Immunology, 1968, 15, 305.

Isolation and Characterization of Two Antigenically ActivePeptides from Bovine p-Lactoglobulin-A

D. H. BING* AND A. B. STAVITSKY

Department of Microbiology, Western Reserve University, Cleveland, Ohio

(Received 1st February 1968)

Summary. Bovine fl-lactoglobulin-A was hydrolysed with trypsin to yield amixture of peptides which would not precipitate with rabbit antibody against thenative protein, but would still inhibit the antigen-antibody reaction. The hydro-lysate was fractionated by ion exchange chromatography and found to containseven antigenically active fractions. Two of these fractions were found to behomogeneous peptides. Each inhibited the reaction ofantigen with antibody againstthe intact protein in haemagglutination, flocculation and passive cutaneousanaphylaxis reaction in guinea-pigs. One of the peptides had a molecular weightof 950 and the other had a molecular weight of 575. Both contained about equalnumbers of polar and apolar amino acids.

INTRODUCTION

The early experiments by Landsteiner with hapten-protein conjugates and enzymaticor acid hydrolysates of proteins indicated that the serological specificity of an antigencould be attributed to the haptenic groups or low molecular weight portions of the immun-izing materials (Landsteiner, 1947). More recently the enzymatic hydrolysis of severalnon-globular proteins such as silk fibroin (Cebra, 1960), lens crystalline (Hara, 1956)oxidized ribonuclease (Brown, 1962) and tobacco mosaic virus protein (Young, Benjamini,Shimizu, Leung and Feingold, 1963; Young, Benjamini, Shimizu and Leung, 1966) hasresulted in the preparation of small antigenically active peptides. Studies on the antigenicdeterminantst of serum albumin were initially limited to the isolation of relatively highmolecular weight fragments (Lapresle, Kaminski and Tanner, 1959; Lapresle and Webb,1965; Press and Porter, 1960) but lately the separation of small antigenically activefragments of albumin (Richard, Beck and Hock, 1960) and another globular protein,thyroglobulin (Metzger, Sharp and Edelhoch, 1962) has been reported. A detailed studyof low molecular weight antigenically active peptides derived from myoglobin has alsobeen reported (Crumpton and Wilkinson, 1965).The purpose of the present study was to isolate and characterize antigenically active

peptides from bovine fl-lactoglobulin-A, a small globular protein found in cow's milk.This protein was chosen as it has a low molecular weight (36,000), contains two identicalpolypeptide chains, is readily purified, and has already been extensively characterized

* Present address: Department of Microbiology and Public Health, Michigan State University, East Lansing,Michigan 48823.

t According to the terminology of Sela (1966a), an antigenic determinant is defined as that portion of the antigenwhich reacts with the antibody. In contrast, the portion(s) of the antigen which induces antibody formation isdefined as the immunogenic determinant(s).

305

D. H. Bing and A. B. Stavitsky(Bell and McKenzie, 1964). The results to be described will show that there are at leastseven peptides in the tryptic hydrolysate of 1-lactoglobulin-A which will specificallyinhibit the reaction of anti-fi-lactoglobulin antiserum with the antigen, and that at leasttwo of these peptides have a molecular weight of less than 1000.

MATERIALS AND METHODS

ANTIGENS

Three times crystallized bovine /3-lactoglobulin-A and B (BLG-AB), bovine serumalbumin (Cohn Fraction V-BSA), and rabbit y-globulin (Cohn Fraction II-RGG) werepurchased from Pentex Laboratories, Kankakee, Illinois. Alum precipitated antigens weremade according to the method described by Kabat and Mayer (1961).

Bovine fl-lactoglobulin-A (BLG-A) was separated from fl-lactoglobulin-B (BLG-B) by ionexchange chromatography on DEAE-cellulose according to the method of Piez, Davie, Folkand Gladner (196 1). The protein was then crystallized according to the method of Larsonand Jenness (1955). This preparation of BLG-A was chromatographed twice and crystal-lized twice before it was used. It was judged to be physically and immunochemicallyhomogeneous according to the following criteria: (1) at a concentration of 40 mg/ml itmigrated as a single band in starch gel electrophoresis in Tris boric acid-ethylenediamineacetic acid buffer, pH 8-6 (Aronsson and Gronwall, 1957), (2) at a concentration of 10mg/ml it sedimented as a single homogeneous peak in the Model E Analytical Ultra-centrifuge with a S20w coefficient of 2-6, and (3) at a concentration of 20 mg/ml it developeda single line of precipitation with an antiserum to bovine raw skim milk according toimmunoelectrophoresis or gel diffusion experiments.

PREPARATION OF ANTISERA

All antisera were raised in adult male or female New Zealand White albino rabbits.All sera were heated at 560 for 30 minutes and stored at -200 until used. Anti-BSAantiserum was prepared by a single injection of 2 mg alum precipitated antigen into bothhind footpads. The animal was bled 2 weeks later by cardiac puncture. Antisera againstBLG-A were produced by the injection of 10 mg of alum precipitated antigen into eachhind footpad three times a week for 3 weeks. At the end of the 4th week the animals werebled from the lateral ear vein every day for 3 days. Sera from the three bleedings of anindividual rabbit were pooled. Anti-bovine raw skim milk serum was purchased fromAntibodies, Inc., Davis, California.

Nitrogen and protein determinationNitrogen was determined by the micro-Kjeldahl method (Kabat and Mayer, 1961).

Protein concentrations were also determined by the Folin Biuret method according toLowry, Rosebrough, Farr and Randall (1951). A solution ofRGG or BLG-A on which aKjeldahl analysis had been done was used for construction of standard curves.

Chromatography of the tryptic peptides ofBLG-AThe tryptic hydrolysate of BLG-A was chromatographed on Dowex 50 with 2 per cent

306

Antigenically Active Peptides

cross-linkage according to the method of Moore and Stein (1951) using 1 8 x 140 cmcolumn for the chromatography of 500 mg of the tryptic hydrolysate. Peptides weredetected in the effluent with the reagent 2,4,6-trinitrobenzene sulphonic acid (NutritionalBiochemical Corporation, Cleveland, Ohio) according to the method of Okuyama andSatake (1960), the extinctions read at 340 myi in a Zeiss Spectrophotometer. The peptideswere desalted by passage through 4 x 1500 cm columns of BioGel P2, 50-100 mesh(BioRad Laboratories, Richmond, California). The samples were eluted with distilledwater at room temperature. The flow rate was 240 ml/hr, and 20-ml fractions werecollected. The fractions from the BioGel columns were assayed for peptides with ninhydrinwithout alkaline hydrolysis, according to the method of Moore and Stein (1948), and theextinctions read in a Klett Colorimeter with a No. 56 filter.

ANALYTICAL METHODS

Gel filtration on BioGel P6Minimal molecular weight estimations were made on a 1 2 x 68 cm column of BioGel

P6, 50-100 mesh (BioRad Laboratories, Richmond, California). The column was a1 2 x 84 cm Kontes Chromaflex column No. 1 (Kontes Glass Company, Vineland, NewJersey). The column was coated twice with 1 per cent dimethylchlorosilane (v/v) inbenzene at 55-60°. The solvent for elution was 01 M NaCl. All chromatography was doneat 40, and 2-ml fractions were collected with a GME volumetric fraction collector (GilsonMedical Electronics, Middleton, Wisconsin). For chromatography, 0-2-0-3 ml of eachsample was mixed with 0-3 ml of 50 per cent aqueous sucrose (w/v) and then layered witha capillary pipette on top of the resin below the level of the solvent. The sample wasallowed to flow into the resin before connecting the column to the solvent reservoir. Theextinction of Blue Dextran (Pharmacia Corporation, Uppsala, Sweden) was read at280 my in a Zeiss Spectrophotometer using 1-ml quartz cuvettes with 1 cm light path.Oxytocin, and Insulin A and B chains were measured by the Folin Biuret reaction accord-ing to the method of Lowry et al. (1951). The peptides were assayed by the ninhydrinmethod (Moore and Stein, 1948). Oxytocin was kindly donated by Dr John P. Gusdon,Jr, Department of Gynecology and Obstetrics, Western Reserve University, School ofMedicine, Cleveland, Ohio. Insulin A and B chains were obtained as the sulpho deriva-tives and were generously given by Dr Y. Yagi, Roswell Park Memorial Institute, Buffalo,New York.

ChromatographyThin layer chromatography was done on 8 x 8 in. plates of Silica Gel HHR (Brinkmann

Company, Great Neck, New York), 250 yu thick, prepared with the Desaga apparatus.Ten to 25 !l of a sample were applied 3 cm from the bottom of the plate and chromato-graphy was done by ascending method in glass tanks containing 125 ml of the solvent.The solvent was allowed to migrate 15 cm from the bottom of the plate. The chromato-grams were allowed to air dry, and the peptides located with a tertiary butyl hypochlorite-starch iodide stain (Nutritional Biochemical Company, Cleveland, Ohio). Solvents usedfor chromatography were propan-1-ol-acetic acid-water (8: 2: 1, by volume), butan-1 -ol-saturated with 5 per cent NH40H (v/v), and butan-l-ol-acetic acid-water (4:5:5, byvolume, upper phase).

307

D. H. Bing and A. B. Stavitsky

Peptide mappingPeptide maps were made on paper according to the method of Helinski and Yanofsky

(1962). Peptide maps on silica gel were made according to the method of Ritschard (1964).

Analytical ultracentrifugeSedimentation coefficients were determined by use of the Beckman Model E Analytical

Ultracentrifuge. All determinations were made with a synthetic boundary cell at 110.The centrifuge was accelerated to 57,280 rev/min. Sedimentation coefficients were calcu-lated according to the method of Schachmann (1957) and corrected to the density ofwater at 200.

Amino acid analysisAmino acid analysis was performed according to the method of Moore and Stein (1963)

using a Beckman Spinco Amino Acid Analyzer. The peptide samples were hydrolysed in6 N HC1 for 18 hours in an evacuated tube at 1 100. The number of amino acid residues wasnormalized to lysine and rounded off to the nearest integer.

PREPARATION OF TRYPTIC PEPTIDES OF fl-LACTOGLOBULIN AND BOVINE SERUM ALBUMIN

One gram of twice crystallized 13-lactoglobulin-A or bovine serum albumin (CohnFraction V) was digested with three times crystallized trypsin (Worthington BiochemicalCorporation, Freehold, New Jersey) at an enzyme-substrate ratio of 1: 100 (w/w) in 100ml of 0-05 M (NH4)2CO3, pH 8-6. Digestion was carried out for 24 hours at 370 withshaking. At this time another aliquot of trypsin equal to the original amount was addedand the digestion continued at 370 with shaking for another 15 hours. A drop of toluenewas added to prevent bacterial growth. The digest was lyophilized to remove the buffersalts, and the dry powder stored at 40 until used.

IMMUNOCHEMICAL METHODS

Precipitin testPrecipitin tests were performed with whole serum or the y-globulin fraction according

to the method ofKabat and Mayer (1961). The diluent was 0 15 M NaCl-0 02 M phosphatebuffer, pH 6f4 (Kabat and Mayer, 1961). Protein in the precipitate was determined bythe Folin biuret method (Lowry et al., 1951).

Gel diffusion and immunoelectrophoresisImmunoelectrophoresis was done according to the method of Scheidegger (1955). Gel

diffusion was performed in 1 per cent (w/v) agar (Difco Purified Agar, Difco Co., Detroit,Michigan), buffered with 0f15 M NaCl-0 02 M phosphate buffer, pH 6f4.

Haemagglutination and haemagglutination inhibitionHaemagglutination and haemagglutination inhibition assays employed glutaraldehyde

preserved sheep erythrocytes to which BLG-A was conjugated with the bifunctionalreagent bis-diazotized benzidine (Bing, Weyand and Stavitsky, 1967). Bovine serumalbumin was coupled to formalin preserved erythrocytes by the tannic acid procedure(Daniel, Weyand and Stavitksy, 1963). Antisera and inhibitors were diluted in 0-02 Mphosphate buffer-0f15 M NaCl buffer, pH 5.3, containing 0'25 per cent (v/v) normal

308

Antigenically Active Peptides 309rabbit serum (Cappel Laboratories, West Chester, Pennsylvania) which had beenpreviously absorbed with an equal volume of glutaraldehyde or formalin preserved sheepred blood cells and heated at 560 for 30 minutes. Haemagglutination assays were performedand settling patterns read as previously described (Stavitsky, 1954). The inhibition assaywas done as follows: Inhibitor was serially diluted by two-fold dilutions, and a constantamount (0.1 ml) of antibody was added to each tube. The amount of antibody added(0.01-0-015 ug antibody nitrogen) had been previously determined by haemagglutinationassay to be twice the minimal amount required for reproducible complete agglutinationof 0 05 ml of a 2 per cent (v/v) suspension of antigen conjugated cells (see Stavitsky, 1954).Antibody and inhibitor were routinely incubated for 30 minutes at room temperaturebefore addition of erythrocytes. In all cases the final volume of the reaction mixture was0-5 ml. The pH of 5-3 was determined empirically to be the optimal pH for measuringinhibition by the peptide fractions. Specificity was determined in every assay by testingfor inhibition by the peptide hydrolysate ofhaemagglutination ofBSA conjugated erythro-cytes by anti-BSA serum, or measuring the ability of the BSA tryptic digest to inhibithaemagglutination of BLG-A conjugated erythrocytes by anti-BLG-A serum. Althoughagglutination methods employing doubling dilutions of antiserum usually are consideredreproducible only within one tube above or below a given dilution, in practice, haemag-glutination or inhibition titres varied by no more than one tube.

Flocculation assayThe flocculation inhition assay employed the y-globulin fraction of the antiserum pre-

pared by precipitation three times at 33 per cent saturation of ammonium sulphate.Antigen was added to each tube, followed by 0-15 M NaCl-0 02 M phosphate buffer, pH5 3, to bring each tube to constant volume, and, finally, a constant volume of antibodyto make a total volume of 0*7 ml. The tubes were mixed, incubated 15 minutes at 370and the turbidity determined at 660 my in a Zeiss Spectrophotometer. For inhibition offlocculation, a different amount of peptide was added to each tube, 200 pg antibodynitrogen and, finally, 0-15 M NaCl-0 02 M phosphate buffer, pH 5.3, to make the volumeup to 0*7 ml. After mixing and incubation for 20 minutes at room temperature, 2-5 pgofantigen nitrogen were added, the tubes mixed again and incubated at 370 for 15 minutes.Two hundred micrograms of antibody nitrogen had been previously determined empiric-ally to give maximal turbidity with 2-5 pg of antigen under identical conditions. Percentinhibition equalled: (E1 cm of antibody plus antigenl- cm antibody plus inhibitor plusantigen/E, cm antibody plus antigen) x 100. All assays were done in duplicate, andextinctions of duplicate samples did not vary by more than 5 per cent. The specificity ofevery assay was determined by testing the ability of a comparable amount of the tryptichydrolysate of BSA to inhibit the reaction of BLG-A with its antibody.

Passive cutaneous anaphylaxisThe passive cutaneous anaphylaxis (PCA) reaction was done according to the method

ofOvary (1958). Inhibition of the reaction was accomplished by mixing a constant amountof antibody with varying amounts of the inhibitor before intradermal injection. All testswere done in duplicate. The size of the area of blueing was reproducible within the limitsof +10 per cent from guinea-pig to guinea-pig and from one set of determinations toanother.

D. H. Bing and A. B. Stavitsky

RESULTS

PHYSICAL AND IMMUNOCHEMICAL CHARACTERIZATION OF THE TRYPTIC

HYDROLYSATE OF BLG-A

The hydrolysate was heterogeneous according to its sedimentation in the ultracentrifuge.At a concentration of 25 mg/ml, it exhibited a broad peak with an S20W coefficient betweenthe values of 0 4 and 1 0. Peptide mapping of 10 mg of the digest on paper and stainingwith ninhydrin indicated a minimum of twenty peptides. This material still reacted withantibody; at pH 5.3, 16 pug ofnitrogen of the digest specifically inhibited the haemagglu-tination of BLG-A conjugated erythrocytes by 0-012 jig of BLG-A-antibody nitrogen. Thetryptic hydrolysate at concentrations up to 30 mg/ml did not precipitate with anti-BLG-Aserum in interfacial ring, gel diffusion or immunoelectrophoretic reactions. Electro-phoresis on paper of50 mg ofthe tryptic digest and 10 mg ofBLG-A at 2500 V and stainingwith ninhydrin and tert-butyl hypochlorite-starch iodide indicated there was no nativeprotein in the digest, eliminating the possibility that small amounts of undigested BLG-Awere causing inhibition of haemagglutination.

E0 {

0O4r

0 3

02

0*1

f Start 2 N, pH 6-6 550

4200 4400 4600 4800 5000 5200 5400Effluent (ml)

FIG. 1. Chromatographic pattern of the tryptic digest of BLG-A on Dowex 50. The solid line is theextinction of each fraction at 340 mg after reaction with trinitrobenzene sulphonic acid. The hatchedareas represent those fractions which inhibited haemagglutination by 0-012 pg nitrogen of BLG-Aantibody. Experimental details are given in the text.

310

-- E-l.I-

Antigenically Active Peptides 311

SEPARATION AND CHARACTERIZATION OF PEPTIDES IN THE TRYPTICHYDROLYSATE OF BLG-A

The peptides were initially separated into eighteen fractions by chromatography onDowex 50 (Fig. 1). Ninety-five per cent ofthe material added to the column was recoveredin terms of colour at 340 myi applied and recovered as the trinitrobenzene derivative ofthe peptides. Peaks were pooled and desalted by passage through the BioGel P2 column.The shaded areas indicate those fractions which inhibited the haemagglutination ofBLG-A conjugated erythrocytes by 0-012 yg nitrogen BLG-A antibody (Table 1). Inhibi-tion was specific as 200-500 ug of the BLG-A tryptic digest failed to cause any inhibitionof haemagglutination of BSA conjugated erythrocytes by 0 1 ml of a 1:400 dilution ofanti-BSA serum (haemagglutination titre 5000).

TABLE 1THE INHIBITION OF ANTI-BLG-A MEDIATEDHAEMAGGLUTINATION BY THE DowEx 50-BIOGEL P2 FRACTIONS OF THE TRYPTIC DIGEST

OF BLG-A

NitrogenFraction causing inhibition

(,ug)3 207 77a 10

10 25]Oa 1312 2413 613a 713b 2016a 3017b 20

BLG-A tryptic digest 2BLG-A 4-6x 10-5

The peptide fractions were diluted in 0-02 Mphosphate0- 15 M NaCl buffer, pH 5 3, con-taining 0-25 per cent normal rabbit serum,0-012 pg BLG-A-antibody nitrogen was addedto each dilution, tubes were incubated for 30minutes at 20-25°, and finally 0 05 ml of2 percent BLG-A conjugated erythrocytes wereadded to each mixture. Settling patterns wereread as described in the text. The results wererecorded as the minimal pg nitrogen ofinhibitor causing inhibition of agglutination.

Analysis of the desalted fractions by the peptide mapping technique on paper andsilica gel indicated that with the exception of fractions 3 and 7 all were mixtures of threeor more peptides. Fraction 3 was homogeneous and fraction 7 was a mixture of a majorpeptide and a minor contaminating peptide according to the peptide mapping results.The major peptide in fraction 7 was resolved by chromatography on the BioGel P2column. The homogeneity of the desalted peptides 7 and 3 was confirmed by chromato-graphy on silica gel in three different solvents (Table 2). Both were excluded after

312 D. H. Bing and A. B. StavitskyTABLE 2

CHROMATOGRAPHY OF PEPTIDE FRACTIONS 3 AND 7 ONSILICA GEL

RFSolvent

Peptide 3 Peptide 7

Butan-I-ol-acetic-H20 0-167 0-173Propan-I-ol-acetic-H20 0 347 0 267Butan-I-ol-5 per cent 0 300 0 280NH40H

Experimental details are given in the text.

oxytocin on the BioGel P6 column (Table 3) indicating they had a molecular weight ofless than 900. Neither contained aromatic amino acids according to spectral analysis atpH 7 between 220 and 325 mp. The amino acid analysis indicated that peptide 3 contained

TABLE 3THE EXCLUSION VOLUMES (Vs) OF KNOWN PEPTIDES ANDOF THE TRYPTIC PEPTIDE FRACTIONS ON BIOGEL P6

Molecular weight V, (ml)

Blue dextran 1 X 106 35-6Insulin B chain 3500 44 0Insulin A chain 2471 46-2Oxytocin 995 50-0Peptide 3 76-5Peptide 7 74-6

Experimental details are given in the text. The exclu-sion volume was calculated by plotting the elutionprofile versus ml eluted and extrapolating to the volumeof exclusion. All the materials chromatographed toexactly the same positions even after rechromatographythree times.

lysine, aspartate, glutamate, alanine and leucine, and had a molecular weight of 575;peptide 7 contained lysine, aspartate, serine and isoleucine, and had a molecular weightof 903 (Table 4).

FURTHER IMMUNOCHEMICAL CHARACTERIZATION OF THE TRYPTIC

HYDROLYSATE AND PEPTIDES 3 AND 7

The whole digest and peptides 3 and 7 were first tested further to determine moreprecisely the extent of their antigenic activity. Two additional immunological assays wereemployed; the inhibition of flocculation of BLG-A by BLG-A antibody and inhibition ofthe PCA reaction of BLG-A antibody. The results of these experiments are summarizedin Table 5 and Fig. 2. The amounts of peptides 3 which inhibited the PCA reaction of10 yg nitrogen, BLG-A antibody with 62 yg nitrogen of BLG-A were comparable to theamount which inhibited the haemagglutination reaction (cf. Table 1). On the other hand

Antigenically Active PeptidesTABLE 4

THE AMINO ACID COMPOSITION OF PEPTIDE FRACTIONS 3 AND 7

Peptide 7 Peptide 3Amino acid

pmoles pmoles/fmole x 2 Nearest jpmoles gumoles/,pmole Nearestlysine integer lysine integer

Lysine 0 019 1 2 2 0-0186 1 1Aspartate 0 0094 0-448 0-967 1 0-014 0-78 1ThreonineSerine 0-016 0-827 1-66 2Glutamate 0-021 1*13 1Proline - -GlycineAlanine 0-015 0-79 1ValineIsoleucine 0-029 1-5 3 3Leucine 0-016 0-96 1

100 per cent recovery nitrogen 98 per cent recovery nitrogenEight amino acids Five amino acidsMolecular weight = 903 Molecular weight = 575

The peptides were hydrolysed for 24 hours at 110 in sealed tubes and umoles of amino acids were determinedas described in the text.

peptide 7 only partially inhibited the reaction at 90 pg nitrogen, about ten times theamount which inhibited the haemagglutination reaction. The concentration of peptide 3which inhibited the flocculation test was comparable to amounts found inhibitory in theother assays; 10 yg nitrogen inhibited the floccuation of BLG-A about 30 per cent. In

TABLE 5THE INHIBITION OF THE PCA REACTION BY THE PEPTIDE

FRACTIONS

Nitrogen Size of reactionPeptide (mg) (mm)

None 20BSA tryptic digest 125 20Peptide 3 6 11

13 1027 740 3-557 267 Trace

Peptide 7 45 2090 15180 10270 7360 10451 10

BLG-A tryptic digest 20BLG-A 0*07

The size of the reaction is recorded as mm diameter ofreaction. A negative (-) indicates no blueing occurred atthe site of injection. Experimental details are given in thetext.

313

D. H. Bing and A. B. Stavitsky

contrast, 30 ,ug of nitrogen of peptide 7 caused only about 10 per cent inhibition offlocculation. The inhibition in each assay was determined to be specific; a BSA tryptichydrolysate inhibited neither the PCA reaction nor the flocculation reaction.

It was also noted that none of the peptides were as active as the whole digest in termsofweight of material which inhibited the reaction ofBLG-A with BLG-A antibody. Thus,up to ten times as much of a single peptide was required to inhibit the haemagglutinationreaction (cf. Table 1) or the flocculation reaction (cf. Fig. 2). No estimation was possible

80 _

60 -0

C.' 4

a .2-

° - rL

20 40 60 80,Ug N i nhibitor

FIG. 2. Inhibition of flocculation ofBLG-A by BLG-A antibody. The peptide fractions were incubatedat pH 5*3 with 200 jig of BLG-A antibody for 20 minutes, 2-5 pg nitrogen BLG-A was added and allmixtures were incubated for 15 minutes at 370 before reading the extinctions at 660 mpg. o, BLG-Atryptic digest; *, peptide 3; *, peptide 7; cl, BSA tryptic digest. Per cent inhibition was calculated asdescribed in text.

in the PCA test, as no end point in inhibition of the reaction was found with as little as20 /g nitrogen of the whole digest and 0 07 /g nitrogen of the native protein. The relativeresults of all of these assays, however, indicated that the two peptides and whole digestwere antigenically active.

DISCUSSION

The tryptic hydrolysate of highly purified JJ-lactoglobulin-A yielded fragments whichdid not precipitate with rabbit antibody against the native protein, but did inhibit thereaction of native protein with the same antiserum in three distinct immunological assays.This hydrolysate seemed particularly suitable for the isolation of antigenically activepeptides because: (1) trypsin splits the peptide chain at arginine and lysine residues, thelatter of which is not important in the antigenicity of this protein (Singer, 1957), and(2) the apparent absence of precipitating fragments in the digest suggested that the anti-genically active peptides might be univalent (Campbell and McCasland, 1944). In fact,at least seven distinct peptide fractions were found, and two homogeneous peptides hadmolecular weights of less than 1000.

314

Antigenically Active PeptidesThe antigenic activity of all the peptide fractions was very low when compared on a

weight basis to inhibitory capacity of the native protein (cf. Tables 1 and 5). We believe,however, that the amount of activity was significant, based on the reproducibility of theresults within a given test, and the specificity of inhibition as judged by the inability of aBSA tryptic digest to inhibit the BLG-A-anti-BLG-A reaction, and the inability of theBLG-A tryptic digest to inhibit the BSA-anti-BSA reaction. The finding of minimalantigenic activity recalls the results of experiments with other low molecular weightantigenic determinants including small oligosaccharides (Mage and Kabat, 1963), smallpeptides from a fibrous protein (Cebra, 1960) or nonfibrous proteins (Metzger et al., 1962;Brown 1962; Crupton and Wilkinson, 1965) and synthetic polypeptides (Gill, Kunz,Friedman and Doty, 1963). The molar ratio of inhibitor to antibody at 50 per centmaximal inhibitory activity in the flocculation assay for peptides 3 and 7 was 91: 1 and324: 1, respectively, a result which is similar to inhibitor to antibody ratios of the anti-genically active peptides of sperm whale myoglobin (Crumpton and Wilkinson, 1965)and certain synthetic polypeptides (Gill et al., 1963).

Certain discrepancies were noted in the behaviour ofthe peptides in the assays employedto measure the antigenic activity. Thus peptide 3 was equally inhibitory in all three assays,but about six times as much of peptide 7 was required to inhibit the flocculation reaction(cf. Fig. 2) and the same material was only marginally active in the inhibition of thepassive cutaneous anaphylaxis reaction (cf. Table 5). The variations were probably dueto a combination of effects. First, each assay employed different amounts of antibody;haemagglutination inhibition required only 0-01 pg antibody nitrogen, whereas passivecutaneous anaphylaxis employed 10 pg antibody nitrogen and 200 pg antibody nitrogenwas used in the flocculation assay. Second, to circumvent the presence of serum proteaseswhich split peptides (Sela, 1966b) short incubation periods at lower pHs (5.3) were usedwith the mixtures of antibody and inhibitor. Thus the inhibitor-antibody reaction maynot have had time to come to equilibrium under optimal conditions of pH and ionicstrength. The main object of the experiments was to determine whether a peptide wasantigenically active; the reproducibility of a given assay and the relative results of allthree assays provided this information.The results of amino acid analysis of two homogeneous peptides indicated that aromatic

residues were absent and both contained about equal amounts of apolar and polar aminoacids. No experiments were done to determine sequence of these peptides, and thus thepossible position of these peptides in the polypeptide chain cannot be related to partialamino acid sequences reported by Townsend (1965) and Greenburg and Kalan (1965).Stahmann, Lapresle, Buchanan-Davidson and Grabar (1959) found that antiserum againsta polyleucine polypeptide reacted weakly with bovine fl-lactoglobulin in interfacial ringtests. It might be noted in closing, that the presence of leucine in peptides 3 and 7 is inaccord with these results.One of the most striking findings in the present study is the presence of seven antigenic-

ally active peptide fractions. f,-Lactoglobulin has eighteen arginine and lysine residuesper polypeptide chain (molecular weight = 18,000) (Piez et al., 1961). Assuming randomdistribution of amino acids, nineteen peptides with a molecular weight of about 1000would be expected after trypsin hydrolysis. In fact, at least twenty were found, and twohad molecular weights of less than 1000. If the molecular weight of each active peptidewas 500-1000, then it can be estimated that over a third of the polypeptide chain ofBLG-A was involved in the antigenically active sites of the intact protein. It should be

315

316 D. H. Bing and A. B. Stavitskynoted, however, that only hyperimmune antisera were used in these experiments, and theintense immunization of the animals may have resulted in the production of antibody to alarger portion of the protein molecule.

ACKNOWLEDGMENTS

This investigation was supported by Research Grant AI-01865 and Training Grant5T1-GM-171 from the United States Public Health Service. The authors are grateful forthe excellent technical assistance of Mr Weldon W. Harold. They are also pleased toacknowledge the help of Dr L. Astrachan in performing the determinations in theAnalytical Ultracentrifuge, Miss Violet Forgach for help in performing the amino acidanalysis, and Dr Paul Mauer, University of Pennsylvania for instruction in the techniqueof passive cutaneous anaphylaxis.

REFERENCES

ARONSON, T. and GRONWALL, A. (1957). 'Improvedseparation ofserum proteins in paper electrophoresis:a new electrophoresis buffer.' Scand. 7. Clin. lab.Invest., 9, 338.

BELL, K. and McKENZIE, H. A. (1964). 'The betalactoglobulins.' Nature (Lond.), 207, 1275.

BING, D. H., WEYAND, J. G. M. and STAVITSKY, A. B.(1967). 'Hemagglutination with aldehyde fixederythrocytes for assay of antigens and antibodies.'Proc. Soc. exp. Biol. (N.r.), 124, 1166.

BROWN, R. K. (1962). 'Studies on the antigenicstructure of ribonuclease. III. Inhibition by peptidesof antibody to performic acid-oxidized ribonuclease.'_7. biol. Chem., 237, 1162.

CAMPBELL, D. H. and MCCASLAND, G. E. (1944). 'Invitro anaphylactic response to polyhaptenic andmonohaptenic simple antigens.'_7. Immunol., 49, 315.

CEBRA, J. (1960). 'Studies on combining sites ofprotein antigen silk fibroin. III. Inhibition of thesilk fibroin-anti-silk fibroin system by peptidesderived from the antigen.' 7. Immunol., 86, 205.

COLE, W. H. (1958). Serological and Biochemical Com-parison of Proteins, p. 155. Rutgers University Press,Brunswick, New Jersey.

CRUMPTON, M. J. and WILKINSON, J. M. (1965). 'Theimmunological activity of some of the chymotrypticpeptides of sperm-whale myoglobin.' Biochem. I., 94,545.

DANIEL, T. M., WEYAND, J. G. M. and STAVITSKY,A. B. (1963). 'Micro methods for study of proteinsand antibodies. IV. Factors involved in preparationand use of stable preparation of formalinized, tannicacid treated, protein sensitized erythrocytes fordetection of antibody.'_7. Immunol., 90, 741.

GILL, T. S., III, KUNZ, H. W., FRIEDMAN, E. andDOTY, P. (1963). 'Studies on synthetic polypeptideantigens. VIII. The inhibition of the antibody-synthetic polypeptide reaction by amino acids,dipeptides, amino acids, alcohols, and dicarboxylicacids.'_7. biol. Chem., 238, 108.

GREENBURG, R. and KALAN, E. B. (1965). 'Studies on,f-lactoglobulins A, B, and C. II. Preparation ofmodified proteins by treatment with carboxypeptidase A.' Biochemistry, 4, 1660.

HARA, T. (1956). 'Chemical studies on the organspecificity.' Jap. Y. Biochem., 43, 263.

HELINSKI, D. R. and YANOFSKY, C. (1962). 'Peptidepattern studies on the wild-type A protein of trypto-phan synthetase of Escherichia coli.' Biochim. biophys.Acta (Amst.), 63, 10.

KABAT, E. A. and MAYER, M. M. (1961). ExperimentalImmunochemistry. Thomas, Springfield, Illinois.

LANDSTEINER, K. (1947). The Specificity of SerologicalReactions, pp. 55-67. Harvard University Press,Cambridge, Massachusetts.

LAPRESLE, C., KAMINSKI, M. and TANNER, C. E. (1959).'Analysis of the antigenic structure of human serumalbumin.' 7. Immunol., 82, 94.

LAPRESLE, C. and WEBB, T. (1965). 'Isolation andstudy of a fragment of human serum albumincontaining one of the antigenic sites of the wholemolecule.' Biochem. J., 95, 245.

LARSON, B. L. and JENNESs, R. (1955). 'Beta lacto-globulin.' Biochemical Preparations (Ed. by W. W.Westerfield), Vol. 4, p. 23. Wiley, New York.

LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L. andRANDALL, R. J. (1951). 'Protein measurement withthe Folin phenol reagent.'_7. biol. Chem., 193, 265.

MAGE, R. G. and KABAT, E. A. (1963). 'The com-bining regions oftype III pneumococcus polysacchar-ide and homologous antibody.' Biochemistry, 2, 1278.

METZGER, H., SHARP, G. C. and EDELHOCH, H. (1962).'Properties of thyroglobulin. VII. The immunologicactivity of thyroglobulin fragments.' Biochemistry, 1,205.

MOORE, S. and STEIN, W. H. (1948). 'Photometricninhydrin method for use in the chromatography ofamino acids.'_7. biol. Chem., 176, 367.

MOORE, S. and STEIN, W. H. (1951). 'Chromatographyof amino acids on sulfonated polystrene resins.'J.biol. Chem., 192, 663.

MOORE, S. and STEIN, W. H. (1963). 'Determinationof amino acids.' Methods in Enzymology (Ed. by S. P.Colowick and N. 0. Kaplan), Vol. 6, p. 819.Academic Press, New York.

OKUYAMA, T. and SATAKE, K. (1960). 'On thepreparation and properties of 2,4,6-trinitrophenyl-amino acids and peptides.' Jap. J. Biochem., 47, 454.

OVARY, Z. (1958). 'Immediate reactions in the skinof experimental animals provoked by antigen-anti-body reaction.' Progr. Allergy, 5, 459.

PIEz, K. A., DAVIE, E. W., FOLK, J. E. and GLADNER,

Antigenically Active Peptides 317J. (1961). ',f-Lactoglobulins A and B. I. Chromato-graphic separation and amino acid composition.'J. biol. Chzem., 236, 2912.

PRESS, E. M. and PORTER, R. R. (1960). 'Isolation andcharacterization of fragments of human serumalbumin containing some of the antigenic sites of thewhole molecule.' Biochem. J., 83, 172.

RICHARD, A. J., BECK, S. and HOCH, H. (1960). 'Theisolation of serologically active peptides of lowmolecular weight from a hydrolysate of bovineplasma albumin.' Arch. Biochem. Biophys., 90, 309.

RITSCHARD, W. J. (1964). 'Thin-layer techniques formaking peptide maps.' J. Chromatogr., 16, 327.

SCHACHMAN, H. K. (1957). 'Ultracentrifugation, diffu-sion and viscometry.' Methods in Enzymology (Ed. byS. P. Colowick and N. 0. Kaplan), Vol. 4, p. 32.Academic Press, New York.

SCHEIDEGGER, J. J. (1955). 'Une micro-method deimmuno-6lectrophoreise.' Int. Arch. Allergy, 7, 103.

SELA, M. (1966a). 'Immunological studies withsynthetic polypeptides.' Advanc. Immunol., 5, 29.

SELA, M. (1966b). 'Combining sites of antibodies withL-alanine and D-alanine peptide specificity and theeffect of serum proteolytic activity on their estima-tion.' Biochim. biophys. Acta (Amst.), 127, 438.

SINGER, S. J. (1957). 'Physical-chemical studies on the

nature of antigen-antibody reactions.' J. CellPhysiol., 50 (Suppl. 1), 51.

STAHMANN, M. A., LAPRESLE, C., BUCHANAN-DAVID-SON, D. R. and GRABAR, P. (1959). 'Immuno-chemistry of synthetic polypeptides and polypeptidylproteins. I. Qualitative studies on the modifiedproteins.'J. Immunol., 83, 534.

STAVrrSKY, A. B. (1954). 'Micromethods for study ofproteins and antibodies. I. Procedure and generalapplications of hemagglutination with tannic acidand protein-treated red blood cells.'J. Immunol., 72,350.

TOWNSEND, R. (1965). ',f-Lactoglobulins A and B: theenvironment of the Asp/Gly difference residue.'Arch. Biochem. Biophys., 109, 1.

YOUNG, J. D., BENJAMINI, E., SHIMIZU, M., LEUNG,C. Y. and FEINGOLD, B. F. (1963). 'Antigenic activityof tryptic peptides of tobacco mosaic virus protein.'Nature (Lond.), 199, 831.

YOUNG, J. D., BENJAMINI, E., SHIMIZU, M. and LEUNG,C. Y. (1966). 'Immunochemical studies on thetobacco mosaic virus protien. III. Degradation of animmunologically active tryptic peptide of tobaccomosaic virus protein and the reactivity of thedegradation products to the antibodies to the wholeprotein.' Biochemistry, 5, 1481.