JOURNAL OF BACTERIOLOGY, Dec. 1968, p. 1953-1960Copyright @ 1968 American Society for Microbiology

Preparation and Description of High-Molecular-Weight Soluble Surface Antigens from a

Group A StreptococcusRICHARD W. BESDINE AND LEO PINE

National Communicable Disease Center, U.S. Public Health Service, Atlanta, Georgia 30333

Received for publication 18 September 1968

High-molecular-weight proteins having M protein reactivity were isolated withoutacid or alkaline digestion. Treatment of a heat-killed group A Streptococcus withsonic vibration released antigens which reacted strongly and specifically withabsorbed type-specific antiserum. This antigen preparation was released withoutdiminishing the total yield of acid-extractable M protein of the original heat-killed cells. Fractionation of the sonic preparation on a sucrose gradient yieldedfour peaks of M reactivity. When these fractions were placed on Sephadex G-200columns, the M reactive material of three fractions appeared in the void volumes,suggesting that the active material in each had a molecular weight greater than300,000. The reactivity of the fourth fraction followed closely the void volume ofSephadex G-100. Chemical analysis revealed heterogeneity of the fractions. Spectralanalysis showed virtual absence of nucleic acid in three of the fractions and a mod-erate amount in the fourth. Bactericidal inhibition tests showed activity of three ofthe four fractions. Analysis of the fractions by Ouchterlony double-diffusion tech-nique revealed that each of the four fractions had several antigenic constituents. Allfour contained M antigen. T antigen and a third unnamed antigen were present insome of the fractions. Group reactivity was present in all fractions, but did not resideon the M molecule. The enhanced potential of sonically released antigens to inducehigh-titer specific precipitating antibodies to M protein is discussed.

M protein of group A streptococci, one of theorganism's many surface antigens (9), has longbeen recognized as the best correlate of virulence(15). It is a phagocytosis-inhibiting factor, andits loss from an organism greatly reduces theorganism's pathogenicity and ability to survive innormal human blood. This protein is also thebasis of typing of streptococci.

Since its original description by Lancefield(10) in 1928, M protein has been obtained byboiling the streptococcal cell in acid. Acid-extracted M protein, however, elicits a very poorprecipitating antibody response. Whole cells,therefore, are used for immunization of animalsto obtain potent precipitating antiserum to Mprotein, but absorption to remove nonspecificcross-reactions in this serum generally results inthe precipitous decline of the desired specificantibody.

It seemed likely that the harsh acid treatmentused for its extraction must impair the integrityof the M protein released. The following studieswere carried out to obtain M protein in a form

more native than that available from acid extrac-tion. It was hoped that, by using physical ratherthan chemical methods to release M protein, a

better immunogen could be isolated. In thispaper, we report the isolation and the serological,chemical, and antigenic properties of soluble,high-molecular-weight surface antigens from a

group A Streptococcus.

MATERIALS AND METHODS

Bacterial strains. S. pyogenes, group A, type 1,strain 0001/B1, obtained from W. K. Harrell, Bio-logical Reagents Section, National CommunicableDisease Center, was used in all of our studies. Thestrain is virulent for man and is a good producer ofMprotein. After one passage through Todd-Hewittbroth (THB) enriched with defibrinated rabbit blood(THBB), stock cultures were divided into 1-ml por-tions and stored at -60 C.

Culture techniques. After inoculation with 2 ml ofa 6-hr THBB culture, several 30-ml cultures in THBBwere grown overnight at 37 C. Reagent bottles (4liters) containing 2.7 liters of THB were each inocu-lated with a 30-ml culture. A supplement (300 ml,

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containing 100 g of glucose/liter, 80 g of NaHCO3/liter, and 10 g of Na2PO4/liter) was added 2 hr afterinoculation. After 18 hr of growth at 37 C, cultureswere heat-killed in a water bath (60 C) for 1 hr. Thecells were allowed to settle overnight at 4 C, werecentrifuged at 10,000 X g for 20 min, and were washedtwice with 50 volumes of buffered saline (0.9% salinein 0.01 M K2HPO4-KH2PO4, pH 7.0). All manipula-tions were carried out at 5 C.M protein isolation. Acid extraction was carried

out by adjusting a 20% suspension (v/v) of heat-killed cells in buffered saline to pH 2.0 with 1.0 NHCl, heating the suspension in a boiling-water bathfor 10 min, cooling it and adjusting it to pH 7.0 with1.0 N NaOH (15). After the suspension was cen-trifuged at 38,000 X g for 30 min, the supematantfluid containing the acid-extracted M protein (Macid) was collected and stored at 4 C.

Braun extraction was carried out by cell disintegra-tion with glass beads by the method of Bleiweisset al. (1). To three volumes of a 33% suspension(v/v) of heat-killed cells, one volume of glass beads(3M microbeads, class IV, type 2740 WP, MinnesotaMining and Manufacturing Co., St. Paul, Minn.) wasadded. The suspension was shaken in a Braun ho-mogenizer for 8 min in 2-min bursts with cooling bycompressed CO2. The glass beads were removed by a10-min centrifugation at 1,000 X g, and then the sus-pension was centrifuged at 38,000 X g for 30 min.The supernatant fluid was aspirated and stored at4 C. The precipitates were acid-extracted.

Sonic release of M protein was accomplished bysubjecting a 20% suspension (v/v) of heat-killed cellsin buffered saline to high-frequency sonic vibrationat 150 w with the Sonifier Cell Disruptor (HeatSystems Co., Melville, N.Y.) in 1-min bursts. Thesuspension was kept cold with a dry ice-alcohol bath.To avoid entering the overload zone, the intensitywas reduced as the viscosity of the suspension in-creased with time. After sonic treatment for 15 to 18min, the suspension was centrifuged at 38,000 X gfor 30 min at 5 C. The precipitate was washed oncewith an equal volume of cold buffered saline andcentrifuged at 38,000 X g for 30 min; the supematantfluids were combined. This fraction of M protein,released by sonic treatment, is called sonic M (Mson).

Serology. Absorbed type-specific antisera and groupA antiserum were supplied by W. K. Harrell. Type 1absorbed serum is known to contain anti-T antibodyas a contaminant. Type-specific quantitative capillaryprecipitin tests were performed according to themethod of Cohen and Pine (4). Results were expressedas units ofM protein by reference to a standard curve.All quantitative M protein data were expressed asunits of M per milligram (dry weight) of cells in theoriginal suspension. One unit of M protein is definedas that amount of precipitate resulting from thereaction of 0.008 ml of a standard absorbed antiserumwith 0.008 ml of a standard acid-extracted M proteinantigen. Precipitin tests were performed in the stand-ard manner (14) and graded 0 to +5. Group-specificqualitative ring tests were performed in a standardmanner (17) and were graded 0 to +5. Immuno-

diffusion tests were performed in a medium (developedby Paul Nichols) of the following composition: 2.25 gof NaCl, 2.25 g of Noble Agar, 0.50 g of K2HPO4,0.50 ml of 10% MgCI2, 1.0 g of sodium citrate, 0.50ml of 80% phenol, made up to 250 ml with distilledwater. Glass slides (2 by 3 inches; 5.1 by 7.6 cm) wereused and developed in a moist chamber at roomtemperature (24 to 48 hr).

Density gradientt centrifugation. Linear sucrosegradients of 11 ml were prepared with a small gradientmachine. Each gradient was loaded with 0.5 to 1.0ml of test material and was centrifuged 4.5, 6, or 20hr in the Spinco L-2 ultracentrifuge by using theT-50 rotor (2, 3) or 25.2 swinging-bucket rotor.Fractions of 0.4 to 0.5 ml were collected from eachgradient after centrifugation by puncturing the bottomof the tube. The average per cent of sucrose in eachfraction was determined by using an Abbe refractom-eter.

Column chromatography. Sephadex G-100 andG-200 dextran gels were suspended in 0.01 M phos-phate (pH 7.2) with 0.02% sodium azide. The columnswere equilibrated and eluted with buffered saline.Fractions of approximately 2 ml were collected at arate of one drop every 11 sec. Fractions were moni-tored at 254 nm with a continuous recorder.

Miscellaneous. Dry weights were determined on asemimicro Mettler B balance (h0.02 mg). Fractionswere concentrated by dialyzing against Carbowax20 or by directing a jet of dry air on the solutions.Optical densities (OD) and absorption spectra weredetermined with a Gilford 2000 spectrophotometerhaving Beckman optics. Bactericidal inhibition testswere performed according to the method of Stoller-man (7). Nucleic acid content was estimated by the260 to 280 nm OD ratios (16). Hexose was determinedby the cysteine-sulfuric acid method of Dische (5).Methylpentose was determined by the cysteine-sulfuric acid method of Dische and Shettles (6). Pro-tein was determined by the micromethod of Lowryet al. (11).

RESULTS

Timed release ofM son and M acid from cells.The relationship of M son to M acid and thedynamics of the liberation of M son were deter-mined first. Figure 1 shows that M son wasreleased from heat-killed cells in increasingamounts as the time of sonic treatment increasedand that the amount ofM acid in the cell residuewas not reduced.

In repeated experiments, amounts of M sonand M acid obtained from a given lot of cells wereapproximately equal. These results indicate thatM son is a different molecular species from Macid. It makes no contribution to traditional Macid. With several type-specific absorbed antiseraother than type 1, M son showed no precipitinreaction, but crude M son showed a +3 reactionwith group A antiserum. When M son wassubjected to the conventional acid extractionmethod, its reactivity was decreased by 70%.

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0L 600/3 JMson

500/) 400-z

300-

200-

100-

50-25-0

0 3 6 9 12 15 18MINUTES

FIG. 1. Time release curve ofM son and M acid.A 33-ml amount of a 20% suspension (v/v) of heat-killed cells in buffered saline (dry weight, 39.2 mg/ml)was sonically treated for 18 min; J-ml samples were

removed at time-zero and every 3 min thereafter. Eachsample was centrifuged for 30 min at 38,000 X g, andthe supernatant fluids were collected. The precipitateswere washed once with I ml of buffered saline, cen-

trifuged, acid-extracted twice, and washed once. Eachsample was diluted 10-fold, and quantitative precipitintests were performed in quadruplicate.

Effects of acid extraction on subsequent sonicrelease of M. The above data suggested eitherthat acid extraction destroyed M son or that,after acid extraction, M son was left intact on thecell. This experiment attempted to distinguishbetween these alternatives. Table 1 gives theamount of M son released from whole cellsbefore and after acid extraction of the same cells.The results show that prior acid extraction ofwhole cells drastically reduced the amount of Mson available from the cells and suggest stronglythat the standard acid extraction proceduredestroyed M son

Release of soluble M by mechanical disintegra-tion. To determine whether soluble M reactivematerial could be released by other physicalmethods, the effect of mechanical disintegrationon M son and M acid was studied. Table 2 givesthe quantitative data obtained by disintegratingcells with glass beads in the Braun shaker. Thedata show that cell disintegration with the Braunshaker released soluble M reactive material("Braun sup") but that the quantity ofM reactivematerial obtained by cell disintegration was only30% of the amount of the reactive material

TABLE 1. Quantitative effects of acid extraction onthe release of M son from heat-killed cells

Total units Mg (dry Uits ofTreatment of M weight) (Mdrwt)g

Sonic treatmentbefore acidextraction-... 0.20 X 105 30 650

Acid extrac-tionb......... 7.20 X 105 1,006 715

Sonic treatmentafter acid ex-tractionc. 0.53 X 105 1,006 53

a A 20-ml amount of a 15 to 20% suspension(v/v) of heat-killed cells (30 mg/ml, dry weight)was treated with sonic vibration for 18 min.

b A 33-ml amount of the original suspension wasacid-extracted.

c The residue of acid extraction was sonicallytreated.

TABLE 2. Quanititative effects of mechanicaldisintegrationa on the release of solubleM protein from heat-killed cells

Mg Units ofTreatment Total units (dry /smgof M wt) (dry

wt)

Braun supernatantfluid............. 1.69 X 105 774 218

Braun supernatantfluid (acid-ex-tracted) .......... 0.31 X 10' 774 40

Acid extraction ofBraun precipitate. 3.08 X 105 774 398

Sonic treatment ofBraun precipitate. 0.65 X 10' 774 84

a A suspension of 5.5 g, wet weight (774 mg,dry weight) of heat-killed cells and 5.5 ml of glassbeads in 11 ml of buffered saline was subjected toBraun disintegration for 8 min in 2-min bursts.After the addition of 15 ml of buffered saline, thesuspension was centrifuged for 10 min at 1,100 X gto remove the glass beads. The beads were washedtwice with 5 ml of buffered saline, and the super-natant fluids were combined and centrifuged for30 min at 38,000 X g. The supernatant fluid wassaved for quantitative precipitin tests. A portionwas acid-extracted. One portion of the precipitatewas acid-extracted, and one portion was sonicallytreated.

released by acid extraction. Furthermore, dis-integration of cells with the Braun shaker de-creased by about 40% the amount of M reactivematerial available from them by acid extraction.This decrease is approximately the amount of Mreactivity in the supernatant fluid after Braundisintegration. When the Braun sup was subjected

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to the conventional acid extraction method, itsreactivity was decreased by 80%. The M sonavailable from the cells after Braun disintegrationis reduced by almost 90%. These findings sug-gested that Braun disintegration as well as acidextraction destroys M son.

Comparison ofM son andM acid by centrifuga-tion on sucrose gradients. To determine whetherM acid and M son had qualitative and quantita-tive physical differences, the two preparationswere centrifuged in identical sucrose gradients(Fig. 2). Two 11-ml linear sucrose gradients from10 to 55% sucrose were loaded with 0.5 ml ofcrude M son and 0.5 ml of crude M acid, re-spectively. Each was centrifuged for 20 hr at150,000 x g (average) by using the T-50 rotor.M reactivity of the two preparations was clearlyseparated on the gradients. M son reactivityappeared in 30% sucrose, peaked in 40%, anddisappeared in 52%, whereas M acid reactivityappeared in 20% sucrose, peaked in 25%, anddisappeared in 30%. The behavior of M acidobserved in this test is in general agreement withprevious measurements (8). Contrary to theresults obtained with M acid, the M reactivematerial in the sonically treated preparation wasseparated from the great bulk of 280-nm absorb-ing material.

Partial purification and resolution of M sonobtained by sucrose gradient centrifugation. Todefine the M reactive fractions present in M son,10 ml of the crude material was centrifuged for6.5 hr in sucrose gradients (Fig. 3). Four peaks ofM reactivity were distributed over the gradient.The peaks were collected as follows. Fraction Iincluded 6.8 to 17.9% sucrose; fraction II, 18.0 to31.4% sucrose; fraction III, 31.5 to 41.0% su-crose; and fraction IV, 41.1 to 49.1 % sucrose.After fractions I through IV were dialyzed over-night against water to remove sucrose and salts,

0.1-5 D/\3

4 03E

FRACTION FRACTION FRACTION I FRACTIONI I U m + z

78910 15 20 25 30 35 40 45 50% SUCROSE

FIG. 3. Separation of M son reactive componentsby sucrose gradient centrifugation. Ten 1-mil linearsucrose gradients from 10 to 55% sucrose were eachloaded with 1.0 ml ofcrude M son and were centrifuigedfor 6.5 hr at 150,000 X g by using the T-50 rotor.Fractions (0.5 ml) were collected separately from eachof the 10 gradients by puncturing the bottoms of thegradient tubes. The average per cent of sucrose in eachfraction was read in the Abbe' refractometer. Fractionswere combined on the basis of sucrose concentration(±1%); they were diluted 10-fold; the OD at 280 nmwas determined; and quantitative precipitin tests wereperformed.

each fraction was concentrated to 1 to 2 ml inCarbowax.To further purify the concentrated fractions

I through IV, each was placed on a SephadexG-100 column and was eluted with bufferedsaline. Each fraction showed an OD peak at thevoid volume of the column. Fraction I, which wastaken from the least dense sucrose, showed sub-stantial disparity between the location of Mreactivity and the void volume OD peak. Frac-tions II, III, and IV, however, showed coincidenceof the M reactive tubes and the OD peaks at thevoid volume.

Within each fraction eluted from the SephadexG-100 column, the tubes having M reactivitywere combined and concentrated in Carbowax 20to a final volume of 1.0 to 1.5 ml. Each fractionwas placed on a Sephadex G-200 column andeluted with buffered saline. Fraction I demon-strated no OD peak at the void volume whenpassed through Sephadex G-200, and the Mreactivity of this fraction was not recovered fromthe column. Each of the elution curves of frac-tions II, III, and IV from Sephadex G-200, how-ever, showed a coincident OD and M reactivitypeak at the void volume.

Spectral analysis ofpurified fractions. To obtaininformation about the chemical composition ofthe M reactive fractions from either SephadexG-100 or G-200 and to obtain an indication oftheir purity, continuous spectral analyses were

%/. SUCROSE

FiG. 2. Comparison of M soilantd M acid by cei-trifugation on sucrose gr-adients.

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performed on the tubes that showed the greatestM reactivity. The spectral curves showed thatfractions I, II, and III were essentially free ofnucleic acid and that the tubes comprising theactive peaks within each fraction were homogene-ous. Fraction IV, however, had a significantamount of nucleic acid, and the tubes comprisingthe M reactive peak were not homogenous.

Chemical analysis of the M reactive fractions.Because of the similarity of the spectral curves ofthe tubes of fraction II from Sephadex G-200,tubes 13 to 17 were combined and concentratedby evaporation to 1.0 ml. Similarly, tubes 10 to12 of fraction III from Sephadex G-200 werecombined and concentrated. The individual tubesof fraction IV from Sephadex G-200 were keptseparate because of their obvious heterogeneity,and tube 10, the one with the greatest activity,was analyzed. The most active tube of fraction Ifrom Sephadex G-100 was also analyzed. Table3 gives the results of analyses of the fractions forprotein, hexose, and methylpentose. Protein,hexose, and methylpentose were found in thefractions. The constancy of the hexose-methyl-pentose ratios and the variation of the protein-hexose and protein values showed that the chemi-cal differences among the three fractions were dueto variation in their protein content.

Group-specific reactivity in M active prepara-tions. The comparative group and type reactionsof the fractions of M son in various stages ofpurification are presented in Table 4. Althoughthe strength of the M reactivity changed littlewith purification, group reactivity in the prepara-tions decreased. However, group reactivity waspresent in all of the fractions which were isolated.

Bactericidal inhibition tests. Bactericidal inhibi-tion tests showed that all but fraction III inacti-vated type-specific immune serum and that thegreatest activity was in fraction I. The lack ofactivity in fraction III, however, may reflect aconcentration phenomenon rather than im-potence of this fraction.

Antigenic analysis of fractions by immuinodif-

TABLE 3. Chemical analysis ofM reactive fractionsfrom Sephadex

Meth- Protein- Hexose-Fraction Protein Hexose meth-Fratio /Omi) (,,g/l.) yl-etos he*s ylpentose,jg,mn, ratios ratioa

II, G-200 131.5 70.8 2.40 1.86 29.5III, G-200 90.4 85.6 2.76 1.06 31.0IV, tube 10, 9.0 70.2 2.07 0.13 29.5G-200

I, G-100 144.0 41.0 NDa 3.50 NDa

a Not determined.

fusion studies. The antigenic composition of eachpurified fraction was studied by the Ouchterlonydouble-diffusion technique. Figure 4 shows thereaction of three antigen preparations, M son, Macid, and M son treated with acid, with type-specific absorbed antiserum.A band of identity which represents M protein

was present in all three antigens, M acid, M son,and fraction I, Sephadex G-100. Two other bandswere present in M son and were acid-labile,since they were not found in M acid and acid-treated M son. Because acid lability is a classicfeature of T protein and because anti-T antibodywas known to be in the absorbed M antiserum,it appeared that one of the two additional bands

TABLE 4. Comparative group andoffractions

type reactions

Fraction Group reaction Type reaction

M son +3 +5I +3 +5II +3 +5III +3 +5IV +3 +4G-100

I +3 +4II +2 +4III +1 +4IV +3 +4

G-200I +1 +4II trace +4III trace +4IV, T 10 trace +3

FIG. 4. Ouchterlony reactions of M son, M acid,and M son-acid against M antiserum. M son-acid wasprepared by heating crude M son adjusted to pH 2.0with HCl for 10 min at 95 C in a water bath.

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in M son was T protein. The band closest to theantigen well, by its continuation against T anti-serum, was positively identified as T protein(Fig. 5). The third band of reactivity in M sonwas not present in fraction I, Sephadex G-100.

Figure 6 shows the reaction of fraction Itpurified on Sephadex G-200 with M and T anti-sera. Like fraction I, Sephadex G-100, fractionII, Sephadex G-200, contains an M band and aT band. In addition, however, fraction II alsocontains the third, unidentified band ofM son.

Figure 7 shows the reaction of fraction IIIpurified on Sephadex G-200 with M and T anti-sera. This fraction has the same antigenic con-stitution as fraction I1, Sephadex G-200, and

FIG. 5. Ouchlterlony reactioni of fractioni I, Sepha-dex G-100, against M af?d T anitiser-a.

FIG. 6. Ouchterloniy reactioni of fraction II, Sepha-dex G-200, against M and T antisera.

contains all three bands of M son-M protein,T protein, and the third, unknown antigen.

Figure 8 shows the reaction of fraction IVpurified on Sephadex G-200 with M and T anti-sera. This fraction has no T band. It shows aband of partial identity with M and a band oftotal identity with the third, unnamed bandpresent in M son.

Figure 9 shows the reaction of fractions Ithrough IV against type-specific absorbed Mantiserum and group A antiserum. Each fractionshowed group reactivity. The broadness of thebands makes interpretation difficult, but itappears that the M band of M son shows noidentity with group A antiserum.

FIG. 7. Ouchterlony reaction offraction III, Sepha-dex G-200, against M and T antisera.

FIG. 8. Ouchterlony reactioni offraction IV, Sepha-dex G-200, against M and T antisera.

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FIG. 9. Ouchterlony reactions offractions I throughiIV with M and group A antisera.

DIscussIoN

The prime objective of these studies was torelease from group A streptococci an antigenwhich would induce an immune response thatincluded a precipitating antibody of high titer andspecificity for M protein. The disparity betweenthe effectiveness of whole heat-killed cells andacid-extracted M protein in eliciting such aresponse is convincing evidence that such animmunogenic molecule exists. Because the acidextraction method probably involves hydrolysisof a native protein, it seemed likely that themolecule sought was of relatively high-molecuilarweight. The task was to depart from traditionalacid extraction and find the molecule whereinthe desired properties resided.The early investigation of Mudd and his

associates (12) demonstrated that a complexsubstance with M protein and group A reactivitycould be released from live streptococci by acrude form of sonic vibration. Slade (13) con-firmed these observations and suggested thatsonic oscillation seemed more likely to releasenative M protein than did acid extraction. Ourstudies show that M protein is released fromheat-killed cells in increasing amounts with in-crease in time of sonic treatment; but, moreimportant, they show that the M protein releasedby sonic energy came from a molecular poolwhich was destroyed by acid extraction andwhich made no quantitative contribution toclassical acid M. The clear difference in sedimen-tation characteristics between M son and M acidin a sucrose gradient suggested that all of theM protein reactive constituents of M son had

higher molecular weights. This evidence suggeststhat M son is more likely to be an undegradedprotein of the streptococcal cell than is acidextracted M.The fractionation of M son on a sucrose gra-

dient yielded four distinguishable fractions.Physical, chemical, and serological analysisconfirmed that these fractions are different fromone another. Fraction I was retained on Sepha-dex G-100. It was the only fraction whose Mreactivity was not confined to the void volume ofSephadex G-100 and G-200. Since the reactivityof fraction I was eluted immediately after thevoid volume of Sephadex G-100, its molecularweight can be estimated at less than 100,000.Although this molecular weight is in the rangereported for acid-extracted M protein (8), thetwo preparations cannot be considered equivalentsince only one of them has been subjected to acidhydrolysis. In addition, the sedimentation char-acteristics of fraction I and M acid are different.The M reactivity of fractions II, III, and IV wasconfined to the void volume of Sephadex G-100and G-200, suggesting that the M reactive ma-terial in each fraction had a molecular weight inexcess of 300,000. Their relative positions in thesucrose gradient suggest that fraction IV has adensity greater than fraction III which is denserthan fraction II.

Antigenic analysis of the fractions by Ouch-terlony double-diffusion tests revealed that eachfraction is composed of at least two differentantigens. Fraction I contains M and T antigen.Fractions II and III contain M, T, and a thirdunknown antigen. Fraction IV contains M andthe unknown antigen, but no T. M son is not asingle antigenic species, but rather is three familiesof antigens of increasing density and probably ofincreasing molecular weight. M antigen appearsin each fraction; T appears in all but the mostdense fraction, and the third antigen, in all butthe least dense fraction.

All the fractions had some group A activity,although it diminished with purification. It wasnecessary to determine if the group activity wasattached to one of the antigens or if it existed asa separate molecular species. Analysis of theOuchterlony double-diffusion test showed thatthere was no band of identity between M anti-serum reacting with M son and group A antiserumreacting with M son. This meant that groupactivity did not reside on the same molecule as Mreactivity. As yet, it is not possible to be sure ifgroup reactivity is attached to another antigen inM son.The overall results show that sonic treatment, as

previously reported by Mudd et al. (12), specifi-

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cally released soluble M antigens from a heat-killed group A Streptococcus. These antigens,which include a spectrum of at least four differentM antigens, also include T and group A antigens.The M antigens differ in their chemical andphysical properties. Since they have not beentreated with acid in the usual manner and are ofhigh molecular weight, they merit further studyas potential antigens for use in the production ofbiological reagents or in other immunologicalactivities. The fraction with the highest specificactivity in the bactericidal inhibition test wouldappear to be the most promising for further study.

LITERATURE CITED

1. Bleiweiss, A. S., W. W. Karakawa, and R. M.Krause. 1964. Improved technique for thepreparation of streptococcal cell walls. J.Bacteriol. 88:1198-1200.

2. Brentani, R., M. Brentani, and I. Raw. 1967.Zone sedimentation analysis of RNA in anglehead rotors. Anal. Biochem. 20:361-363.

3. Charlwood, P. A. 1963. Applications of radio-actively labelled marker proteins in densitygradient ultracentrifugation. Anal. Biochem.5:226-245.

4. Cohen, J. O., and L. Pine. 1968. Quantitativeaspects of the M protein capillary precipitintest. Appl. Microbiol. 16:122-127.

5. Dische, Z. 1949. Spectrophotometric method forthe determination of free pentose and pentosein nucleotides. J. Biol. Chem. 181:379-392.

6. Dische, Z., and L. B. Shettles. 1948. A specificcolor reaction of methylpentoses and a spectro-photometric micromethod for their determi-nation. J. Biol. Chem. 175:595-603.

7. Ekstedt, R. D., and G. H. Stollerman. 1960.Factors affecting the chain length of Group Astreptococci. HI. Quantitative M-anti-M rela-tionships in the long chain test. J. Exptl. Med.112:687-698.

8. Fox, E. N., and M. K. Wittner. 1965. The multiplemolecular structure of the M proteins of groupA streptococci. Proc. Natl. Acad. Sci. U.S.54:1118-1125.

9. Hahn, J. J., and R. M. Cole. 1963. StreptococcalM antigen location and synthesis studied byimmunofluorescence. J. Exptl. Med. 118:659-666.

10. Lancefield, R. C. 1928. Antigenic structure ofStreptococcus hemolyticus. I. Demonstrationof a type-specific substance in extracts of S.hemolyticus. J. Exptl. Med. 47:91-103.

11. Lowry, 0. H., N. J. Rosebrough, L. Farr, andR. J. Randall. 1951. Protein measurement withthe Folin phenol reagent. J. Biol. Chem. 193:265-275.

12. Mudd, S., E. J. Czarnetzky, D. Lackman, andH. Pettit. 1938. The antigenic structure ofhemolytic streptococci of Lancefield group A.I. The preparation of a labile, type-specificantigen; its identification as the Griffith type-specific agglutinogen and as a substance fromwhich a group-specific and type-specific haptenare derivable. J. Immunol. 34:117-153.

13. Slade, H. D., and J. K. Vetter. 1956. Studies onStreptococcus pyogenes. I. Observations onthe microscopical and biological aspects of thedisintegration and solubilization of a type 6strain by sonic oscillation. J. Bacteriol. 71:236-243.

14. Swift, H. F., A. T. Wilson, and R. C. Lancefield.1943. Typing group A hemolytic streptococciby M precipitin reactions in capillary pipettes.J. Exptl. Med. 78:127-133.

15. Todd, E. W., and R. C. Lancefield. 1928. Variantsof hemolytic streptococci; their relation to type-specific substance, virulence, and toxin. J.Exptl. Med. 48:751-767.

16. Warburg, O., and W. Christian. 1942. Isolationand crystallization of enolase. Biochem. Z.310:384-421.

17. Williams, R. E. 0. 1958. Laboratory diagnosis ofstreptococcal infections. Bull. World HealthOrgan. 19:153-176.

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