the free polypeptides were added to diluted antiserum followed by additions of complement and conjugated 'test antigen' (RSA-angio or polylysine-angio). After incubation at 4 ° for 16 hr, sensitized red cells were added and hemolysis measured. Thus, hapteninhibition was a measure of the ability of free polypeptides to prevent fixation of complement by antiserum and a complex antigen similar to the immunizing antigen. The inhibitions were specific for the antigen-antibody system described herein. Angiotensin failed to inhibit complement fixation in several other systems, and other polypep~ide hormones failed to inhibit complement fixation by antiangiotensin.~a~ Materials. Synthetic 1-asparagine, 5-valine angiotensin was the gift of Dr R. Gaunt, Ciba, Inc. Rabbit serum albumin was purchased from Pentex, Inc.; complete Freund's adjuvant from Difco Laboratories; 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride from Ott Chemicals, Muskegon, Mich. ; and 1-cyclo-hexyl-3-(2-morpholinyl (4) ethyl)-carbodiimide metho-p-toluene sulfonate from Aldrich Chemical Co., Milwaukee, Wis. The N-ethylbenzisoxazolium reagent was prepared by one of us (D.K., ref. 6). Poly-L-lysine.HCl was the gift of Dr E. Haber, Massachusetts General Hospital, and had a mean molecular weight of 17,000. Analogues of angiotensin were the gift of Dr R. Schwyzer, Ciba, Basle, Switzerland. RESULTS (A) Composition of antigens. The average composition of two of the antigenic compounds containing angiotensin are listed in Table 1. T h e proportion of angiotensin in each was calculated from the relative increase in content of amino TABLE 1. COMPOSITION OF ANTIGENS CONTAINING ANGIOTENSIN
Amino acid residues per mole Compound Polylysine Polylysineangiotensin Albumin (RSA) RSA-angiotensin
Arg
His
0
0
13 13
13 13
Pro
Phe
Val
27.2 32.7 5.5
21.4 27.9 6.5
36-9 50.7 13.8
Mean angiotensin molecules per mole of conjugate
13 6
The moles of amino acids in each carrier molecule, and respective antigenic compound containing angiotensin, were obtained by amino acid analysis using the method of Spackman, Stein and Moore. tT~ In the table, they are listed relative to amino acids which are present in the carrier but not in angiotensin: Lysine for the polylysine compound, and glycine for the albumin (RSA) compound. The figures for RSA-angiotensin using glycine as reference amino acid agree with those obtained when other amino acids are used as reference points. For the calculation of amino acid residues per mole, the number of residues of reference amino acid per mole was taken as 130 for lysines per mole of polylysine (mol. wt. 17,000), and 20 for glycines per mole of albumin (tool wt. 65,000). Note that there are two valine residues per mole of 5-val-angiotensin.
226
THEODOREGOODFRIEND,GERALDFASMAN,DANIELKEMP and LAWRENCELEVlNE
acids comprising angiotensin, compared to those not in the hormone, multiplied by the molar content of the reference amino acids in the carrier molecules. (B) Serologic properties. Antibodies induced by injection of RSA-angio-(Ethyl CDI) fixed complement with RSA-angio-(Morpho CDI), and polylysine-angio(EBIZ), (Fig. 1). Antiserum No. 360 reacted with RSA-angio-(Morpho CDI) at a dilution of 1 : 600, and with the polylysine conjugate at a dilution of 1 : 400. The proportions of reactants giving peak fixation were confirmed by repeated experiments. Antisera from the second immunized animal reacted in a similar way. All further experiments were performed with a single antiserum. I00 A
B
o
~o z
o
60
x
40
20
i
0'01
0"1
I-0 0'01
I
i
I
llil
i
I
i
i
Oq
~ ili~
I.~
~.g ANTIGEN
FIG. 1. Complement fixation by antiserum to RSA-angio-(ethyl CDI) reacting with RSA-angio-(Morpho CDI), Panel A; and with polylysineangio-(EBIZ), Panel B. The dilutions of antiserum No. 360 were 1:600 (A), and 1:400 (B).
Figures 2 and 3 show inhibition by haptens of the reaction between antiserum No. 360 and RSA-angio-(Morpho CDI), or polylysine-angio-(EBIZ). Table 2 shows this data expressed as quantity of hapten required for 50 per cent inhibition of the two complement fixation reactions. In this table a second experiment with RSA-angio is recorded to illustrate the range of experimental variation. The pattern of relative inhibitory potency of the analogues is the same (within experimental error), whether the complement fixing antigen was the RSA or polylysine compound. As. shown by the data in Table 2, angiotensin and 1-asparagine angiotensin were the most active inhibitors of the antibody reaction with both antigens; 1-D-aspartic-angiotensin was less effective in both systems; 1,2-Desangiotensin and 4,4'-tyrosine-angiotensin were another order of magnitude less potent; 4-phenylalanine was next in decreasing order; and 4-D-tyrosine angiotensin was least active in both systems. Although there were variations in absolute inhibition levels for various combinations of antiserum and antigen, it was always possible to detect as little as 0.1 ~g of free angiotensin by inhibition of complement fixation.
Immunochemistry of Angiotensin
227
I00
80 A
7
60
x
40
20
I
0
0"1
I
1
1
0'2
0"3
0.4
I
0"5
0
I
1
I
I
25
50
75
I00
/.z.g INHIBITOR
FIG. 2. Inhibition of complement fixation by free polypeptides. T h e reaction mixture contained the proportions of antiserum and R S A - a n g i o - ( M o r p h o C D I ) which gave maximum fixation in Fig. 1A, in a final volume of 7"0 ml. ~a) • = 5-val-angiotensin; o = 1-asparagine, 5-val-angiotensin; • = 1-Dasparagine, 5-val-angiotensin; x = 1,2-des, 5-val-angiotensin; [] = 4 , 4 homo-tyr, 5-val-angiotensin; • = 4-phe, 5-val-angiotensin; and ~ = 4-Dtyr, 5 val-angiotensin.
I00
80
Z
O
60
Z
#
40
20
0
I
1
l
I
0'25
0'5
0"75
I'0
^
0
~
I0
'~
I
I
I
.I
20
30
40
50
/./.,g INHIBITOR
FIC. 3. Inhibition of complement fixation by free polypeptides. T h e reaction mixture contained the proportions of antiserum and polylysine-angio-(EBIZ) which gave maximum fixation in Fig. 1B, in a final volume of 7"0 ml. is) T h e symbols refer to the same analogues as in Fig. 2.
2
Asp Asp
Arg Arg
Asp Arg Asp-NH, Arg D-Asp Arg . . . . . . Asp Arg
H~N-1
Val Val
Val Val Val Val Val
3
5
Tyr Val Tyr Val Tyr Val Tyr Val Tyr-Val Tyr Phe Val D-Tyr Val
4
His His
His His His His His
6
Pro Pro
Pro Pro Pro Pro Pro
7
Phe Phe
Phe Phe Phe Phe Phe
8-COOH
13-0 76-0
0-045 0.055 0-27 3-0 1-0
14-5 > 50-0
0-096 0.066 0.54 3-9 3.1
30.0 > 50-0
0.06 0-04 0-12 2-0 6-0
Amount of analogue required for 50 per cent inhibition of complement fixation by anti-angio and: RSA-angio Polylysine-angio (~g) (/~g) Exp. 1 Exp. 2
T h e antigen-antibody reaction was measured by complement fixation, as described in the text. T h e hapten-inhibition curves from which the data were derived are illustrated in Figs. 2 and 3.
6 7
1 2 3 4 5
Analogue No.
ANALOGUES
T A B L E 2. I N H I B I T I O N OF THE REACTION BETVCEEN R S A - - A N G I O T E N S I N OR POLYLYSINE--ANGIOTENSIN AND A N T I - - A N G I O N T E N S I N BY A N G I O T E N S I N
fl
~
~ ~ ~ ~O ~
0 0I~
O
229
Immunochemistry of Angiotensin
(C) Biological data. Comparison of the serologic activities of analogues of angiotensin in the hapten-inhibition test with their activities in two biological systems is seen in Table 3. The serologic activity was calculated as per cent relative to 5-val-angiotensin from the data of Table 2 (Exp. 1). The biological activities were obtained from the literature. ~9) The pattern of relative activities of the analogues is roughly the same in each test system. TABLE 3. RELATIVE SEROLOGIC AND BIOLOGICAL ACTIVITIES OF ANGIOTENSIN ANALOGUES a
Analogue
5 rval-angiotensin
1-asparagine, 5-val-angiotensin 1-D-asp, 5-val-angiotensin 1,2-des, 5-val-angiotensin 4,4 homo-tyr, 5-val-angiotensin 4-phe, 5-val-angiotensin 4-v-tyr, 5-val-angiotensin
Relative pressor activity (nephrectomized rat) (%) 100 100 150 <0.1 1 10 0
Relative oxytocic activity (rat uterus) (%) 100 27 <0.1 0.33 4.9
Immunologic activity (inhibition of complement fixation)~ (%) 100 150 50 3 1 0.2 <0.08
The data are calculated from Table 2, by referring all activities to 5-val-angiotensin, and from ref. (11). ~ Native angiotensin is: NHs-asp-arg-val-tyr-ileu-his-pro-phe-COOH. The analogues are abbreviated by noting the substitutent which replaces the normal amino acid in the positions indicated: e.g. 1-asparagine, 5-valine-angiotensin is an octapeptide containing asparagine in the first (amino terminal) position and valine in the fifth position, with the remainder of the residues the same as native angiotensin. ~ The antigen-antibody reaction inhibited was between anti-angiotensin and polylysineangiotensin.
DISCUSSION Production of complement fixing antibodies to angiotensin, illustrates the usefulness of the two peptide coupling reagents described in this report. This use of a carbodiimide was reported in a previous publication, t3~ The analogue of Woodward's Reagent K has not been utilized before for immunochemical investigation. In contrast to carbodiimides, it has the advantage of effecting a two-stage coupling reaction, beginning with the activation of carboxyl groups. This enables better prediction of the exact products of synthesis. The polylysine-angio-(EBIZ) synthesized using benzisoxazolium salt contained the same molar proportion of angiotensin to lysine (1:10) as an antigen previously synthesized by Haber using m-xylylene diisocyanate, t4~ This may reflect an upper limit of the possible conjugation of these two molecules imposed by the stereochemistry of the reactants and products. Thus, the free ~-amino groups of polyL-lysine may have heterogeneous reactivities due to the conformation of this polypeptide at the reaction pH. txo~ Also, the size of the angiotensin molecule and its spatial orientation to polylysine may limit the number of hormone molecules that can be attached to polylysine. Similar limitations may account for the fact that an average of only six moles of angiotensin coupled to each molecule of albumin, despite an excess of reagents and theoretically available reactive groups.
230
TI-IEODOREGOODFRIEND, GERALD FASMAN, DANEIL KEMP and LAWRENCE LEVINE
Analogues of angiotensin have been used in hapten inhibition studies to investigate requirements for the antigen-antibody reaction. The antiserum was obtained from a rabbit immunized with RSA-angio. It is probable that this immunizing antigen contained angiotensin coupled to albumin in at least two different ways: via its terminal amino group, or its terminal carboxyl group. Therefore, the antiserum may contain antibodies which are heterogeneous with respect to the spatial orientation of the hapten and carrier. To minimize the ambiguity introduced by this possible heterogeneity, we tested the antiserum with an antigen containing angiotensin coupled to a carrier in only one way: the carboxyl group of the hormone to the ~-amino groups of polylysine. Thus, we favored reactions of a population of antibodies directed toward angiotensin linked to a carrier via its carboxyl group. Despite the possible complexity of the antiserum caused by immunization with RSA-angio, the reactions of antiserum with RSA-angio and with polylysine-angio showed the same pattern of hapten inhibition with the analogues of angiotensin. For example, in the reaction with RSA-angio, 1-D-asparagine-angio was a better inhibitor than 4-phenylalanine-angio. The same relative inhibitions were seen in the reaction with polylysine-angio (Table 2). The inhibition data show the relative importance, for serologic activity of angiotensin, of the phenolic hydroxyl at position 4, the two amino acids at the N-terminus of the polypeptide, and the conformation of the chain as determined by the optical isomer of the amino acid at position 4. In contrast, the data show the relative unimportance of the free fl-carboxyl at position I, and the optical isomer at position I. The structural requirements for serologic activity in angiotensin may be the same as the .requirements for biological activity, as indicated in Table 3. One exception is the optical isomer at position I. 1-D-asparagine-angiotensin is more active than angiotensin in biological systems, but less active immunologically. It must be noted that bioassays reflect penetration, attachment, and activation of the target organ by the peptide, followed by release and degradation of the hormone. On the other hand, immunological activity involves fewer steps. Possibly, the alteration by substitution of D-asparagine at position I causes slower destruction of the peptide in the bioassay system, which over-compensates for its reduction in reactivity at other steps. The complexity of biological systems is further illustrated by the difference in activity of the analogues between the two biological systems. The overall conclusion that antibody and biological receptor sites require similar structural features of angiotcnsin would lead to the prediction that antibody would inhibit biological activity of angiotensin. Using the rabbit aortic strip, Haber et al. were unable to show inhibition of angiotensin by rabbit antiserum. 14~ Deodhar obtained weak inhibition by rabbit antiserum of the effect of angiotensin on dog blood pressure. ~sl The results of preliminary experiments with our antiserum to angiotensin suggest that the biological effects of the free polypcptide on the blood pressure of the rat are partially neutralized (Roger Hicklcr, personal communication). The antiserum also neutralizes the biological activity of angiotensin in the isolated rat uterus (.M. Webster, personal communication).
/Ic/¢noz~leclgements--Thc authors thank Drs Robert Gaunt and Robert Schwyzer, elba, Inc., for generous gifts of angiotensin and analogues. We are indebted to Miss Eleanor Wasserman and Mr Fred Castillo for technical assistance.
Immunochemistry of Angiotensin
231
REFERENCES
t SCHACHTER M. (Ed.), Muscles and Blood Vessels. Pergamon Press, New York (1960). z DEODH^R S. D., ft. exp. Med. 111, 429 (1960). ~ GOODFmEND T. L., LEVlNE L. and FASM~a'qG., Science, N . Y . 144, 1344 (1964). 4 HABEI~ E., PAGE L. B. and JAconY G. A., Biochemistry, N . Y . 4, 693 (1965). 5 UTIGER R., Personal communication. 6 KEMP D. S., Ph.D. thesis, Harvard University, 1964. Dissertation Abstracts 25, 3845 (1965). 7 SP^CKM~W D. H., STEIN W. H. and Moop~ S., Analyt. Chem. 30, 1190 (1958). 8 WA$$ERMAN E. and LEVlNE L., )t. Immunol. 87, 290 (1961). 9 SCHWYZEa R., Pure appl. Chem. 6, 265 (1963). 10 APPLEQUIST J. and DOTY P., In Polyamino Acids, Polypeptides and Proteins, (Edited by STAH1VIANM.), p. 161. University of Wisconsin Press (1962).
STUDIES
OF
ANGIOTENSIN*
THEODORE GOODFRIEND,~fGERALDFASMAN,:~DANIEL KEMP § and LAWRENCELEVlNE 11 Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts
(Received 14 October 1965) Abstract--Water-soluble carbodiimides and a benzisoxazolium salt were used to synthesize conjugates containing angiotensin coupled to albumin or poly-L-lysine. Antibodies to angiotensin were induced with a conjugate containing the hormone bound to rabbit serum albumin..The antigenic determinants of angiotensin were investigated by inhibition of complement fixation by analogues of the polypeptide. Among the structural features of angiotensin tested, those which had greatest importance for serologic activity were conformation as determined by an optical isomer in the middle of the hormone molecule; the presence of the two amino acids at the N-terminus ; and the single phenolic hydroxyl. The structural features shown to be important for serologic activity were those previously shown to be important for biological activity. INTRODUCTION THE SMALL p o l y p e p t i d e h o r m o n e s w h i c h affect s m o o t h muscles and blood vessels have b e e n studied extensively f r o m the point of view of their biological activity. Ill I m m u n o l o g i c a l investigations have b e e n h a m p e r e d b y the small size, widespread occurrence, and biological lability of these molecules. Successful i n d u c t i o n of antibodies to angiotensin, 12-~1 bradykinin, ~3~ and vasopressin 151 have been reported. W e have used conjugates containing angiotensin c o u p l e d to a l b u m i n to induce antibodies to angiotensin in rabbits, and have m e a s u r e d inhibition of these antibodies b y analogues of the natural polypeptide to s t u d y the antigenic determinants of angiotensin. Previous investigations, b y others, of the biological properties of the analogues enabled us to c o m p a r e biological and serologic properties of this polypepfide h o r m o n e . * Presented in part at the American Federation for Clinical Investigation, Atlantic City, N.J., May 1964. Publication No. 407 from the Graduate Department of Biochemistry, Brandeis University. ~f Work performed during tenure of a fellowship from the Helen Hay Whitney Foundation. Present address: Departments of Medicine and Pharmacology, School of Medicine, University of Wisconsin, Madison, Wis. ~: This work was performed during the tenure of an Established Investigatorship of the American Heart Association, and was supported in part by research grants from the National Institutes of Health (AM-05852) and the National Science Foundation (GB-428). § Department of Chemistry, Massachusetts Insitute of Technology, Cambridge, Mass. II American Cancer Society, Professor of Biochemistry (PRP-21). This work was supported in part by a research grant from the National Institutes of Health (AI-01940). Abbreviations used: RSA = rabbit serum albumin; Ethyl C D I = 1-Ethyl-3-(3-dimethylaminopropyl)ocarbodiimide hydrochloride; Morpho CDI = 1-Cyclohexyl-3-(2-morpholinyl (4) ethyl)-carbodiimide metho-p-toluene sulfonate; EBIZ = N-ethyl-benzisoxazole fluoborate; angio = 1-asparagine, 5-valine angiotensin; asp = aspartic acid; arg = arginine ; val = valine ; tyr = tyrosine ; his = histidine ; pro = proline ; phe = phenylalanine; asp-NH2 = asparagine; ileu = isoleucine; Des 1,2 = omitting the first two amino acids; polylysine = poly-L-lysine. The conjugates are abbreviated to include the coupling reagent used in their synthesis, e.g. RSA-angio (Morpho CDI) = the conjugate containing albumin and angiotensin, synthesized with Morpho CDI. 223
224
TH~ODOR~GOODFRIEND,GERALDFASMAN,DANII~LKEMP and LAWRENC~LEVINE
M A T E R I A L S AND M E T H O D S
Synthesis of the immunizing antigen: rabbit serum albumin-angiotensin (RSt1anglo). Immunogenic compounds of angiotensin and rabbit serum albumin (RSA) were synthesized with the water soluble carbodiimide 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (Ethyl CDI). The proportions of reagents were: 1-asparagine, 5-valine-angiotensin (angio) 10 mg (10/~moles); RSA 5 mg (0.1/~moles); Ethyl CDI 150 mg (1 m-mole) in 0.2 ml water. The reaction mixtttre was allowed to stand at room temperature for 1-12 hr. Appearance of a cloudy or colloidal precipitate was taken as the end-point of the reaction. (With some syntheses the precipitation end-point did not occur, and an overnight incubation was arbitrarily chosen.) Upon appearance of precipitate, or at the end of incubation, the reaction mixture was dialysed against water for 48 hr. The reaction yielded roughly 5 mg of prodtlct using the above proportions. Test antigens. Two 'test antigens' were synthesized. (1) The first was produced by a reaction similar to that used for the immunizing antigen, but a different carbodiimide was used, 1-cyclohexyl-3-(2-morpholinyl (4) ethyl)-c~rbodiimide metho-p-toluene sulfonate (Morpho CDI). The second test antigen contained angiotensin coupled to polylysine. This was synthesized by the use of an anlogue of 'Woodward's reagent K', the fluoborate salt of N-ethyl-benzisoxazole (EBIZ).t6~ The reaction was performed in two stages as follows: 10 mg of angiotensin was dissolved in 0.1 ml water at pH 5.0. To this solution was added 20 mg EBIZ, and the mixture warmed slightly to dissolve the reagent. Polylysine 5 mg, and 0.15 ml of dimethylformamide were added immediately, and the pH adjusted to 8.5 with concentrated sodium hydroxide. The mixture was allowed to stand at room temperature for 2 days, after which, water (1.0 ml) was added, and the pH brought to 2.0 with concentrated hydrochloric acid. Benzene, 2 ml, was added and the mixture shaken to extract the side-products of the EBIZ reagent. The aqueous layer was dialysed to purify the antigen. Five milligrams of soluble conjugate were produced by this procedure from the above reagents. The composition of the antigens was determined by amino acid analysis, using the method of Spackman, Stein, and Moore. tT~ All complexes synthesized with the carbodiimides and albumin contained carbodiimide derivatives (substituted ureas), linked via side-chain carboxyl groups of the albumin. Therefore all such complexes were in fact doubly-substituted albumin molecules:, angiox-RSA(CDI-urea)y. The quantity of carbodiimide thus bound was not determined. Immunization. One third of the product RSA-angio (Ethyl CDI), whether precipitated or soluble, roughly 2 mg of protein, was suspended in water, 0.5 ml, with an equal volume of complete Freund's adjuvant, emulsified, and injected into the toe pads and leg muscles of albino New Zealand rabbits. Three weeks later, a booster of the same amount was mixed with Freund's adjuvant and injected intraperitioneally. Ten to fourteen days later the animals were bled and boosted again as indicated. Two rabbits received 1LSA-angio, and both produced detectable antibodies after the first booster injection. The single antiserum used in the studies reported here was obtained after a total immunization schedule of 8 weeks, including two booster injections.
Immunochemistryof Angiotensin
225
Serologic procedures. Antibodies were detected by semi-micro-complement fixation, as described by Wasserman and Levine. ~s~ This method uses a final reaction volume of 7.0 ml. Hapten inhibition of complement fixation was also performed in the semimicro system. Varying amounts of the free polypeptides were added to diluted antiserum followed by additions of complement and conjugated 'test antigen' (RSA-angio or polylysine-angio). After incubation at 4 ° for 16 hr, sensitized red cells were added and hemolysis measured. Thus, hapteninhibition was a measure of the ability of free polypeptides to prevent fixation of complement by antiserum and a complex antigen similar to the immunizing antigen. The inhibitions were specific for the antigen-antibody system described herein. Angiotensin failed to inhibit complement fixation in several other systems, and other polypep~ide hormones failed to inhibit complement fixation by antiangiotensin.~a~ Materials. Synthetic 1-asparagine, 5-valine angiotensin was the gift of Dr R. Gaunt, Ciba, Inc. Rabbit serum albumin was purchased from Pentex, Inc.; complete Freund's adjuvant from Difco Laboratories; 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride from Ott Chemicals, Muskegon, Mich. ; and 1-cyclo-hexyl-3-(2-morpholinyl (4) ethyl)-carbodiimide metho-p-toluene sulfonate from Aldrich Chemical Co., Milwaukee, Wis. The N-ethylbenzisoxazolium reagent was prepared by one of us (D.K., ref. 6). Poly-L-lysine.HCl was the gift of Dr E. Haber, Massachusetts General Hospital, and had a mean molecular weight of 17,000. Analogues of angiotensin were the gift of Dr R. Schwyzer, Ciba, Basle, Switzerland. RESULTS (A) Composition of antigens. The average composition of two of the antigenic compounds containing angiotensin are listed in Table 1. T h e proportion of angiotensin in each was calculated from the relative increase in content of amino TABLE 1. COMPOSITION OF ANTIGENS CONTAINING ANGIOTENSIN
Amino acid residues per mole Compound Polylysine Polylysineangiotensin Albumin (RSA) RSA-angiotensin
Arg
His
0
0
13 13
13 13
Pro
Phe
Val
27.2 32.7 5.5
21.4 27.9 6.5
36-9 50.7 13.8
Mean angiotensin molecules per mole of conjugate
13 6
The moles of amino acids in each carrier molecule, and respective antigenic compound containing angiotensin, were obtained by amino acid analysis using the method of Spackman, Stein and Moore. tT~ In the table, they are listed relative to amino acids which are present in the carrier but not in angiotensin: Lysine for the polylysine compound, and glycine for the albumin (RSA) compound. The figures for RSA-angiotensin using glycine as reference amino acid agree with those obtained when other amino acids are used as reference points. For the calculation of amino acid residues per mole, the number of residues of reference amino acid per mole was taken as 130 for lysines per mole of polylysine (mol. wt. 17,000), and 20 for glycines per mole of albumin (tool wt. 65,000). Note that there are two valine residues per mole of 5-val-angiotensin.
226
THEODOREGOODFRIEND,GERALDFASMAN,DANIELKEMP and LAWRENCELEVlNE
acids comprising angiotensin, compared to those not in the hormone, multiplied by the molar content of the reference amino acids in the carrier molecules. (B) Serologic properties. Antibodies induced by injection of RSA-angio-(Ethyl CDI) fixed complement with RSA-angio-(Morpho CDI), and polylysine-angio(EBIZ), (Fig. 1). Antiserum No. 360 reacted with RSA-angio-(Morpho CDI) at a dilution of 1 : 600, and with the polylysine conjugate at a dilution of 1 : 400. The proportions of reactants giving peak fixation were confirmed by repeated experiments. Antisera from the second immunized animal reacted in a similar way. All further experiments were performed with a single antiserum. I00 A
B
o
~o z
o
60
x
40
20
i
0'01
0"1
I-0 0'01
I
i
I
llil
i
I
i
i
Oq
~ ili~
I.~
~.g ANTIGEN
FIG. 1. Complement fixation by antiserum to RSA-angio-(ethyl CDI) reacting with RSA-angio-(Morpho CDI), Panel A; and with polylysineangio-(EBIZ), Panel B. The dilutions of antiserum No. 360 were 1:600 (A), and 1:400 (B).
Figures 2 and 3 show inhibition by haptens of the reaction between antiserum No. 360 and RSA-angio-(Morpho CDI), or polylysine-angio-(EBIZ). Table 2 shows this data expressed as quantity of hapten required for 50 per cent inhibition of the two complement fixation reactions. In this table a second experiment with RSA-angio is recorded to illustrate the range of experimental variation. The pattern of relative inhibitory potency of the analogues is the same (within experimental error), whether the complement fixing antigen was the RSA or polylysine compound. As. shown by the data in Table 2, angiotensin and 1-asparagine angiotensin were the most active inhibitors of the antibody reaction with both antigens; 1-D-aspartic-angiotensin was less effective in both systems; 1,2-Desangiotensin and 4,4'-tyrosine-angiotensin were another order of magnitude less potent; 4-phenylalanine was next in decreasing order; and 4-D-tyrosine angiotensin was least active in both systems. Although there were variations in absolute inhibition levels for various combinations of antiserum and antigen, it was always possible to detect as little as 0.1 ~g of free angiotensin by inhibition of complement fixation.
Immunochemistry of Angiotensin
227
I00
80 A
7
60
x
40
20
I
0
0"1
I
1
1
0'2
0"3
0.4
I
0"5
0
I
1
I
I
25
50
75
I00
/.z.g INHIBITOR
FIG. 2. Inhibition of complement fixation by free polypeptides. T h e reaction mixture contained the proportions of antiserum and R S A - a n g i o - ( M o r p h o C D I ) which gave maximum fixation in Fig. 1A, in a final volume of 7"0 ml. ~a) • = 5-val-angiotensin; o = 1-asparagine, 5-val-angiotensin; • = 1-Dasparagine, 5-val-angiotensin; x = 1,2-des, 5-val-angiotensin; [] = 4 , 4 homo-tyr, 5-val-angiotensin; • = 4-phe, 5-val-angiotensin; and ~ = 4-Dtyr, 5 val-angiotensin.
I00
80
Z
O
60
Z
#
40
20
0
I
1
l
I
0'25
0'5
0"75
I'0
^
0
~
I0
'~
I
I
I
.I
20
30
40
50
/./.,g INHIBITOR
FIC. 3. Inhibition of complement fixation by free polypeptides. T h e reaction mixture contained the proportions of antiserum and polylysine-angio-(EBIZ) which gave maximum fixation in Fig. 1B, in a final volume of 7"0 ml. is) T h e symbols refer to the same analogues as in Fig. 2.
2
Asp Asp
Arg Arg
Asp Arg Asp-NH, Arg D-Asp Arg . . . . . . Asp Arg
H~N-1
Val Val
Val Val Val Val Val
3
5
Tyr Val Tyr Val Tyr Val Tyr Val Tyr-Val Tyr Phe Val D-Tyr Val
4
His His
His His His His His
6
Pro Pro
Pro Pro Pro Pro Pro
7
Phe Phe
Phe Phe Phe Phe Phe
8-COOH
13-0 76-0
0-045 0.055 0-27 3-0 1-0
14-5 > 50-0
0-096 0.066 0.54 3-9 3.1
30.0 > 50-0
0.06 0-04 0-12 2-0 6-0
Amount of analogue required for 50 per cent inhibition of complement fixation by anti-angio and: RSA-angio Polylysine-angio (~g) (/~g) Exp. 1 Exp. 2
T h e antigen-antibody reaction was measured by complement fixation, as described in the text. T h e hapten-inhibition curves from which the data were derived are illustrated in Figs. 2 and 3.
6 7
1 2 3 4 5
Analogue No.
ANALOGUES
T A B L E 2. I N H I B I T I O N OF THE REACTION BETVCEEN R S A - - A N G I O T E N S I N OR POLYLYSINE--ANGIOTENSIN AND A N T I - - A N G I O N T E N S I N BY A N G I O T E N S I N
fl
~
~ ~ ~ ~O ~
0 0I~
O
229
Immunochemistry of Angiotensin
(C) Biological data. Comparison of the serologic activities of analogues of angiotensin in the hapten-inhibition test with their activities in two biological systems is seen in Table 3. The serologic activity was calculated as per cent relative to 5-val-angiotensin from the data of Table 2 (Exp. 1). The biological activities were obtained from the literature. ~9) The pattern of relative activities of the analogues is roughly the same in each test system. TABLE 3. RELATIVE SEROLOGIC AND BIOLOGICAL ACTIVITIES OF ANGIOTENSIN ANALOGUES a
Analogue
5 rval-angiotensin
1-asparagine, 5-val-angiotensin 1-D-asp, 5-val-angiotensin 1,2-des, 5-val-angiotensin 4,4 homo-tyr, 5-val-angiotensin 4-phe, 5-val-angiotensin 4-v-tyr, 5-val-angiotensin
Relative pressor activity (nephrectomized rat) (%) 100 100 150 <0.1 1 10 0
Relative oxytocic activity (rat uterus) (%) 100 27 <0.1 0.33 4.9
Immunologic activity (inhibition of complement fixation)~ (%) 100 150 50 3 1 0.2 <0.08
The data are calculated from Table 2, by referring all activities to 5-val-angiotensin, and from ref. (11). ~ Native angiotensin is: NHs-asp-arg-val-tyr-ileu-his-pro-phe-COOH. The analogues are abbreviated by noting the substitutent which replaces the normal amino acid in the positions indicated: e.g. 1-asparagine, 5-valine-angiotensin is an octapeptide containing asparagine in the first (amino terminal) position and valine in the fifth position, with the remainder of the residues the same as native angiotensin. ~ The antigen-antibody reaction inhibited was between anti-angiotensin and polylysineangiotensin.
DISCUSSION Production of complement fixing antibodies to angiotensin, illustrates the usefulness of the two peptide coupling reagents described in this report. This use of a carbodiimide was reported in a previous publication, t3~ The analogue of Woodward's Reagent K has not been utilized before for immunochemical investigation. In contrast to carbodiimides, it has the advantage of effecting a two-stage coupling reaction, beginning with the activation of carboxyl groups. This enables better prediction of the exact products of synthesis. The polylysine-angio-(EBIZ) synthesized using benzisoxazolium salt contained the same molar proportion of angiotensin to lysine (1:10) as an antigen previously synthesized by Haber using m-xylylene diisocyanate, t4~ This may reflect an upper limit of the possible conjugation of these two molecules imposed by the stereochemistry of the reactants and products. Thus, the free ~-amino groups of polyL-lysine may have heterogeneous reactivities due to the conformation of this polypeptide at the reaction pH. txo~ Also, the size of the angiotensin molecule and its spatial orientation to polylysine may limit the number of hormone molecules that can be attached to polylysine. Similar limitations may account for the fact that an average of only six moles of angiotensin coupled to each molecule of albumin, despite an excess of reagents and theoretically available reactive groups.
230
TI-IEODOREGOODFRIEND, GERALD FASMAN, DANEIL KEMP and LAWRENCE LEVINE
Analogues of angiotensin have been used in hapten inhibition studies to investigate requirements for the antigen-antibody reaction. The antiserum was obtained from a rabbit immunized with RSA-angio. It is probable that this immunizing antigen contained angiotensin coupled to albumin in at least two different ways: via its terminal amino group, or its terminal carboxyl group. Therefore, the antiserum may contain antibodies which are heterogeneous with respect to the spatial orientation of the hapten and carrier. To minimize the ambiguity introduced by this possible heterogeneity, we tested the antiserum with an antigen containing angiotensin coupled to a carrier in only one way: the carboxyl group of the hormone to the ~-amino groups of polylysine. Thus, we favored reactions of a population of antibodies directed toward angiotensin linked to a carrier via its carboxyl group. Despite the possible complexity of the antiserum caused by immunization with RSA-angio, the reactions of antiserum with RSA-angio and with polylysine-angio showed the same pattern of hapten inhibition with the analogues of angiotensin. For example, in the reaction with RSA-angio, 1-D-asparagine-angio was a better inhibitor than 4-phenylalanine-angio. The same relative inhibitions were seen in the reaction with polylysine-angio (Table 2). The inhibition data show the relative importance, for serologic activity of angiotensin, of the phenolic hydroxyl at position 4, the two amino acids at the N-terminus of the polypeptide, and the conformation of the chain as determined by the optical isomer of the amino acid at position 4. In contrast, the data show the relative unimportance of the free fl-carboxyl at position I, and the optical isomer at position I. The structural requirements for serologic activity in angiotensin may be the same as the .requirements for biological activity, as indicated in Table 3. One exception is the optical isomer at position I. 1-D-asparagine-angiotensin is more active than angiotensin in biological systems, but less active immunologically. It must be noted that bioassays reflect penetration, attachment, and activation of the target organ by the peptide, followed by release and degradation of the hormone. On the other hand, immunological activity involves fewer steps. Possibly, the alteration by substitution of D-asparagine at position I causes slower destruction of the peptide in the bioassay system, which over-compensates for its reduction in reactivity at other steps. The complexity of biological systems is further illustrated by the difference in activity of the analogues between the two biological systems. The overall conclusion that antibody and biological receptor sites require similar structural features of angiotcnsin would lead to the prediction that antibody would inhibit biological activity of angiotensin. Using the rabbit aortic strip, Haber et al. were unable to show inhibition of angiotensin by rabbit antiserum. 14~ Deodhar obtained weak inhibition by rabbit antiserum of the effect of angiotensin on dog blood pressure. ~sl The results of preliminary experiments with our antiserum to angiotensin suggest that the biological effects of the free polypcptide on the blood pressure of the rat are partially neutralized (Roger Hicklcr, personal communication). The antiserum also neutralizes the biological activity of angiotensin in the isolated rat uterus (.M. Webster, personal communication).
/Ic/¢noz~leclgements--Thc authors thank Drs Robert Gaunt and Robert Schwyzer, elba, Inc., for generous gifts of angiotensin and analogues. We are indebted to Miss Eleanor Wasserman and Mr Fred Castillo for technical assistance.
Immunochemistry of Angiotensin
231
REFERENCES
t SCHACHTER M. (Ed.), Muscles and Blood Vessels. Pergamon Press, New York (1960). z DEODH^R S. D., ft. exp. Med. 111, 429 (1960). ~ GOODFmEND T. L., LEVlNE L. and FASM~a'qG., Science, N . Y . 144, 1344 (1964). 4 HABEI~ E., PAGE L. B. and JAconY G. A., Biochemistry, N . Y . 4, 693 (1965). 5 UTIGER R., Personal communication. 6 KEMP D. S., Ph.D. thesis, Harvard University, 1964. Dissertation Abstracts 25, 3845 (1965). 7 SP^CKM~W D. H., STEIN W. H. and Moop~ S., Analyt. Chem. 30, 1190 (1958). 8 WA$$ERMAN E. and LEVlNE L., )t. Immunol. 87, 290 (1961). 9 SCHWYZEa R., Pure appl. Chem. 6, 265 (1963). 10 APPLEQUIST J. and DOTY P., In Polyamino Acids, Polypeptides and Proteins, (Edited by STAH1VIANM.), p. 161. University of Wisconsin Press (1962).