Eur. J. Biochem. 18 (1971) 561-572

Distinct Antigenic Specificities of Alanine Peptide Determinants Attached to Protein Carriers via Terminal Amino or Carboxyl Groups

Israel SCHECHTER, Enrico CLERICI, and Etti ZAZEPITZKI Department of Chemical Immunology, The Weixmann Institute of Science, Rehovot

(Received September 2/November 26, 1970)

L-Alanine chains, serving as haptenic groups were coupled to proteins in opposite directions. There was no cross-precipitation reaction between “anti-amino-alanyl” antibodies (obtained by immunization with conjugates containing alanine chains with free a-amino groups) and “anti- alanyl-carboxyl” antibodies (obtained by immunization with conjugates containing alanine chains with free a-carboxyl groups). The properties of the combining sites of these antibodies were evaluated by means of the hapten-inhibition method. For the inhibition of the precipitin reaction, series of free peptides and peptide derivatives (blocked in the amino or carboxyl func- tion) composed of L- and D-alanine residues were employed. The results led to the conclusion that the overall properties of the combining sites in both systems, but not their specificity, were quite similar. The size of the combining sites of “anti-amino-alanyl” antibodies is such as to accommo- date a tripeptide, whereas that of “anti-alanyl-carboxyl” antibodies is such as to accommodate tri-to a tetrapeptide. I n both systems the most exposed portion of the antigenie determinant is immunodominant . Thus, “anti-amino-alanyl” antibodies bind very poorly peptides in which the amino terminal end was altered (e.g., by replacing the N-terminal L-alanine residue by a D-residue, or by N-acetylation), but will combine very effectively with amides of L-alanine pep- tides. Correspondingly, “anti-alanyl-carboxyl” antibodies react very poorly with alanine peptides in which the carboxyl terminal end was altered, e.g., they do not bind amides of L-alanine peptides. Both types of antibodies can react with free L-alanine peptides, but in their combining sites these peptides are probably bound in opposite directions. Thus, antibodies with distinctly different specificities can be elicited against L-alanine peptide determinants, depending on their mode of attachment in the immunogen.

It is also shown that more precise information on the combining sites is obtained by comparing pairs of haptens of equal size which differ from one another in a particular feature, rather than from studying the effect of elongation in a homologous series of haptens.

Antibodies specific to alanine chains can be easily obtained in various animal species by injecting poly- alanyl proteins [a-61, the alanyl moieties in the conjugates serving as haptenic groups. The antigens used in these studies were prepared either by poly- merization of N-carboxy-L(or D)-alanine anhydride on the &-amino groups of lysine residues in the pro- tein [2--41, or by reacting the protein with the N-hydroxysuccinimide ester of alanine peptides [5]. In both procedures the attached alanine chains carry free N-terminal a-amino groups. The anti- alanyl antibodies formed were found to be stereo- specific, as expected. From the capacity of various alanine peptide diastereoisomers to inhibit the precipitin reaction it was concluded that the size of the combining site is such as to accommodate 3 to 4 alanine residues. Furthermore, the region of the

Note. Abbreviations for amino acid derivatives and peptides follow CBN rules, see Eur. J . Biochem. 1 (1967) 375.

antigenic determinant furthest removed from the protein carrier (the N-terminal alanine residue) was of paramount importance in determining the speci- ficities of the antibodies formed [3,5].

Lendsteiner et al. [7] used peptide-azoproteins as antigens. The peptides (composed of glycine and leucine) were nitrobenzoylated, reduced to the amino compounds and then coupled to proteins via an azo linkage. By this procedure the most exposed portion of the conjugated peptide is the C-terminal a-carb- oxyl group. On the basis of cross-precipitation ex- periments and qualitative inhibition studies, Land- steiner concluded that the specificity of the anti- peptidyl antibodies formed was determined by the amino acid carrying the free a-carboxyl group, and to a lesser degree by the second amino acid.

It was now of interest to investigate the properties of antibodies specific to alanine chains carrying free C-terminal a-carboxyl groups and to compare them to antibodies directed to alanine chains carrying free

Distinct Antigenic Specificities of Alanine Chains Determinants Eur. J. Biochem. 562

NH3 H3C-b H3C-iH NH i " t H3c3; HE$

H3C-b H3C-iH NH i " t

1 J

H$-f

NH

A B

Fig. 1. Schematic representation of two polyalanyl proteins containing alanine chains which are bound in opposite direc- tions. (A) Poly-L-alanine-human serum albumine, prepared by polymerization of N-carboxy-L-alanine anhydride. The chains are of somewhat variable size and carry free a-amino groups. (B) Human serum albumin-tetra-L-alanine, prepared by reaction with iodoacetyl-tetra-L-alanine. All chains are of identical size and carry free a-carboxyl groups. Amow represents an alanine residue with the head pointing to its C-terminus. Bold face -NH stands for e-amino groups of lysine residues of the protein. HSA = human serum albumin

N-terminal a-amino groups. For this purpose protein conjugates containing L-alanine chains carrying free C-terminal a-carboxyl groups were prepared and injected into rabbits. The size and nature of the combining sites of the antibodies formed were evaluated from cross-precipitation reactions and from inhibition experiments (using series of free and blocked alanine peptides to inhibit the precipitin reaction). The results led to the conclusion that antibodies with distinctly different specificities can be elicited against L-alanine peptide determinants, depending on the mode of presentation in the immu- nogen. Fig. 1 shows schematically two immunogens containing L-alanyl peptide determinants : in one, the exposed portions of the attached alanine chains carry free a-carboxyl groups, in the other they carry free a-amino groups.

A preliminary report on this work was submitted a t the Second Meeting of the Israel Immunological Society [l].

MATERIALS AND METHODS

Human serum albumin (crystallized), and rabbit serum albumin (crystallized) were obtained from Mann Research Laboratories (New York). Ribo- nuclease A, 5 x recrystallized, and N-acetyl-L-alanine were purchased from Sigma (St. Louis, Mo.). L-Ma- nine amide hydrochloride, L-alanine methyl ester hydrochloride and L-alanine benzyl ester hydro-

chloride were obtained from Miles-Yeda (Rehovot, Israel). Other reagents and solvents were of reagent grade.

The p-nitrobenzyl esters of L-alanine peptides and the various diastereoisomers of free alanine pept>ides, used as inhibitors of the precipitin reactions, were described before [S].

Benzyloxymrbonyl-L-ahnine Peptide Amides These were synthesized stepwise by coupling the

N-hydroxysuccinimide ester of benzyloxycarbonyl- L-alanine (m.p. 123") reported [9] 123-123.5") to the a-amino group of the corresponding L-alanine peptide amide. As all peptides were prepared in a similar manner, the synthesis of only one, Z-Ala-Ala- Ala * NH,, is given.

A solution of 10mmoles of the N-hydroxy- succinimide ester of benzyloxycarbonyl-L-alanine in 10 ml dimethylformamide was added to a solution of 10 mmoles L-alanyl-L-alanyl amide hydrobromide and 10 mmoles triethylamine in 10 ml dimethyl- formamide and 3 ml water (water was necessary to solubilize completely the peptide amide). Within a few minutes the product started to precipitate out in the reaction mixture and after 1 h a t room temper- ature water (100 ml) was added t o achieve complete precipitation. The product was collected on a sintered glass filter, washed with water, 0.5 M KHCO,, water, 0.02N HC1 and water, and dried in vacuo over sulfuric acid and potassium hydroxide. Yield of Z-Ala-Ala-Ala * NH,, 3.2 g (73O/,).

This method gave good yields (70 to 9O0/,) in all cases, and the purity of the compounds a t this stage was satisfactory (correct elementary analysis). Nevertheless, in order to avoid trace contamination with reactants, the compounds (1 g) were refluxed for 10 min in alcohol (30 ml). The material was not completely solubilized. The alcoholic suspension was kept overnight at 4" and then the compound was collected and dried. Yield was over 9501,. Analytical data of the benzyloxycarbonyl-L-alanine peptide amides are given in Table 1.

I;-Alanine Peptide Anaide Hydrobromides The benzyloxycarbonyl group was removed from

benzyloxycarbonyl-L-alanine peptide amide by the hydrogen bromide method [lo]. N-blocked peptide amides (1 g) were dissolved in 30°/, (w/v) HBr in acetic acid (6 ml) and kept a t room temperature for 15min. Anhydrous ether (60ml) was added, the precipitate formed was allowed to settle, and the supernatant was decanted. The crude product (usually a semisolid) was twice shaken with ether and dried in vacuo for 30min. It was dissolved in 5 to 10 ml water, reprecipitated by 50 ml alcohol and 50 ml ether, collected on a sintered glass filter, and

563 VoLi8, h'o.4,1971 I. SCHECHTER, E. CLERICI, and E. ZAZEPITZEI

Table 1. Analytical data of amides of benzyloxycarbonyl-L-alanine peptides ~

Molecular Calcd. Found

C H N C H N weight M.p. Compound Formula

Z-Ala, . NH, C,,H19N,04 293 225 57.4 6.5 14.3 57.5 6.5 14.3 Z-Ala, . NH, Cl,H,,N,O, 364 266 56.0 6.6 15.4 55.9 6.6 15.6 Z-Ala, . NH, C,,H,9N,0,, 435 275 55.1 6.7 16.1 54.9 6.7 16.4

Table 2. Analytical data and specific rotations of amides of L-alanine peptides Halogen was determined by aqueous titration with mercuric chloride [ill. a-Amine was determined by anhydrous titration in acetic acid with perchloric acid in the presence of excess mercuric acetate [12]. The ratio of total nitrogen (micro-Kjeldahl) to (n + 1) times (n = 1-5) amino nitrogen (Van Slyke) should equal unity. Specific rotation was memured witha Cary 60

spectrophotopolarimeter

Compound Neutral equivalents

Halogen u-Amine

Molecular from titration of Nt0tC.l r u1;* weight (n + 1)Namim (in 0.1 N HCI)

HCl H . Ala - NH, 124.5 126 124 0.96 + 9.8 (c 1.5) HBr H . Ah, * NH, 240 236 239 1.01 - 14.6 ( C 1.0)

- 59.0 (C 0.6) HBr H * Ah, * NH, 382 379 380 0.96 - 96.8 ( C 0.6) HBr H * Ale, * NH, 453 464 451 0.96 -119 (C 0.6)

HBr H - Ala, - NH, 311 308 308 0.99

dried in vacuo over sulfuric acid and potassium hydroxide. Yields were between 70°/, and 95O/,. The purity of the compounds was determined in four ways: (a) by high voltage paper electrophoresis a t pH 1.4 [S] where the peptide amides are separated according to their size, thus contamination by the reactants in the synthesis would show up as addi- tional spots. With ninhydrin reagent only one spot was detected on the electrophoresis sheet a t the appropriate distance from the application point (400 nmoles peptide load). (b) By chromatography in n-butanol-acetic acid-water, one spot was detected. (c) By determining the neutral equivalent; using mercuric chloride for tritation of the halogen in aqueous medium [ill, and perchloric acid for titra- tion of the a-amino group under anhydrous condi- tions [12]. (d) By determining the ratio of total nitrogen (micro-Kjeldahl) to amino nitrogen (Van Slyke), which should be equal to n + 1, where n = number of alanine residues in the peptide. The data for (c) and (d), as well as the specific rotations of the peptides, are given in Table 2.

N- Acetyl-L-ahnine Peptides The following compounds were prepared : Ac-Ala-

Ala OH, Ac-Ala-Ala-Ala - OH, Ac-Ala-Ala-Ala-Ala * OH. The compounds were prepared by reacting the free L-alanine peptides with acetic anhydride in water and the products were then recrystallized. The compounds did not react with ninhydrin (showing the absence of free a-amino groups) and by anhy- drous titration with sodium methoxide, the neutral 38 Eur. J. Biochem., Voi. 18

equivalent values obtained were within 201, of the theoretical ones. Further characterization and ana- lytical data of these peptides will be published elsewhere [13].

Iodoacetyl-hydroxysuccini~ide Ester To a solution containing 50 mmoles iodoacetic

acid and 50 mmoles N-hydroxysuccinimide in 50 ml dioxane and 20 ml ethylacetate a t +4" was added 50 mmoles N,N'-dicyclohexylcarbodiimide. After 12 h a t + 4" the N,N'-dicyclohexylurea formed (11.1 g, m.p. 230") was removed by Gltration, the volume of the filtrate was reduced to 20 ml by flash evaporation and then 30ml isopropanol was added. After 18h a t +4" the crystals which appeared were collected on a sintered glass filter, washed with isopropanol and ether and dried in vmuo. Yield, 7.7 g (540/,), m.p. 147". Analysis: calcd. for C,H,INO, (283): C, 25.4; H, 2.1; N, 4.95; I, 44.9. Found: C, 25.3; H, 2.2; N, 4.95; I , 45.10/,.

Iodoacetyl-tetra-L-alanine Tetra-L-alanine ( 2 mmoles) and 4 mmoles

NaHCO, in 50 ml water were mixed with 2 mmoles iodoacetyl-hydroxysuccinimide ester in 20 ml diox- ane. After 12 h a t +4" the dioxane in the reaction mixture was removed by flash evaporation, and 20 ml water and 5 mmoles HC1 were added. Gelatin- ous matter which was formed was collected on a sintered glass filter, washed with 0.02N HC1 and water and dried in vacuo. Yield, 0.55 g (59O/,).

564 Distinct Antigenic Specificities of Alanine Chains Determinants Eur. J. Biochem.

Table 3. Characterization of polypeptidyl protein prepared by wupling of preformed peptides HSA = human serum albumin; RSA = rabbit serum albumin

PeDtide attached

Designation Iodoacetyl-tetra- A,anine attached From From reduction alanine in lysine and attached histidine

and no. of derivative L-alanine

glg protein moleslmole protein

HSA-(LAla,),, (1355) 0.28 145 36 34

RNase-(LAla,),., (1357) 0.35 29 7.3 7.3 RSA- (~Ala,),, (1356) 0.22 87 22 26

Table 4. Characterization of polypeptidyl proteins prepared by polymerization technique HSA = human serum albumin; RSA = rabbit serum albumin

Designation and no. of derivative

N-Carboxy- L-alanine anhydride Alanine attached Peptide chains

Average no. of amino acid

residues

no./chain g/g protein moleslmole protein moles/mole derivative

Poly-L-Ala-HSA (1330) 0.8 Polv-L- Ala-RSA (569) 0.8

120 222

25 26

4.8 8.5

Analysis: calcd. for C,,H,,IN,O, (470): N, 11.9; I, 27.0. Found: N, 11.9; I, 24.5O/, (no reaction with ninhydrin, incidating blocking of a-amino groups). No effort was made to purify this compound and it was used as such for further synthesis.

Peptidyl Proteins Prepared with Preformed Peptides. General Procedure

A solution (18 ml) containing 0.5 g protein, 8 M urea and 0.1 M NaHCO, was brought to pH 10 with 1 N NaOH. Iodoacetyl-tetra-L-alanine (0.11 -0.18 g) was added and it dissolved immediately. After 3 days a t 37" the reaction mixture was dialyzed, and then lyophilized. Yield: 0.5 g. The protein conjugates prepared, details of the synthesis, and analytical data are given in3Table 3. By this procedure the attached peptides carry free C-terminal a-carboxyl groups.

Peptidyl Proteins Prepared by Polymerization Technique

The N-carboxy-L-alanine anhydride was reacted with the protein in a water-dioxane mixture as de- scribed before [3]. Analytical data of the protein conjugates prepared are given in Table 4. In this procedure the attached poly-L-alanine chains carry free N-terminal a-amino groups.

Immunoadsorbents Conjugates (0.3 g) of poly-L-alanine or tetra-r,

alanine with rabbit serum albumin were reacted with bromoacetyl cellulose (I g) as described previous- ly [14]. The physical adsorption was performed a t pH 4.6. About 150 mg of pursed antibodies could

be extracted from the appropriate antiserum by means of 1 g immunoadsorbent.

Immunization and Bleeding Procedures Randomly bred rabbits (2.5-3.5 kg) of both

sexes were used. Four injections of the antigen (IOmg per injection) in complete Preund's adju- vant [3] were administered a t fortnightly intervals. The first injection was divided among the four foot- pads, the other three were injected intramuscularly. Bleeding was started after the second injection and then a t weekly intervals for another two months. Quantitative studies were carried out on pooled antisera (P-1 to P-5) obtained from groups of four to six animals.

Purified Antibody Practions The immunospecific isolation of antibodies from

the serum was achieved by means of adsorption on the appropriate immunoadsorbent according to published procedure [14,15]. The adsorbed anti- bodies were dissociated from the immunoadsorbent stepwise : by tetra-L-alanine dissolved in phosphate- buffered saline (0.9O/, sodium chloride, 0.01 M phos- phate buffer pH 7.3) to yield Ab-4 fraction, and then by 0.1 M acetic acid to yield Ab-Ac fraction.

Immunoadsorbent (2 g) was suspended in serum (200 to 300ml), kept with stirring overnight a t 4') and then the serum was removed by centrifugation. This was repeated until the absorbance a t 280nm of the washing fluid (phosphate-buffered saline) was less than 0.04. The first elution was carried out by suspending the immunoadsorbent-antibody com- plex in 100 ml of 0.01 M tetra-L-alanine in phosphate-

voi.18, No.4, 1971 1. SCHECEPTER, E. CLERICI, and E. ZAZEPITZKI 565

Table 5. Purified antibody fractions Antisera were adsorbed on water insoluble immunoadsorbent and the purified antibodies were obtained by stepwise extraction, first by 0.01 M tetra-L-alanine in phosphate-buffered saline to yield Ab-4 fraction, and then by 0.1 M acetic acid to yield Ab-Ac fraction. RSA-(L-Ala),-cellulose conjugate was used for adsorption of P-1, P-2 and P-3. Poly-L-Ala-RSA-cellulose conjugate

was used for adsorption of P-4 and P-5. HSA = human serum albumin: RSA = rabbit serum albumin Ab-4 fraction Ab-Ac fraction

extracted by antigen extracted by antigen

Serum Antibody pool Immunogen in serum Protein Precipitated Protein Precipitated Total yield

mg mg "1. mg "I* "1.

P-1 HSA-(L-Ala), 660 308 64 370 65 103 P-2 HSA-(L-Ala), 304 103 55 197 56 99 P-3 HSA-(L-Ala), 580 250 76 312 73 97

P-5 poly-L-Ala-RSA 380 137 72 231 62 97 P-4 poly-L-Ala-RSA 710 334 77 270 58 85

buffered saline, stirring for 30 min a t 37", and collect- ing the supernatant after centrifugation. The second elution was performed as before except that 60 ml of tetra-L-alanine solution was used. The amount of protein extracted in this step was 15-30°/0 of that extracted in the previous step (these fractions were combined). The third elution was carried out with 100 ml of 0.1 M acetic acid. The tetra-L-alanine and acetic acid were removed from the eluates by ex- haustive dialysis against phosphate-buffered saline a t 4" (4 days with frequent changes of the buffered saline). The dialyzed solutions were centrifuged to re- move precipitates which were formed and then con- centrated (to about 2 mg/ml) by ultrafiltration [16]. The amount of antibody eluted from the immuno- adsorbent was calculated from the absorbance of the solution at 280 nm, using the value E1,:Em = 1.4 for 1 mglml of IgG. The amounts of antibodies obtained and their precipitability with antigens are given in Table 5 .

Since L-alanine peptides are readily degraded in serum [3] the purified antibodies were kept with 0.01 M tetra-L-alanine under the conditions of inhibition experiments (see below). The reaction mixtures were then analyzed by high voltage paper electrophoresis at pH 1.4 [8] and by paper chromatog- raphy in butanol-acetic acid-water. It was found that Ab-4 and Ab-Ac fractions were devoid of any proteolytic activity. Ultracentrifugal analyses of the fractions obtained from antisera P-1 and P-4 showed one peak with values of ~ 2 0 , ~ = 6.2-6.4 S, corre- sponding to that of IgG.

Quantitative Precipitin Studies To a constant volume of antiserum or of pursed

antibody fraction (0.2 to 0.5 ml) increasing amounts of the precipitant (the amount of precipitant was determined by Kjeldahl nitrogen analysis, using the factor of 6.25 for conversion from pg N to pg pro- tein) dissolved in phosphate-buffered saline were added, and the h a 1 volume was brought to 1.7 ml with phosphate-buffered saline. The reaction mix- 38'

ture was kept at 37" for 30min and then a t 5" for 18 h. Precipitates formed were separated by centri- fugation, washed twice with 2.0 ml of phosphate- buffered saline, dissolved in 1.1 ml of 0.1 N sodium hydroxide, and the absorbance a t 280 nm was deter- mined.

The percent of purified antibody fraction pre- cipitated by antigen was calculated from the ab- sorbance of the dissolved precipitate and of the supernatant, obtained a t the optimal zone of the precipitin curve, and both values agreed within 50/,. In this region the amount of antigen is small, and it did not exceed So/, of the total absorbance re- corded.

Quantitative Inhibition Studies Inhibition was estimated by measuring the

absorbance of the precipitate (in 0.1 N NaOH) formed in a mixture which contained peptide, purified anti- body and precipitant. Constant amounts of purified antibody and precipitant were used. These were chosen from the optimal zone of the precipitin curve to yield a precipitate which in 0.1 N NaOH solution would have an absorbance a t 280nm i n the range of 0.6 to 0.7. The reagents were mixed as follows: varying amounts of peptide dissolved in phosphate- buffered saline were added to a constant volume of purified antibody, the volume was adjusted to 1.5 ml with phosphate-buffered saline, the mixture was kept at 37" for 30 min, a constant amount of preci- pitant in 0.2ml of phosphate-buffered saline was added, and the reaction mixture was kept for an additional 30 min a t 37", and then a t 4" for 18 h. The precipitates formed were separated by centrifuga- tion and were treated as described above for quanti- tative precipitin studies.

Amino Acid Analysis The protein samples were subjected to hydrolysis

with 6 N hydrochloric acid in sealed tubes a t 110" for 24h. The amino acids were then determined quantitatively [17] with the Beckman-Spinco auto- matic amino acid analyzer Model 120B.

566 Distinct Antigenic Specificities of Alanine Chains Determinants Eur. J. Blochem.

Ultracentrifugal Analysis Solutions of purified antibody fractions (O.go/,)

in phosphate-buffered saline were sedimented (59 780 rev./min) a t 20” in a Spinco Model E ultracentrifuge.

RESULTS Synthesis and Characterization of Protein

Conjugates Prepared with Preformed Peptides The compound iodoacetyl-tetra-L-alanine reacts

with the &-amino groups of lysine residues and with histidine-ring nitrogens in the protein, yielding the corresponding carboxymethyl-tetra-L-alanine deri- vatives. The attached peptides are of equal size and composition, and they carry free C-terminal a-carb- oxyl groups. The average number of peptides coupled to a protein molecule was calculated in two ways. (a) The amount of alanine attached was calculated from amino acid analyses of the native protein and of the conjugate. By dividing this value by 4, the average number of tetraalanine peptides in a con- jugated protein molecule is obtained. (b) The carboxy- methyl derivatives of lysine and histidine could not be determined accurately from the amino acid analysis of the acid hydrolyzate. Therefore, the reduction in the number of moles of lysine and histidine in the acid hydrolyzate of the protein conjugate was used for calculating the number of peptides attached. The agreement between the results obtained by two independent methods (see Table 3) shows that only one peptide was attached to a lysine or histidine residue in the protein. Under the con- ditions employed, in the presence of 8 M urea (see Materials and Methods), the coupling reaction was fairly efficient: 65 to 75O/, of the iodoacetyl-tetra- peptide was attached to the protein. In preliminary experiments it was found that only ZO10 of the peptide was conjugated if the urea was omitted from the reaction mixture.

Precipitin Reactions of Anti-Alanyl Antibodies The rabbit antibodies specific to alanine chains

were detected by using rabbit serum albumin derivatives for precipitation. In this case the protein moiety of the precipitant is incapable of reacting with any antibody formed. For convenience, anti- bodies to alanine chains carrying free a-carboxyl groups (obtained by immunizing with conjugates of human serum albumin and RNase with tetra-L- alanine) will be designated “anti-alanyl-carboxyl” antibodies, whereas antibodies to alanine chains carrying a-amino groups (obtained by immunizing with a conjugate of human serum albumin and poly-L-alanine) will be designated “anti-amino- alanyl” antibodies.

Qualitative precipitin tests were carried out with sera withdrawn from individual rabbits. When sera

Precipitant (Kglml serum)

Fig. 2. Quantitative precipitin reactions of “anti-alanyl- carboxyl” and of “anti-amino-alanyl” antibodies. Absorbances a t 280 nm of solutions in 0.1 N sodium hydroxide (1.1 ml) of precipitates obtained by the addition of rabbit serum albumin-tetra-L-alanine (0) and poly-L-alanine-rabbit serum albumin ( 0 ) to antisera against: (A) human serum albumin- tetra-L-alanine and (B) poly-L-alanine-human serum albumin

(0.5 ml) of animals injected with human serum albumin-tetra-~-alanine or RNase-tetra-~-alanine conjugates were mixed with rabbit serum albumin- tetra-L-alanine, precipitates were formed in all cases ; no reaction was observed by adding poly-L-alanine- rabbit serum albumin. On the other hand, sera of animals injected with poly-L-alanine-human serum albumin reacted well with poly-L-alanine-rabbit serum albumin but not with rabbit serum albumin- tetra-L-alanine. Typical quantitative precipitin cur- ves, obtained on pooled sera, are given in Fig.2. It is seen that there is no cross-precipitation reaction between “anti-alanyl-carboxyl” and “anti-amino- alanyl” antibodies (identical results were obtained with the purified antibody fractions). The qualitative and quantitative precipitin reaction studies showed that the extent offormation of anti-alanyl antibodies, of either type, was of the same order of magnitude.

Inhibition Studies of “Anti-Alanyl-Carboxyl” Antibodies

Sera obtained from three groups of rabbits (P-I to P-3) immunized with human serum albumin- tetra-~-alanine served as the antibody source for the preparation of the purified antibody fractions. Representative inhibition curves of purified anti- bodies are shown in Fig.3. The peptide concentra- tions causing 50°/, inhibition of the precipitin reac- tions are given in Table 6 . It is seen that the efficiency of inhibition increases with the site of ~ a l a n i n e peptides, the pentapeptide being still better than the tetrapeptide by a factor of 3 to 9. The inhibitory capacity of L-alanine peptides is remarkably reduced

Vol.18, N0.4, 1971 I. SCHECHTER, E. CLERICI, and E. ZAZEPITZEI

I I I “7

567

Fig. 3. Hapten inhibition of the “anti-alanyl-carbo~l” system. The precipitin reaction between Ab-4 fraction of P-I and rabbit serum albumin-tetra-L-alanine was inhibited by: w, Ala (L); A, Ala, (LJ; B, Ale, (L,); 4, Ala, (L,); 0, Ala, (L,); A , Ac-Ala

(L); 0, Ac-Ala, ( L ~ ) ; 0, Ac-Ala, (L,); 0, Ac-Ala, ( L ~ ) ; u, Ala, (DL,); +, Ah, (DL,); x, Ah, (DL,)

Table 6. Inhibition of the precipitin reaction of “anti-alanyl-carbo~l’~ antibodies by alanine peptdea Reaction mixtures contained purified antibodies, the precipitating antigen, rabbit serum albumin-(L-Ala),, and peptide. A number of concentrations of peptides were employed to determine the point of 50°/, inhibition. When 50°/, mhibition was

not achieved, the percent inhibition obtained at the maximal peptide concentration used is given in brackets

Peptide concentration at 50°/0 inhibition

Antibody source Alanine peptide

P-1 P-2 P-3

Ab-4 Ab-Ac Ab-4 Ab-4

mM (%) mM (%) mM (%) mM (%)

H .Ala . OH (L) > 6 (0) > 9 (0) > 6 (0) >6.5 (7) > 6 (3 ) H * Ala, . OH (L,) > 6 (10) > 6 (0)

6.5 >4.7 (40) H . Ah,. OH (L~) 2.9 > 6 (16) H . Ala, . OH ( L ~ ) 0.49 6.0 1.4 0.94 H . Ah, . OH ( L ~ ) 0.16 0.65 0.26 0.35

H Ala, . OH (DLJ 0.76 > 6 (40) H . Ala, . OH (DL,) 0.14 0.45 0.24 0.35

H * Ala, * OH ( L ~ D ) >3.5 (3) >3.5 (0) >4.8 (0) > 3 (18) H . Ala, * OH (L,D) > 1.2 (3) >0.7 (0) >0.5 (0) >0.6 (20)

> 6 (0) > 6.5 (14) > 6 (20)

> 6.5 (25) > 6 (10) 2.6 3.9

H . Ala, . OH ( D L ~ ) > G (32) > 6 (8)

H . Ala, . OH (L,D) >3.5 (0) >3.5 (0)

Ac-Ala * OH (L) > 6 (15) > 6 (0) Ac-Ah, . OH (L,) > 6 (47) > 6 (5)

H 1 Ala, . NH, (L,) > 3.5 (0) > 3.5 (0)

2.0 1.2 Ac-Ala, * OH ( L ~ ) 0.32 2.5 Ac-Ala, . OH (L,) 0.14 0.45 0.4 0.35

H . Ah, . NH, ( L3) >3.5 (10) >3.5 (0) > 6 (25) H . Ala, * NH, (L,) > 1.8 (10) >1.8 (0) >2.5 (2) > 3 (15) H . Ala, * NH, ( L ~ ) > 1.8 (20) > 1.8 (10) >0.5 (5)

by introducing changes a t the C-terminal part of the peptide: (a) by converting the free a-carboxyl group into an amide group (e.g. , in Ab-4 of P-1, H - Ala, - OH at 0.49 mM causes 500/, inhibition whereas H -Ala, *NH2 even at 1.8 mM causes only loo/,

inhibition) ; (b) by replacement of the C-terminal L-residue by a D-residue (e.g. in Ab-4 of P-1, the L~ isomer of H - Ala, * OH a t 0.16 mM causes 50°/, inhibition, whereas the L ~ D isomer, even a t 1.2 mM, causes only 3Ol0 inhibition). On the other hand,

568 Distinct Antigenic Specificities of Alanine Chains Determinants

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Eur. J. Biochem.

103 -

0.25 0.50 0.75 0.25 0.50 0.75 Peptide (mM)

Pig. 4. Hapten-inhibition of the “anti-amino-alanyl” system. The precipitin reaction between Ab-4 fraction of P-5 and poly- L-alanine-rabbit serum albumin was inhibited by the following peptides. In (A) ‘I, Ala (L); 0, Ala, (L,); ., Ala, (L,); +, Ala, (L4); 0, A& (L~); A , Ala, (L2D); 0, Ala, (L3D); X , Ala, (LpD); 0, Ala, (DL,); +, Ala, (DL,); A, Ala, (DL~). In (B) 0, Ala . NH, (L) ; W, Ala, . NH, (L~) ; +, Ala, * NH, (L,); x , Ala, * NH, (L,) ; 0, Ala, . NH, (LJ ; ‘I, Ac-Ala, ( L ~ ) ; + , Ac-Ala, ( L ~ ) ; 0, Ac-Ala, (L,)

acetylation of the a-amino groups improved the inhibitory capacity of L-alanine peptides (e .g . , in Ab-4 of P-I, 50OlO inhibition is obtained by 2.9 mM of H * Ala, - O H and by 0.32 mM of Ac-Ala, OH). Replacement of the N-terminal L-residue by a D-re- sidue could reduce the inhibitory efficacy of an alanine peptide. The effect of a D-alanine residue decreased with distance from the C-terminal end of the peptide. Thus, in tripeptides the L~ isomer is much better as an inhibitor than the DL, isomer ; in tetrapeptides the L, isomer is somewhat better than the DL, isomer; in pentapeptides the inhibitory capacities of the L, and DL, isomers are practically identical.

Inhibition Xtudies of “Anti-Amino-Alanyl” Antibodies

Sera obtained from two groups of rabbits (P-4 and P-5) immunized with poly-L-alanine-human serum albumin served as the antibody source. Typical inhibition curves of purified antibodies are shown in Fig.4. The peptide concentrations causing 5O0lO inhibition of the precipitin reactions are given in Table 7. In is seen that the inhibitory capacity of free L-alanine peptides increases with their size up to the tetrapeptide, the Ala, being as good an inhibitor as Ala,. A large reduction in the inhibitory potency of L-alanine peptides occurred when the structure of the N-terminal part of the peptide was altered: (a) by blocking of the a-amino function with an acetyl group (e .g . , in Ab-4 of P-4, H * Ala, - OH a t 0.07 mM causes 50°/0 inhibition whereas Ac-Ala, * OH even a t 1.8 mM causes only 4Ol0 inhibition); (b) by replacement of the N-terminal L-residue by a D-residue (e.g. , in Ab-4 of P-4, the L, isomer of H - Ala, - OH at 0.055 mM causes 50°/, inhibition whereas the DL, isomer even a t 1.8 mM causes only 20°/, inhibition). On the other hand, blocking of the a-carboxyl function by amidation or by esterification improved the inhibitory capacity of L-alanine pep-

tides (e .g . , in Ab-4 of P-5, 50°/0 inhibition is obtained by 8.5 mM of H * Ala, * OH, by 0.32 mM of H - Ala, - NH, and by 0.38 mM of di-alanine p-nitrobenzyl ester). Replacement of the C-terminal L-residue by a D-residue could reduce the inhibitory efficiency ; this effect become weaker the further the replacement from the N-terminal end of the free peptide. Thus, in tripeptides the L, isomer is much better as an inhibitor than the L,D isomer, whereas the corre- sponding isomers of tetrapeptides (L, and L ~ D ) and pentapeptides ( L ~ and L,D) have similar inhibitory potency.

DISCUSSION Alanine chains can be attached to proteins in

“opposite directions”, so that in one case the amino function of the chain is free, and in the other, its carboxyl function is free. By polymerization of N-carboxy-L-alanine anhydride on the e-amino groups of the protein, the resulting polypeptide side chains carry free N-terminal a-amino groups, and the molecular weight distribution of these chains is relatively sharp [18,19]. On the other hand, by reacting the protein with the preformed peptide derivative, iodoacetyl-tetra-L-alanine, the attached peptides are ofthe same size and carry free C-terminal a-carboxyl groups (Fig. I). The anti-alanyl antibodies formed upon immunization with these two types of antigens do not show any cross-precipitation reac- tion (Fig. 2). Further information on the combining sites of these antibodies was obtained from studying the capacity of series of alanine peptides to inhibit the precipitin reaction. The extent and properties of the binding sites are deduced from the correlation between the structural features of the haptens and their strength of interaction with the antibodies (as manifested by their capacity to inhibit the precipitin reaction). The structural changes applied here were : (a) elongation of the peptide chain of the hapten; (b) replacement on an L-alanine residue in the peptide

Vol. 18, No. 4, 1971 I. SCHECHTER, E. CLERICI, and E. ZAZEPITZHI 569

Table 7. Inhibition of the precipitin reaction of LLanti-amina-alany~7’ antibodies by alanine peptides Reaction mixtures contained purified antibodies, the precipitating antigen, poly-L-Ala-rabbit serum albumin, and peptide. A number of concentrations of peptides were employed to determine the point of 50°/, inhibition. When 50°/, inhibition was

not achieved, the percent inhibition obtained at the maximal peptide concentration used is given brackets

Peptide concentration at 50°/0 inhibition

Antibody 8onrce Alanine peptide

P-4 P-5

Ab-4 Ab-Ac Ab-4b

mM (%) mM (*lo) mM ( ”0 )

H . Ala . OH (L) > 6 (5) > 6 (8) > 8.5 (6) H * Ala, . OH (L,) > 6 (43) > 6 (37) 8.5 H - Ala, . OH (L,) 0.30 1.47 0.32

H . Ala, * OH ( L ~ ) 0.055 0.35 0.08 H * Alas . OH (DL,) > 1.8 (0) > 1.8 (0) > 3 (13) H * Ala, . OH (DL,) >1.8 (15) >1.8 (10) > 3 (31) H . Ala, * OH (DLJ > 1.8 (20) > 1.8 (15) > 3 (30) H * Ala, . OH (LD) >8.5 (25) H . Ala, 1 OH (L,D) > 0.6 (20) 2.9 2.4

H . Ala, OH (L,D) 0.06 0.38 0.07

H . Ala, * OH (L,) 0.07 0.40 0.09

H . Ala, . OH (L,D) 0.09 0.71 0.11

Ac-Ala, . OH (L,) > 1.8 (0) >2.4 (0) > 3 (0) Ac-Ala, OH (L,) > 1.8 (0) >2.4 (0) > 3 (10) Ac-Ala, . OH (L,) > 1.8 (4) > 1.8 (0) > 3 (22) H - Ala NH, (L) > 6 (42) 2.0 >8.5 (42) H * Ala, . NH, (L,) 0.26 0.71 0.32 H * Ala, . NH, ( L ~ ) 0.082 0.41 0.08 H * Ah, * NH, (L,) 0.053 0.32 0.07 H * Ah, * NH, (L,) 0.053 0.32 0.07

The inhibitions obtained by 6 mM of ~-8lanine ethyl ester, 6 mM of L-alanine benzyl ester and 0.35 mM of di-b-alanine p-nitrobensyl ester

The p-nitrobenzyl [esters of L-alanine at 8.5 mM, of di-L-alanine at 0.38 mM and of tri-L-alanine at 0.08 mM caused 46O/,, 50°/, and 50°/0 were 38O/,, 43’1, and 50°/0, respectively.

inhibition, respectively.

by its D antipode ; (c) blocking of the amino function or the carboxyl function of the peptide. The influence of these changes on inhibitory power is taken as evidence for interaction of the modified portion of the peptide with the combining site.

Purified antibody fractions which are devoid of any proteolytic activity were used in order to prevent the cleavage of the peptide inhibitors by proteases present in the serum 131. The results obtained with the fractions, (Ab-4), extracted from the immuno- adsorbent by tetra-L-alanine dissolved in phosphate- buffered saline will be discussed f i s t .

Both types of antibodies react with free L-alanine peptides. The efficiency of inhibition increases with the size of the L-alanine peptides. I n both cases the most exposed portion of the antigenic determinant is immunodominant. Effective binding of a peptide occurs only if it contains the exact structural features corresponding to the immunodominant region. Thus, “anti-alanyl-carboxyl” antibodies ex- hibit a high degree of discrimination with respect to the structure of the C-terminus of its ligand. A small change in volume and geometry brought about by converting the oc-carboxyl group of tetra-L-alanine into an amide group, reduced remarkably the binding

of the peptide. Similarly, H * Ala, * OH ( L ~ D ) is a very poor inhibitor as compared to H - Ala, * OH (L,). These examples show that a charged C-terminal L-residue is very important for binding. Non-ter- minal L-alanine residues alone are not effective since both H -Ala, .NH, (L,) and H .Ala, * OH ( L ~ D ) contain an uninterrupted sequence of four L-alanine residues, and yet they are bound very poorly. In the “anti-amino-alanyl” system alterations at the N-terminus of free L-alanine peptides caused a large drop in inhibitory capacity. Here the L-alanine residue carrying the free a-amino group is essential since both H - Ala, - OH ( D L ~ ) and Ac-Ala, - OH ( L ~ ) , which contain an uninterrupted sequence of four L-residues, are very poor inhibitors as compared to H * Ma, - OH ( L ~ ) . Many other examples showing the above points can easily be found in the data given in Tables 6 and 7.

The lack of cross-precipitation reaction between the two systems is in complete agreement with above results. The “anti-alanyl-carboxyl” antibodies cannot react with poly-L-alanine-rabbit serum albumin because in this precipitant the attached alanine chains do not carry free a-carboxyl groups. Similar “incompatibility” exists between the “anti-amino-

570 Distinct Antigenic Specificities of Alanine Chains Determinants Eur. J. Biochem.

alanyl” antibodies and the precipitant rabbit serum albumin-tetra-L-alanine.

The size of the combining site is commonly estimated from studying the effect of elongation in a homologous series of haptens on their inhibitory efficiency [3,20,21]. In the (‘anti-amino-alanyl” sys- tem (P-4 and P-5 in Table 7) the inhibitory efficiency of free L-alanine peptides and of L-alanine peptide amides increases with the length of the peptide, the increment in inhibitory capacity becoming smaller with increase in size. However, in the free peptide series the increase in inhibitory efficiency (on a molar basis) levels off a t the tetrapeptide (that is, H * Ah, - OH is practically as good an inhibitor as H * Ala, - OH), whereas in the alanine amide series levelling off occurs already with the tri-L-alanine amide (H-Ala,.NH, is practically as good an inhibitor as H - Ala, - NH,). Thus, the results with free peptides would lead to the conclusion that the size of the combining site is complementary to a tetrapeptide; on the other hand, the results with peptide amides indicate a combining site comple- mentary to the size of a tripeptide. The conclusion drawn from the alanine amide series is the correct one because of the following considerations. The alanine peptide determinant in the immunogen poly-L- alanine-human serum albumin contains in its most exposed portion a free a-amino group, and is com- posed of a repeating sequence of the polar amide group and the non-polar a and /I carbons of alanine, but it does not contain any carboxylate group (Figs.1 and 5) . The combining site is designed to interact with the antigenic determinant. Since the peptide amides bear closer structural resemblance to the antigenic determinant than the free peptides, data obtained with them are more relevant.

In principle, more precise information on proper- ties of the combining site can be derived from comparing haptens of equal size which differ from one another in one particular feature, rather than from studying the effect of elongation in a homo- logous series of peptides [22]. This is because the stronger binding caused by elongation is not neces- sarily due to the addition of interaction by the extra piece, but can also be attributed to the abolishment of unfavourable interactions in the smaller hapten. Thus, the finding that H - Ah, * OH (L,) is a better inhibitor, by a factor of 4, than H * Ah, - OH (L,) does not prove that the additional alanine interacts with the binding site. It can also be due to the lack of interaction, or even to interference by the carboxy- late of tri-L-alanine. Indeed, the inhibitory capacity of the H - Ala, * NH, (L,) which is equal in size to H * Ala, * OH (L,) but without the carboxylate ion, is much better than H - Ala, - OH (L,) and similar to that of tetra- and penta-L-alanine (see Table 7). The comparison between pairs of peptides of equal size (as well as the effect of elongation) are given in

Table 8. It is seen that the carboxylate ion interferes with binding up to the third residue (counting from the N-terminal alanine residue which is immuno- dominant), as amidation improved the binding of alanine (see Table 7), Ala, and Ala, but not of Ala, and Ma,. Furthermore, substitution of an L-alanine residue by a D-residue reduced binding in the third position but not in the fourth and fifth positions (Table 8).

To sum up, it is shown that diastereoisomeric replacements and the presence of carboxylate ion are felt over three residues from the N-terminus of an L-alanine peptide. In the L-alanine amide series (which are closely related structurally to the antigenic determinant, see above) the effect of elongation is also felt up to the third residue. This means that the size of the binding site of “anti-amino-alanyl” anti- bodies is such as to accommodate a tri-alanine pep- tide.

It may be of interest to mention that other erro- neous conclusions could be drawn from the use of the free peptide series alone. For example, the con- tribution of the third residue is over-estimated by using data obtained with free peptides, since elonga- tion from di- to tri-peptide increased inhibitory efficacy by factors of 23 and 27. The data obtained with the corresponding amides give factors of 3 and 4 (Table 8), which is a better estimate for this elonga- tion.

Inhibition of the ‘(anti-alanyl-carboxyl” anti- bodies (P-I, P-2 and P-3) by free L-alanine peptides would indicate a combining site capable of accom- modating a t least a pentapeptide since H * Ala, - OH was better than H - Ma, - OH by factors of 3-5 (Tables 6 and 9). Since the peptide determinant in this case does not have any charged amino group (Figs.l and 5 ) it may be that this effect is due to blocking of the amino function of tetra-L-alanine and not to additional interaction by the fifth alanine residue. Application of the more rigorous analysis described above indicates that the size of the com- bining site is smaller. From data given in Tables 6 and 9 it is seen that, by comparing peptides of equal size, the effect of structural changes (replacement of an L-alanine residue by its antipode or by an acetyl group) a t various distances from the C-terminal end are strongly felt in the third position and to a lesser extent in the fourth position. In the fifth position these substitutions do not change significantly the inhibitory capacity of the peptides (that is, the L, and DL, isomers of H * Ma, - OH, as well as Ac-Ala,

OH (L,), are equally good as inhibitors). Therefore, the more accurate estimate for the size of the com- bining site of “anti-alanyl-carboxyl” antibodies is that it can accommodate three to four alanine residues.

It was previously shown that significant damage is induced by treatment of antibodies with acetic

Vol.18, No.4, 1971 I. SCHECHTER, E. CLERICI, and E. ZAZEPITZEI 57 1

Table 8. Comparison of the inhibitory capacity of pairs of peptides in the "anti-amino-alanyl" system The numbers are the ratio between two peptide concentrations at 50°/, inhibition, using data given in Table 7. When inhibition was not achieved the peptide concentrations a t a lower degree of inhibition were employed. The percent inhibition

a t this level is given in brackets. L and D represent alanine optical isomers

Fraction Peptide pairs n = 2 n = 3 n = 4 n = 5

3.7 1.3 0.97 1.3 1.1 1.5 1.0

H . Ln-lD * OH H . Ln * OH H * Ln-1. NHJH * ~n . NH, 31 (42'10) 3.2 H . ~ n - 1 . OH/H * Ln * OH 23 (43'10) 4.3 1.3

Ab-4 fraction of P-4 H . Ln . OH/H * L~ . NH, 30 (43'/0) 7 (20'10)

Ab-4 fraction of P-5 H . L~ . OH/H * L~ . NH, 27 4.0

H Ln-1. NHJH . Ln * NH, 35 (42'1,) 4.0 H . Ln-l*OH/H. Ln * OH

3.6 2.0

H . Ln-1. NH,/H . ~n . NH, 2.8 1.7

H . Ln-1D . OH/H Ln * OH 7 (25°/0) 7.5

27

Ab-Ac fraction of P-4 H . L~ * OH/H . L,, . NH, 14 (37'10) H Ln-1D * OH/H. Ln . OH

H . ~ ~ - 1 - OH/H. L~ 9 OH 10 (37'10)

1.3 1.1 1.2 0.88 1.1 1.0 3.6 1.1

1.2 1.8 1.3 3.7

1.1 1.1 1 .o 1.1

Table 9. Comparison of the inhibitory cupacity of pairs of peptides in the "anti-alanyl-carboxyl" system

at this level is given in brackets. L and D represent alanine optical isomers

The numbers are the ratio between two peptide concentrations at 50°/, inhibition, using data given in Table 6. When 50°/, inhibition was not achieved the peptide concentrations a t a lower degree of inhibition were employed. The percent inhibition

Fraction Peptide pairs ~~~

n = 3 n = 4 n = 5

Ab-4 fraction of P-1 H AC-Ln-l.OH/H * L n . OH H * Ln-l.OH/H * ~n * OH

DLn-I * OH/H * ~n * OH 1.5 0.87 0.65 0.87 5.9 3.1

5 (32°/0) 2.5

Ab-4 fraction of P-2 H . DLn-I*OH/H. ~n . OH AC-Ln-l*OH/H * Ln * OH H * Ln-l.OH/H * ~n * OH

4.4 (25'1') 1.8 1.4 4.6

0.92 1.5 5.4

Ab-4 fraction of P-3 H * DLn-l.OH/H. ~n . OH 4.1 1.0 AC-Ln-I.OH/H * Ln * OH 1.3 1.0 H . Ln-l.OH/H * L~ * OH 8 (40°/,) 2.7

Ab-Ac fraction of P-1 H DLn-1*OH/H. ~ n . OH 1.4 (40°/,) 0.69 AC-Ln-l*OH/H * Ln * OH 0.42 0.69 H * Ln-l*OH/H * Ln * OH 9.2

acid. Purified antibodies prepared by extraction with peptide in phosphate-buffered saline were sub- sequently kept in 0.1 M acetic acid for 1 h a t 37". This treatment caused a substantial decrease in the capacity of peptide-haptens to inhibit the precipitin reaction [15]. In the present work, the same reason may account for the finding that a higher peptide concentration was required to achieve 50°/, inhibition in the Ab-Ac fractions as compared to the Ab-4 fractions (see P-1 in Table 6 and P-4 in Table 7). Bearing this reservation in mind, we can proceed to analyze the inhibition results. The general pattern of inhibition by various peptides obtained with the Ab-Ac fractions is similar to that observed with the Ab-4 fractions. Comparing peptide pairs of equal size : in the "anti-alanyl-carboxyl" system (Ab-Ac of P-1) the results indicate that the combining site can accommodate three to four amino acid residues (Tables 6 and 9); in the ((anti-amino-alany1" system

(Ab-Ac of P-4) the combining site can accommodate about three alanine residues (Tables 7 and 8).

Besides the fact that the antibodies of the two systems recognize different immunodominant struc- tures, one can speculate that they differ in another feature. This feature is related to the arrangement of groups involved in hydrogen bond formation. It is reasonable to assume that in the antibody-hapten complex there is a close contact between the inter- acting surfaces. Hydrogen bonds are probably formed between the polar amide groups ofthe alanine chain and appropriate groups in the combining site. It is advantageous to form these bonds, since they compensate for the loss of hydration of the amide groups when free in solution [23,24]. An analogous situation exists in enzyme-inhibitor complex, where X-ray diffraction data showed hydrogen bonds between hydratable groups of the interacting species [25--271. In Fig.5 the complementarity between the

572 I. SCHECHTER et al. : Distinct Antigenic Specificities of Alanine Chains Determinants Eur. J. Bioohem.

- CH - C-N-Y - C-N-CH-c-N-cG- I I l l / I I I / CH3 O H CH3 OH CH3 OH LHg

riJ-fi-$l--r%-FH - N-C- H ~ H J H O CHR H O CH3 H O CH1

I l l F - -

Fig. 5. A very schematic representation of the mplementarity between surfaces of “anti-alanyl-carbo~l” and “anti-amino- alanyl” antibodies, with their respective antigenic determinants. The symbols are: bulging wedge, hydrogen donor group; depressed wedge, hydrogen acceptor group ; bulging round surface, the a and B carbons and hydrogens of an alanine residue; depressed round surface, a region on the combining site capable of interacting favourably (probably by hydro- phobic interaction) with the a and B carbons and hydrogens

of alanine

surfaces of “anti-alanyl-carboxy” and “anti-emino- alanyl” antibodies with their respective antigenic determinants, is presented in a very schematic way. It is seen that a t equal distance from the region occupying the immunodominant part of the anti- genic determinant, one combining site contains a hydrogen donor group, whereas in the other the same position is occupied by a hydrogen acceptor group. Inspection of this scheme reveals that the only way to line up an alanine chain on both types of com- bining sites, so as to achieve favourable interactions, is to rotate it by 180”. Otherwise strong misfits be- tween the interacting surfaces would occur. These include the overlapping of two hydrogen donor groups or of two hydrogen acceptor groups, as well as the juxtapositioning of polar with non-polar areas.

In conclusion, two types of antibodies with dis- tinctly Merent specificities can be elicited against L-alanine peptide determinants, depending on their mode of attachment in the immunogen (Fig. 1). Both types of antibodies can react with free L-alanine peptides. It may well be, however, that in the combining sites the peptides are bound in opposite directions.

The authors wish to thank Prof. M. Sela for discussions.

7.

8.

9.

10.

11.

12.

13.

14.

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20. 21.

22. 23.

24.

25. 26.

27.

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I. Schechter and E. Zazepitzki Department of Chemical Immunology The Weizmann Institute of Science P. 0. Box 26, Rehovot, Israel

E. Clerici’s permanent address: Instituto di Patologia Generale dell’ UniversitQ Via Mangiagalli 31, 1-20133 Milano, Italy