Physical heterogeneity of bovine γ-globulins: Characterization of γM and γG globulins

ARCHIVES

OF

Physical

BIOCHEMISTRY

Heterogeneity

FREDERICK Department

AND

BIOPHYSICS

of Bovine

r-Globulins:

TM and TG

Globulins’

A. MURPHY:

of Veterinary

126-136 (1965)

112,

Microbiology,

JOHN

W. OSEBOLD,

School of Veterinary Davis, California

Characterization

AND

of

OLE AALUND3

Medicine, University

of California,

Received May 22, 1965 The -rG globulins of bovine serum were characterized by anion exchange chromatography, immunoelectrophoresis, zone electrophoresis, ultracentrifugation, and analysis of the products of papain digestion. Their properties were similar to those of analogous components of human serum. Fast and slow rG globulins were distinguished on the basis of differences in electrophoretic migration rates, chromatographic elution positions, and biological activities (complement fixation), Antigenic differences were detectable only in immunoelectrophoretic patterns (spur formation) . Bovine TM was found to have properties quite similar to those of the analogous protein in human serum as determined by the methods of gel filtration, immunoelectrophoresis, anion exchange chromatography, ultracentrifugation, and reduction with mercaptoethanol. Bovine yA was provisionally recognized in immunoelectrophoretic analyses of serum and chromatographic fractions.

To the extent that immunoglobulin functions depend upon unique physicochemical properties, each class may differ in its efficiency as antibody in a particular immune response. Recognition of immunoglobulin classes by protein characterization is necessarily the foundation for understanding species variations in antibody heterogeneity. In order to broaden the basis for considering analogies between species, it has been recommended that as many criteria as possible “be employed to establish whatever similarities in chemical structure may exist between animal and human immungolobulin classes” (1).

In this report a physicochemical characterization of bovine rG and TM globulins is presented which emphasizes the closeness of their relationship to counterpart proteins in human serum. Biological activity was determined by complement fixation (CF) with serum components from bovine animals experimentally infected with the blood parasite Anaplasma marginale. Antibody activity was associated with electrophoretically fast rG and yM globulins. Kinetics of antibody response in experimental anaplasmosis are reported elsewhere (2,3).

1 This investigation was supported in part by a U.S. Public Health Service Postdoctoral Fellowship, IF2 AI 19788, from the National Institute of Allergy and Infectious Diseases. 2 Present address: Virology Section, Communicable Disease Center, Atlanta, Georgia. s Present address : Hygienic-Bacteriological Laboratory of the Royal Veterinary and Agricultural College, Biilowsvej 13, Copenhagen V, Denmark.

Experimental infection with either of two A. marginale isolates (California strain, Florida strain) provided the antigenic stimulus for 5 cattle. Details pertaining to collection of sera from representative phases of the ensuing disease have been presented elsewhere (3). Anion exchange chromatography of bovine antisera on DEAE-Sephadex A-56 medium (particle size of 106-260 mesh; Pharmacia, Uppsala, Sweden) as well as chromatography of zone

MATERIALS

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AND

METHODS

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electrophoretic and gel filtration fractions of these aera was performed according to the method of Fahey and Horbett (4) and Fahey et al. (5) with some modifications (3, 6). Preparative zone electrophoresis in beds of Pevikon C-870 (Stockholm Superfostat Fabriks A.-B., Stockholm, Sweden) was performed according to the methods of Miiller-Eberhard (7) and Fahey and M’cLaughlin (8). Five ml of serum was fractionated per run in a Pevikon block (39 X 6.5 X 1.3 cm). Pizper electrophoresis was carried out according to rgtandard procedures in Durrum cells (Spinco Division, Beckman Instruments, Inc., Palo Alto, California). Immunoelectrophoresis was performed by using the micromethod of Scheidegger (9) with minor modification. Some slides were stained with Amido Black (10) and used as negatives in a photographic enlarger to make prints. Other slides were directly photographed against a dark field. A macro-format was used for Ouchterlony immunodiffusion studies (11). Analytical ultracentrifugation was carried out in a Spinco model E ultracentrifuge (Spinco Division, Beckman Instruments, Inc., Palo Alto, California) at 59,780 rpm. Samples were thoroughly dialyzed against 0.14 M NaCl buffered with barbital to pH 8.0. Experimentally observed sedimentation coefficients were corrected to a water basis at 20°C (12). Gel filtration of bovine antisera on Sephadex G-200 was accomplished in a column (7 X 70 cm) containing approximately 150 gm (dry weight) of the cross-1ink:ed dextran according to the technique of Flodin for large columns (13). Elution was accomplished with tris buffer (0.1 M tris(hydroxymethyl)aminomethane + 1.0 M NaCI, pH 8.0). Mercaptoethanol reduction was performed by incubating protein solutions in a final concentration of 0.1 M mercaptoethanol for 20 minutes at room temperature prior to loading immuno-

electrophoretic wells, or prior to starting dialysis against 0.14 M NaCl for subsequent CF antibody titration. Papain digestion of bovine r-globulins was accomplished according to the method of Porter (14). Anion exchange chromatography of the digestion products was modified from the method of Fahey and Askonas (15) in that DEAE-Sephadex rather than DEAE-cellulose was used as the exchanger. Complement fixing (CF) antibody titration was performed according to a standardized protocol

(16). RESULTS

Immunoelectrophoretic comparison of human and bovine immunoglobulins. Bovine serum and human reference serum were examined for analogous components at one level by mixing equal volumes of serum from each species in an immunoelectrophoretic well. After electrophoresis, antibovine and antihuman sera were added to opposing trenches (Fig. 1). Controls were included to demonstrate that the antihuman sera would not develop bovine protein arcs and vice versa. Only one (Yprotein had detectable common antigenicity. The immunoelectrophoretic similarities between several human and bovine proteins (e.g., albumin) were well demonstrated. The human TM globulin arc appeared virtually identical in shape and position to its bovine counterpart. In addition, it was noted that the major rG globulin spur, undoubtedly dependent upon distinct antigenic determinants on slow and fast components of -yG globulin (17), appeared to be of identical mobility in human and bovine serum. The

: I.__). FIG. 1. Immunoelectrophoret,ic comparison of human and bovine sera. RABS, rabbit antibovine serum; HITS + BS, human serum and bovine serum mixed (1: 1) prior to application to wells; GAHUS, goat antihuman serum; HAHUS, horse antihuman serum. 1, TM; 2, major rG spur; 3, rG.

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bovine serum (Fig. 2). Immunoelectrophoretic analysis of serial chromatographic fractions revealed a spectrum of rG mobilities similar to that described in human serum (5) (Fig. 3a,b). This spectrum of mobilities was confirmed by paper electrophoresis. The breakthrough peak contained only the slowest 7.G globulins. The first part of peak II (Fig. 2, fractions 17-20) contained only yG globulins representing progressively faster bands of the electrophoretic spectrum, the slowest of which corresponded in position to the major spur on the rG arc in whole serum immuno-

slow yG globulin arc which extended cathodally beyond the spur was longer in human than in bovine serum; evidently the former contained some rG molecules that were less electronegative than any in bovine serum. Gamma A arcs were not developed by these antisera. Separation of bovine immunoglobulins by anion exchange chromatography. Sixty serum samples were submitted to anion exchange chromatography on DEAE-Sephadex columns. The elution profiles were similar to those of human serum, the one difference being the greater magnitude of peak II in

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FJG. 2. The electrophoretic spectrum of bovine YG-globulins as fractionated on DEAIZSephadex. Two ml of serum 464-A was applied to a column containing 1 gm of exchanger; the elution gradient progressed from 0.02 M Pod, pH 8.0 (110 ml) to 0.37 M PO,, pH 4.5 (55 ml) as delivered by a cylinder-cone vessel system (4). Gamma G-globulin extends cathodally beyond the major spur of the arc. The arc centered over the well is that of transferrin.

129

FIG. 3. Immunoelectrophoretic analysis of peaks I (a) and II (b) from DEAE-Sephadex chroma,tography of bovine serum. BS. bovine serum; RABS, rabbit antibovine serum. 1, slow rG; 2, fast yG; 3, transferrin.

electrophoresis. In sera taken from cattle in genie relationship between DEAE-Sephadex post-acute phases of A. marginale infection, peak I and II globulins. One-hundred and the mean mobility of the rG CF antibody fifty mg of protein obtained by pooling peak population is represented by fraction 21 of I chromatographic fractions was conFig. 2. In fractions 21-27 another protein centrated and digested with papain and was encountered in association with the 7.G cysteine for 16 hours at 37°C according to globulin. This was identified as transferrin the method of Porter (14). Similarly, the by its color (absorption maximum at 460 same amount of protein from peak II was mp) and by immunoelectrophoretic com- digested. The products of this digestion were parison with its human counterpart. Gamma then dialyzed against a starting buffer of M and rA globulins, as identified by 0.005 M phosphate (pH 8.0) and chromatoimmunoelectrophoretic analysis (Figs. 8a graphed on DEAE-Sephadex (Fig. 4). In and 9b), were localized exclusively in peak each case the resulting chromatogram was III of DEAE-Sephadex chromatograms (2, complex but similar to the elution profiles that have been obtained with human rG6, 19). Two independently prepared samples of globulins by others (20). Immunoelectrophoretically bovine globulins from peaks I and II of identified DEAE-Sephadex chromatograms were sub- Fab(S) fragments (recommended notations, mitted to analytical ultracentrifugation. Ref. 1) from both peak I and peak II The samples from peak II contained only the globulins appeared in the breakthrough peaks of their respective chromatograms. fractions from the first half of the peak. The sedimentation coefficients were S20,W= 6.6 Two smaller peaks located at 45-75 % of for the peak I protein and S20,w = 6.3 for effluent, the expected Fe(F) fragment area, were seen in each of the two chromatograms. the peak II protein. Four different DEAE-Sephadex peak I Each Fe fragment peak appeared distinct in and peak II preparations were compared elution position. Immunoelectrophoresis reby the Ouchterlony method using 4 antisera vealed that most undigested yG-globulin of rabbit origin: anti-whole bovine serum, from peak I was contained in the breakanti-rG globulin (DEAE-Sephadex peak I), through peak (Fig. 5, pool I&), but some antimacroglobulin (Sephadex G-200 peak I) also contaminated Fc fragment fractions. and anti-y globulin (Sephadex G-200 peak Undigested rG-globulin left from the peak II). In each case complete arc confluence II digestion was located in the Fc area fractions, its normal elution position. This was noted between peak I and peak II y-globulins. was anticipated since Hsaio and Putnam Papain digestion of bovine yG-globulin. (21) had shown that the conditions of digesAnalysis of papain digestion fragments was tion developed by Porter do not reduce the -yG-globulins of the bovine species to 3.5 S used in an attempt to characterize the anti-

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FIG. 4. Chromatography of papain digests of DEAE-Sephadex peak I protein (slow TG-globulin) and peak II protein (fast TG-globulin) on DEAE-Sephadex. Elution gradient progressed from 0.005 M PO?, pH 8.0 (110 ml) to 0.30 M PO.,, pH 8.0 (55 ml) as delivered by a cylinder-cone vessel system. Fractions were pooled and labeled for further study as shown under the chromatograms. Fab(S) fragments were eluted in break-through peaks and Fc (F) fragments in subsequent fractions.

(Fab , c) fragments as efficiently as they do the globulins of man and rabbit. Immunoelectrophoretic comparison of the Fab fragments from the peak I and peak II globulins revealed identical mobilities (Fig. 5, pool IS1 vs. II&). The Fc fragments were found to be immunoelectrophoretically heterogeneous (Figs. 5 and 6). The major Fc fragment of globulin from peak II was accompanied by a very fast component analogous to the Fc’(F’) fragment encountered in human r-globulin (17). In addition, a “midcomponent” arc was noted in the immunoelectrophoretic patterns of several of the bovine Fe fragment pools (Fig. 6, pool IF2). Papain digestion fragments from peak I and peak II globulins were compared with each other and with each of the undigested

TG-globulins by the technique of Ouchterlony. With the antisera used, the Fab fragments of peak I and peak II globulins were antigenically indistinguishable as were the Fc fragments (Fig. 7). In each case where a papain fragment was compared with one of the undigested 7G globulins, there was a reaction of partial intersection signifying partial nonidentity or more exactly antigenie insufficiency. The spur formation on the arc of the undigested TG-globulin indicated, as expected, that all of its antigenic determinants were not present on the fragment with which it was being compared. That most antibody was directed against determinants on Fe fragments was suggested by minimal spur formation when Fe fragments were compared with whole -yGglobulins (Fig. 7, Nos. 7, 8, 11, and 12). In

BOVINE

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contrast, the brightness of the yG-globulin arcs was not visibly diminished beyond the point of intersection with the Fab fragment arcs (Fig. 7, Nos. 5, 6, 9 and 10). These findings were consonant with those of Porter (14), wherein it was concluded that most of the antibody activity of a serum was directed against antigenic determinants on the group III fragment, the rabbit’s equivalent of Fc fragment. Attempts to demonstrate fixation of complement or i:nhibition of complement fixation by chromatographically isolated papain fragments of bovine y-globulins with antibody activity were unsuccessful. Gel Jiltration of bovine serum. Fractionation of bovine sera on Sephadex G-200 yielded 3 protein peaks comparable in size and position to those obtained with human serum as described by others (13). The cathodal side of the immunoelectrophoretic pattern produced with peak I filtrate is shown in Fig. 8c; the single cathodal arc was that of ?.Mglobulin. The CF antibodies of all antisera were found to be distributed proportionally between peaks I and II of Sephadex G-200 gel filtrations just as they were distributed between peaks III and II from DEAESephadex chromatography. Efhuent frac-

FIG. 5. Immunoelectrophoretic analysis of pooled fractions obtained from chromatography of fast and slow TG-globulin papain digests on DEAE-Sephadex as shown in Fig. 4. RABTG, rabbit antibovine r-globulin (anti-DEAE-Sephadex peak I). Fab(S), Fe(F), Fc’(F’) and undigested -rG-globulin arcs are evident.

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FIG. 6. Tracings of immunoelectrophoretic arcs of pooled fractions (Fig. 5) obtained from chromatography of fast and slow TG-globulin papain digests on DEAE-Sephadex as shown in Fig. 4. Fab(S), Fe(F), Fc’(F’) and midcomponent (M) arcs are evident as is the spectrum of F fragment heterogeneity.

tions from the two chromatographic techniques were further related by rechromatographying fractions from Sephadex G-200, peak I, on DEAE-Sephadex. The rMprotein and associated CF antibody activity were eluted terminally just as they were in whole serum chromatography. Fractions from this terminal position were concentrated by dialysis against 20 % polyvinylpyrrolidinone and submitted to analytical ultracentrifugation. The protein sedimented as a single symmetrical peak with a sedimentation coefficient of SzO,W= 18.3. Immunoelectrophoresis of the same terminal fractions demonstrated a cathodal arc analogous in shape and position to human TM-globulin (Fig. 8d). The identity of this arc with its counterpart in whole serum was shown when trenches were treated for 10 minutes with protein from peak I of a Sephadex G-200 filtration prior to loading them with antiserum. Precipitation in the walls of the trenches of antibody specific for protein contained in G-200 peak I filtrate prevented development of the yMarc in the following preparations: whole serum, DEAE-Sephadex peak III, and

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AND AALUND

FIG. 7. Ouchterlony double-diffrlsion comparison of chromatographically separated papain fragments (Fig. 4) of bovine slow and fast YG-globulins and the native proteins developed with rabbit antibovine +globulin. Antigens (upper left well vs. upper right well): 1, pool II& vs. IS,; 2, pool IF, vs. IIF2; 3, pool IF, vs. IF,; 4, pool IIF1, vs. IIF2; 5, pool I& VS. DEAE-Sephadex peak 1; 6, pool I& vs. DEAE-Sephadex peak II; 7, pool IF2 vs. DEAE-Sephadex peak I; 8, pool IF, vs. DEAE-Sephadex peak II; 9, pool II& vs. DEAESephadex peak I; 10, pool II& vs. DEAE-Sephadex peak II; 11, pool IIF? vs. DEAESephadex peak I; 12, pool IIR vs. DEAE-Sephadex peak II.

FIG. 8. Immunoelectrophoresis of: (a) fraction from DEAE-Sephadex peak same fraction after reduction with 0.1 M mercaptoethanol (+ME); (c) fraction of whole serum gel filtration on Sephadex G-200, reduced with mercaptoethanol treated; and (d) fraction eluted at 90yC effluent from macroglobulin (G-200 chromatographed on DEAE-Sephadex. BS, bovine serum references; RABS, bovine serum. 1, TM; 2, yA; and 3, -,G.

Sephadex G-200 peak I (Fig. 9). The same antiserum clearly developed the TM-arc of whole serum in unabsorbed controls and the provisionally identified yA.-arc in un-

III; (b) the from peak I and unpeak I) rerabbit anti-

absorbed or absorbed whole serum and chromatographic fractions. Mercapteethanol sensitivity of bovine serum proteins. Reduction with mercaptoethanol

BOVINE

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FIG. 9. Immunoelectrophoresis of: (a) bovine serum (BS); (b) a DEAE-Sephadex peak III fraction; and (c) a Sephadex G-200 peak I fraction. Prior to adding rabbit antibovine serum (RABS), macroglobulin (G-200 peak I fraction) was applied to trenches for 10 minutes and then removed. Gamma M arc development was prevented in all preparations, but rA (DEAI-Sephadex peak III and whole serum) and rG (whole serum) arcs developed normally. 1, TM; 2, ?A; 3, olzM; and 4, rG.

has been shown to split, human -y&1-globulin (19s) into smaller units (7-85) and to cause a loss of antibody activity (22). The same treatment of human, rabbit and rat rGantibodies does not destroy antigen binding capacity but does abolish the ability to fix complement, when reacting with antigen (23-25). Bovine globulins appeared to possess similar properties. The cathodal arc seen in immunoelectrophoresis of Sephadex G-200 peak I was abolished after preincubation -with mercaptoethanol (Fig. SC). The arc provisionally identified as that of yA by analogy to its human counterpart was eliminated, but -yG was unaffected (Fig. Sb). After mercaptoethanol reduction the ability to fix complement was abolished in both whole antisera and chromatographic fractions containing either yM- or fast rGglobulins. Separation of bovine immunoglobulins by zone electrophoresis. In a study to localize particular immunoglobulin components and their associated CF antibody activities within the electrophoretic continuum of serum proteins, four serum samples from cattle infected with A. marginale were electrophoresed in Pevikon blocks. Figure 10 is a comparison of the electrophoretic separation of a serum sample on paper with

that obtained in Pevikon. The separation of immunoglobulins in Pevikon was always incomplete since the curves for CF antibody were always simple, even in serum samples known to contain two antibody populations. The incompleteness of the separation was also apparent when serial effluent. fractions were examined by immunoelectrophoresis. Electrophoretic localization of rlL’I and yG was recognized when y or p segments from zone electrophoresis were chromatogrammed on DEAE-Sephadex (shaded area of Fig. 10). The yM-antibody activity was divided equally between the Pevikon segments denoting a distribution centered on the line used to divide the two electrophoretic areas. This electrophoretic position corresponded closely to that found for human TMglobulin (l&26). The fast rG, making up the first half of the DEAE-Sephadex peak II and its associated CF antibody, was exclusively localized within the y-electrophoretic area. Transferrin, identified by color (absorption maximum at 460 mp) and immunoelectrophoretic comparison with the human protein, made up the second half of peak II, and like the r&I, it was equally distributed in the y and fi electrophoretic areas.

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DISCUSSION

In relating the early CF antibody activity to the -yM component of bovine serum, cognizance was taken of the known physicochemical and immunochemical properties of macroglobulin antibodies of other species. The shape and position of the immunoelectrophoretic arc produced by the bovine protein was identical to that encountered with -yM-globulin of human serum (8, 22, 26). Similarity in electrophoretic mobility between human and bovine TM was encountered since zone electrophoresis of whole serum resulted in a distribution of CF antibodies with the mean located in the valley between the y and p areas (18, 26). The bovine protein was eluted in peak I when whole serum was subjected to gel filtration on Sephadex G-200. Upon chromatography on DEAE-Sephadex this eluent reproduced its distribution pattern (peak III CF activity) which in turn reproduced the characteristic arc of YM-globulin on immunoelectrophoretic examination. The bovine TM-globulin was altered to such an extent by mercaptoethanol treatment that it no longer developed an arc on immuno-

electrophoretic examination. When subjected to ultracentrifugation, the native protein sedimented as an 18 S peak, which is characteristic of isolated macroglobulin. The experiments with zone electrophoresis demonstrated that the method does not separate the heterogeneous CF antibodies of bovine serum. In this instance the fast ?Gand yM-globulins, though differing markedly in molecular size, were migrating in the same region to produce a single peak of antibody activity. In instances where yG-antibody activity is associated with electrophoretically slow y-globulin, the presence of 7-19 s antibody heterogeneity has been correlated with a bimodal antibody activity peak following zone electrophoresis of whole serum. Such heterogeneity has been reported as an antibody response pattern of rabbits to several antigens (41-43). However, the disclosure of a bimodal antibody activity curve does not identify the nature of the antibodies involved since fast and slow rG globulins may produce such a bimodal curve from some species (27, 44). Electrophoretic techniques alone are not likely to separate the antibodies of the

BOVINE

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bovine species when activity is found in the fast yG- and r&I-molecular groups. Division of bovine yG-globulins into fast and slow electrophoretic groups is in accord with studies in several species (27, 28, 33-36). Progressive elution of bovine rGglobulins from the anion exchanger, which correlated .with increasing electrophoretic migration rates, paralleled the findings by others with. human TG-globulins (4, 18). Immunoelectrophoretic recognition of slow yG in fractions from peak I and fast rG from peak II of DEAE-Sephadex chromatograms was partially based on comparisons with analogous proteins of other mammalian species. The sedimentation coefficients of the bovine slow and fast +globulins were similar to values obtained with other species (4, 27, 28). The mediation of particular antibody activities within the electrophoretic spectrum of immunoglobulins has broadened the basis for considering the independence of the component proteins. For example, in the guinea pig distinctions have been shown between slow and fast +yG-globulins which were correlaked with the nature of the antigenie stimul.us, age of the animals, and the stage in the immune response (27, 37, 38). In the current study on bovine serum the fast -@globulin was found to contain antibodies that fixed complement following reaction with A. marginale antigen, whereas that capacity was lacking in the slow -rGglobulin group. This was of special interest as an indication of specialized function and a demonstration of difference among species since the fixation of complement in guinea pig serum has been associated with the slow ,yG globulins and not with the fast 7Gglobulins (27, 39, 40). Similarity in molecular weight and a major degree of common antigenicity of the bovine fast and slow TG-globulins, and fragments from papain digestion, indicated a close relationship between these 2 rGglobulin groups. In this study distinct spur formation of fast over slow YG-globulin was seen in immunoelectrophoretic analysis with whole proteins. Antigenic differences were not demonstrated with papain fragments studied by the Ouchterlony immunodiffusion

method. Minor antigenic differences between comparable papain fragments of fast and slow TG-globulins were probably masked by the reactions of identity between major common antigens. Nussenzweig and Benacerraf (29, 30) have shown that purified guinea pig fast and slow yGglobulin antibodies of the same specificity, when digested with papain, yielded common Fab(S) fragments as well as Fe(F) fragments which differed in electrophoretic mobility and antigenicity. Analysis of papain digestion products from the bovine globulins indicated that the physical properties of the major fragments were quite similar to those of human globulins. Fe fragment heterogeneity encountered in the bovine globulins was apparently analogous to similar heterogeneities found in normal and myelomatous TG-globulins by other investigators (15, 31, 32). Evidence for distinction between bovine slow and fast TG-globulins included differences in electrophoretic migration rate, chromatographic elution position of the native proteins and their respective papain fragments, spur formation on immunoelectrophoretic analysis and distinct biological activity as indicated by the presence or absence of ability to fix complement in the presence of A. marginale antigen, REFERENCES 1. COMMITTEE ON NOMENCLATURE OF HUMAN IMMUNOGLOBULINSOFW.H.O.,BU~l.W.H.O. 30,447 (1964). 2. MURPHY, F. A., PH.D. Thesis, University of California, Davis. University Microfilms, Inc., Ann Arbor Michigan (1964). 3. MURPHY, F.A., OSEBOLD, J.W., .~NDAALUND, O., in preparation. 4. FAHEY, J. L., AND HORBETT, A. P., J. Biol. Chem. 234, 2645 (1959). 5. FAHEY, J. L., MCCOY, P. F., AND GOULIAN, M. J., J. Clin. Invest. 37, 272 (1958). 6. MURPHY, F.A., AALUND,O.,OSEBOLD, J.W., AND CARROLL, E., Arch. Biochem. Biophys. 103, 230 (1964). 7. MUELLER-EBERHARD, H. J., &and. J. Clin. Lab. Invest. 12, 33 (1960). 8. FAHEY, J. L., AND MCLSUGHLIN, C., J. Immunol. 91, 484 (1963). 9. SCHEIDEGGER, J. J., Int. Arch. Allergy ‘7, 103 (1955).

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p. 304. 10. CROWLE, A., “Immunodiffusion,” Academic Press, New York (1961). 11. OUCHTERLONY, O., “Progress in Allergy,” Vol. 6, p. 48. S. Karger,.Basel (1962). 12. SCHACHMAN, H. K., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. IV, p. 32. Academic Press, New York (1957). 13. FLODIN, P., “Dextran Gels and Their Applications in Gel Filtration.” Meijels Bokindustri, Uppsala, Sweden (1962). 14. PORTER, R. R., Biochem. J. 73, 119 (1959). 15. FAHEY, J. L., AND ASKONAS, B. A., J. Exptl. Med. 116, 623 (1962). 16. ANONYMOUS, A Manual for Conducting the Complement-fixation Test for Anaplasmosis.“’ Agricultural Research Service, U.S. Department of Agriculture (1958). G. M., HEREMANS, J. F., 17. EDELMAN, HEREMANS, M.-TH., AND KUNKEL, H. G., J. Exptl. Med. 112, 203 (1960). 18. FAHEY, J. L., “Advances in Immunology” (W. H. Taliaferro and J. H. Humphrey, eds.), Vol. 2, p. 41. Academic Press, New York (1962). 19. MURPHY, F. A., AALUND, O., AND OSEBOLD, J. W., Proc. Sot. Exptl. Biol. Med. 117, 513 (1964). 20. OLINS, D. E., AND EDELMAN, G. M., J. Exptl. Med. 116, 635 (1962). 21. HSAIO, S., .~ND PUTNAM, F. W., J. Biol. Chem. a36, 122 (1964). 22. KUNKEL, H. G., in “The Plasma Proteins” (F. W. Putnam, ed.), Vol. 1. p. 279. Academic Press, New York (1960). 23 WIEDERMANN, G., MIESCHER, P. A., AND FRANKLIN, E. C., Proc. Sot. Exptl. Biol. Med. 113, 609 (1963). 24. WIEDERMANN, G., NAGASHIMA, H., OVARY, Z., AND MIESCHER, P., Federation Proc. 23,

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