Molecular weight of a purified chicken antibody

Immunochemistry. Pergamon Press 1969. Vol.6, pp. 199-206. Printedin Great Britain

M O L E C U L A R W E I G H T OF A P U R I F I E D C H I C K E N ANTIBODY* JOAN S. GALLAGHER and EDWARD W. VOSS, Jr. Department of Microbiology, University of Illinois, Urbana, Ill. 61803, U.S.A.

(First received5July 1968; in revisedform 23 September 1968) A b s t r a c t - Chicken antibody to the 2,4-DNP group was purified by an immuneadsorbent.

Chemical, physical and immunological studies indicated that this antibody was a single pure component and essentially 100 per cent active. Molecular weight of the antibody calculated from the equation of Svedberg and Pedersen and determined by the high speed equilibrium method of Yphantis was 182,750 and 174,916 respectively. Physical parameters of S20.w,D20.wand partial specific volume were experimentally determined. INTRODUCTION Molecular weights of chicken IgG antibodies prepared by isolation of the gamma globulin fraction or purification of antibody by solubilization of immune precipitates have been reported from 150,000 [1] to 206,000 [2]. In contrast, molecular weight determinations on IgG antibody from a wide range of mammalian species yield a narrow range of 150,000-160,000 [3]. Discrepancies in the wide range of values reported for chicken IgG can be attributed to various factors. Two types of IgG frequently exist and isolation of the respective components is difficult. Failure to separate these components results in a heterogeneous population which influences critical molecular weight determinations. Investigators have assumed values for pertinent physical parameters potentializing erroneous molecular weight calculations. In this study chicken antibody to the 2,4-dinitrophenyl group was specifically purified by an immuneadsorbent. Purification studies revealed this antibody to be a single component and suitable for physicalchemical studies. Molecular weight was determined by sedimentation velocity studies according to the equation of' Svedberg and Pedersen[4] and the high speed equilibrium method of Yphantis [5]. Physical parameters such as $20.,,,, D20.,; and partial specific volume were measured. MATERIALS AND METHODS

Preparation of antigens Dinitrophenylated bovine gamma globulin (DNP-BGG) was prepared and characterized as described by Eisen [6]. The reaction mixture was passed through a column of Dowex I-X8, 200-400 mesh, CI- form (J. T. Baker Chemical Co.) equilibrated in water. Low molecular weight impurities (2,4-DNB-sulfonate and 2,4-dinitrophenolate) are bound to the anion exchange resin, but the substituted protein is excluded. Determination of the number of DNP groups per molecule of protein was based on dry weight analysis and absorbance at 360 m/x. *This investigation was supported by grant AI-08288-01 from the National Institutes of Health, Public Health Service. 199

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Immunization Fifty chickens (White Rock) were immunized intramuscularly with 5.0 mg of DNP51BGG in complete Freunds adjuvant. Prior to injection an emulsion test was made by placing a drop of the antigen solution in contact with water. If antigen and adjuvant are well dispersed in the emulsified droplet rapid phase separation with subsequent dissolution of the droplet does not occur. Sample bleedings were obtained periodically after the primary injection to determine the extent of the primary antibody response (I°). Chickens were boosted 33 days after the primary injection with 2"0mg of DNP51BGG in complete Freunds adjuvant. Eight days after the booster injection (II °) the chickens were bled by cardiac puncture. Hyperimmune sera from all chickens were pooled and served as a source of anti-DNP antibodies for all studies reported below.

Preparation of immuneadsorbent Bromoacetyl cellulose was prepared by the method of Robbins et al.[7]. To 10.0 g of derivatized cellulose was added 3.0 g of DNP2a human serum albumin (DNP23HSA) in 0"5M acetate buffer pH 5.5. Essentially 100 per cent of the DNP23HSA was physically adsorbed at pH 5.5. The cellulose-protein conjugate was centrifuged and 0-10 M bicarbonate (pH 9.0-9.5) was added to the insoluble pellet allowing formation of covalent bonds. Extensive washing in bicarbonate, 8.0 M urea and 0.05 M PO4, pH 8"0 preceeded use of the adsorbent. An average of 250-300 mg of DNP23HSA were covalently linked to 1.0 g of cellulose by this method. Preparations of adsorbent varied, but an immuneadsorbent with the degree of substitution stated above specifically adsorbed antibody on a 0.5-1.0:1 weight ratio.

Purification of antibody Antibodies were purified as described by Voss and Eisen [8]. To 50.0 ml of hyperimmune sera was added 1.0 g of immuneadsorbent. EDTA was added to a final concentration of 0.001 M to inhibit the complexing of complement components. Reaction mixtures were incubated 2 hr at room temperature under mild stirring conditions. The immuneadsorbent was washed 4-5 times with 0.05 M PO4 buffer pH 8.0 followed by 1-2 incubations at 37°C for 1 hr in 0.05 M PO4 pH 8.0, and 1-2 incubations 37°C for 1 hr, with 0.10M N-CBZ-glycine (carbobenzoxy-glycine, Sigma Chemical Co.) in 0.05M PO4 pH 8.0. These incubations were Mock Elutions or controls for temperature, buffer, time and ionic conditions. All supernates were monitored at 278 m/z and 360 m/z. AntiDNP antibody was eluted with 0.10M DNP-glycine in 0.05 M PO4 pH 8.0 at 37°C for 1 hr.

Purification of lgG antibodies DNP-glycine was removed from the eluted antibody solution by passage over a Dowex l-X8 column equilibrated in 0-05 M PO4 pH 8.0. Antibodies were further purified into IgG and IgM fractions by chromatography on DEAE cellulose equilibrated in 0.05 M PO4 pH 8.0. Pure IgG was eluted in the first fraction and IgM was eluted at 0.3 M PO4 pH 8.0[8].

Molecular Weight of Chicken Antibody

201

Preparation of chicken normal IgG Chicken plasma (Pel Freeze Biologicals, Inc.) was treated with three successive sodium sulfate precipitations of 18%, 14% and 12-5% at room temperature. Precipitated protein from the last salt fractionation was chromatographed on DEAE-cellulose equilibrated in 0.05 M PO4 pH 8.0. Purity of the resulting IgG fraction was determined by radioimmunoelectrophoresis and acrylamide gel disc electrophoresis.

Extinction coefftcient Micro-Kjeldahl analyses at pH 8.0 resulted in an extinction coefficient

(~wl~ r~,em,~78m~/ of 15"8, assuming a N content of 16 per cent[9]. A value of 14.4 was

obtained by dry weight analyses. Controls for salt and water content were included in all determinations. The average value of 15.1 was used in all experiments.

Radioimmunoelectrophoresis Microscope glass slides (Lapine Scientific Co.) were coated with 1% Ionagar No. 2 (Colab Laboratories, Inc.) dissolved in barbital buffer pH 8.6, u = 0.05. Proteins were electrophoresed in a field strength of 10 V/cm for 60 min. Rabbit anti-chicken plasma sera was placed in a central trough to develop the precipitin bands. ~25I-DNP2-RNAase was added to the trough after diffusion of the antiserum to demonstrate specific antigen binding. After 24hr development of precipitin bands the slides were washed in 0.05 M PO4 pH 8-0 for 24-48 hr, then in water to remove salts. Slides were dried and placed in contact with Kodak X-ray film (RB-54). X-rays were developed 48-72 hr later.

Preparation of'25I-DNP-RNA ase DNP2 RNAase was labeled with 125I by the IC1 method[10] designed for 10 I groups to be substituted onto the protein. Unreacted excess '25I was removed by Sephadex G-10 gel filtration equilibrated in 0.05 M PO4 pH 8.0. The labeled product was 93 per cent precipitable by 5% trichloroacetic acid with a specific activity of 15.2 × 106 (counts/min)/mg.

Disc gel electrophoresis Disc gel electrophoresis according to the method of Ornstein [11] and Davis [12] was performed at pH 9.5. Gels were electrophoresed at 4 ma/tube for 1.5 hr at 4°C. A Densicord Recording Electrophoresis Densitometer was used to graph the location and intensity of bands.

Ultracentrifugation Analytical ultracentrifugation was performed with a Spinco Model E ultracentrifuge equipped with automatic temperature control. Both Schlieren and interference optics were utilized. All proteins analyzed were dissolved in 0.05 M PO4, pH 8.0 at 20°C. Analysis of photographic plates was done with a Gaertner Microcomparator. Simultaneous determinations of partial specific volume and molecular weight

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were done by the Yphantis sedimentation equilibrium method in H20 and 50% D20 according to Edelstein and Schachman [13]. A double sector cell was used at speeds of 17,980 rev/min using 3 mm column heights, sapphire windows and protein concentration of 0.5 mg/ml. Calculations were made with the aid of an IBM 7094 computer. Sedimentation coefficients were determined from sedimentation velocity experiments at 59,780 rev/min using a double sector standard and wedge cell. Corrections were made to convert S observed to S20.w.

Determination of diffusion coeffcients Diffusion coefficients were determined according to the method of Lundgren and Ward[14] in a synthetic boundary cell at 9,341 rev/min. Corrections were made to convert D observed to D20.w.

Determination of density of solvents Density of solvents were determined at 20°C with a 10-0 ml thin neck pycnometer employing a Cannon constant temperature bath. The pycnometers were calibrated with water in order to relate volume of solvent to weight of solvent.

Amino acid analysis Hydrolysis of chicken anti-DNP antibody was performed in constant boiling HC1 in sealed evacuated tubes at ll0°C for 22 hr. A Beckman Model 120 Amino Acid Analyzer was used and 200/zg of protein was placed on both the long and short columns. A Beckman standard mixture of amino acids was analyzed for calibration. RESULTS

Purification of lgG antibody with immuneadsorbent Pooled hyperimmune sera of 50 chickens (8 Day II °) when adsorbed with the DNP-immuneadsorbent yielded a total of 435 tLg/ml of specifically purifiable antibody. One adsorption (50-75 ml aliquot) yielded 324/zg/ml or 74.5 per cent of the total. Readsorption of the serum supernate yielded 111/zg/ml or 25.5 per cent. A third adsorption gave less than 1 per cent of the total. Purified antibody from the first and second adsorptions were pooled and the IgG fraction obtained by successive passages over Dowex l-X8 and DEAE-cellulose as described in Materials and Methods. Parallel control adsorptions (50-75 ml) of normal chicken plasma resulted in about 11 tcg/ml of adsorbable protein. If this eluted material is non-specific and represents the average amount obtained then the purified antibodies are only 2.3 per cent contaminated. Radioimmunoelectrophoresis of purified chicken antibody eluted in the 0-05 M fraction from DEAE-cellulose gave one distinct line with a mobility characteristic of fast gamma globulin. This higher electrophoretic mobility of chicken IgG in pH 8.6 buffer relative to most mammals confirms a previous report[2]. Figure 1 shows this band reacted specifically with the 125I-DNP2 RNAase antigen. Controls with normal serum or purified normal IgG developed distinct precipitin lines but did not bind 125I-DNP2 RNAase. It should be emphas-

Fig. 1. Radioimmunoelectrophoresis of 0.05 M DEAE fraction of purified chicken anti-DNP antibody (upper well). Lower well contains normal chicken serum. Rabbit anti-chicken plasma and ’ *51-DNPZ RNAase were used as diffusing reagents from the trough. Purified antibody concentration was 7.95 mg/ml.

Fig. 2. (a) Disc gel electrophoresis of normal chicken serum, purified chicken anti-DNP antibody and purified rabbit anti-DNP antibody (left to right). (b) Densitometer tracing of the purified chicken anti-DNP antibody ( -) and purified rabbit anti-DNP antibody (. . . . . .).

(Facing page 202)

Fig. 4. Ultracentrifugation patterns of purified chicken anti-DNP antibody 40 min after reaching speed @59,780 rev/min. Bar angle was 60”. Upper pattern (wedge window) ; protein concentration was 4.05 mg/ml. Lower pattern; protein concentration was 7 *95 mg/ml.

Molecular Weight of Chicken Antibody

203

ized that only one gamma globulin species was evident with the hyperimmune antibody. Purified antibody from bleedings obtained after the primary immunization indicated two distinct components. Disc gel electrophoresis of both purified chicken antibody and purified normal gamma globulin gave single bands as shown in Fig. 2(a). Direct observation indicated that the purified antibody band was less diffuse than the normal gamma globulin band. Figure 2(b) shows a densitometer tracing depicting a relatively symmetrical peak obtained with the purified chicken antibody superimposed over a tracing from purified rabbit anti-DNP antibody. This comparison shows the relative homogeneity of the chicken antibody. Percent antibody activity Determination of percent activity was tested by readsorbability of the purified antibody to the immuneadsorbent. Normal chicken IgG and buffer were used as controls. To 1.0 ml of immuneadsorbent was added 1.5 ml of antibody or a total of 2.337 O.D. units (278 mp). To separate tubes equal volumes of buffer and normal IgG (3.750 O.D. units) were added. Tubes were incubated for 1 hr at room temperature and centrifuged to collect the supernates. Supernates were clarified and read at 278 mp. After proper correction for controls the antibody supernate contained O-034 O.D. units, indicating the purified antibody to be 98.5 per cent active. Sedimentation coe&ient Concentration dependency of the sedimentation rate of the purified antibody in 0.05 M PO, pH 8-O was determined at four different concentrations. Figure 3 shows that extrapolation to zero protein concentration gives an S,,., value of 7.4. Figure 4 shows a sedimentation pattern of the purified antibody from a sedimentation velocity experiment (upper wedge cell = 4.05 mg/ml and lower standard cell = 7.95 mg/ml). The leading shoulder indicates IgG aggregates since IgM antibody has been removed by DEAE chromatography. Molecular weight determined from the Svedberg and Pedersen equation using this experimentally determined S,,., and the D,,., and v reported below was 182,750.

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Fig. 3. The concentration dependence of sedimentation of purified chicken anti-DNP in 0.05 M PO,, pH 8.0.

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J. S. GALLAGHER

Density of solvent

The density of 50% deuterated buffer was derived from the experimentally measured densities of the 0.05 M potassium phosphate buffer (pH 8.0) at 20°C (l-00562 g cmA3) and 90% deuterated buffer (1*10500 g cme3). The density of the 90% deuterated buffer is in good agreement with Edelstein and Schachman [ 131 who reported a 10 per cent increase. Partial specijk volume (5)

Partial specific volume determined by the Edelstein and Schachman method [13] in 50% DzO gave a value of 0.728 cm3 g-l. This value agreed closely with O-725 cm3 g-l obtained by amino acid analysis uncorrected for carbohydrate content. The close correlation of these two values is surprising since at least a 2 per cent deviation of v would be expected in 50% D,O. The value of O-728 cm3 g-l was considered most accurate and was used throughout these studies. [email protected] coefjkient

Concentration dependency of the diffusion rate of the purified antibody in 0.05 M PO, pH 8-O at three different concentrations is shown in Fig. 5. The extrapolated value of D20.wat zero protein concentration is 3.68 X lo-’ cm2sec-1. Concentration dependency of the diffusion coefficient suggests that this is a -critical parameter for molecular weight determination.

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.4

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Fig. 5. The concentration dependence of diffusion of purified chicken antiDNP antibody in 0.05 A4 PO,, pH 8.0. DZO.m X lo7 in cm*sec?. Sedimentation equilibrium

Figure 6 shows a plot of the logarithm of the concentration vs. the radial distance squared from the center of rotation according to the Yphantis sedimentation equilibrium method. A straight line is apparent from this plot (slope = 0*339030) and indicates a homogeneous protein in relation to molecular size. A molecular weight of 174,900 was calculated by the equation as stated by Yphantis [5]. Purified rabbit anti-DNP (IgG) studied under identical conditions gave a molecular weight of 154,300.

Molecular Weight of Chicken Antibody

205

Fig. 6. Yphantis sedimentation equilibrium determination. Representative plot of the logarithm of the concentration vs. the radial distance squared. The line is a least-squares fit of the data. DISCUSSION

to the 2,4_dinitrophenyl group by use of a high capacity immuneadsorbent. The antibody studied was pure within the limits of sensitivity of the assays used. Radioimmunoelectrophoresis and sedimentation velocity patterns at high concentration revealed a single component. Results of sedimentation equilibrium studies at low concentrations confirmed a pure protein. The stained slide in Fig. 1 indicated splitting of the precipitin line. However, repeated experiments revealed a single component and a uniform radioactive arc was always observed. Disc gel electrophoresis showed one component. The purified antibody requires high salt concentrations (1.5-2.0 M NaCl) to precipitate with antigen and gives a negative passive cutaneous anaphylaxis (PCA) reaction in guinea pigs[l6]. Estimations of the molecular weight are dependent on the accuracy of values used for critical parameters such as the extinction coefficient, partial specific volume and diffusion coefficient. Large deviations in the extinction coefficient values were obtained by microKjeldahl (15.8) and dry weight analyses (14.4). The former value was not corrected for percent carbohydrate since an accurate percentage had not been measured for this antibody. The extinction coefficient from the dry weight analysis would not reflect carbohydrate content. However, using the average value of 15.1 in the computation of S20.W, a molecular weight of 182,750 was obtained. This compared closely with the value of 174,900 measured in sedimentation equilibrium studies. Preliminary evidence suggests that the average Chicken

antibody

was purified

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J. S. GALLAGHER

and E. W. VOSS, Jr.

value of 15.1 gives appropriate protein concentrations in ligand binding studies using the equilibrium dialysis method. Tenenhouse and Deutsch[2] reported a value of 13.5 for a purified chicken gamma globulin fraction. The difference in these values may be explained by the tryptophan content of purified anti-DNP antibody. McQuigan and Eisen [ 17, 181 reported a relationship of the extinction coefficient with the tryptophan content and the average intrinsic association constant (K,) of purified rabbit anti-DNP. The average value of 15.1 falls within their range of 14 to 16.5. Using the average extinction coefficient means that variations in S would result in k4.6 per cent variations in the molecular weight of 182,750 as computed by the Svedberg and Pedersen equation. Deviations in V measurements would influence both methods of determining molecular weight. If one assumes at least a 2 per cent deviation in 8[13] a range of 0.714-0.742 would be expected. This would influence the two calculations of molecular weight -+5 per cent. Considering these variations and computing the average molecular weight from the two values measured the size of this purified antibody would be 178,000-179,000. Both methods result in a weight-average molecular weight. Relative to the molecular weight determined for mammalian gamma globulin, chicken antibody is significantly larger. The molecular weight of 178,000-179,000 agrees with the value of 180,000 reported by Orlans et aZ.[19] using purified chicken antibodies. However, this value is significantly lower than the value of 206,000 reported by Tenenhouse and Deutsch [2] working with normal gamma globulin. Acknowledgements- We are grateful to Dr. Richard Prairie, University of Cincinnati

College of Medicine for providing the computer programs for analysis of sedimentation equilibrium data. We also thank Dr. Gary S. David for expert advice and helpful discussions throughout these studies. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

11. 12. 13. 14. 15. 16. 17. 18. 19.

Van Orden D. E. and Teffers H. P.,J. Zmmun. 100,659 (1965). Tenenhouse H. S. and Deutsch H. F., Zmmunochemistry3,11(1966). Putnam F. W., The Plasma Proteins, Vol. 1. Academic Press, New York (1960). Svedberg T. and Pedersen K., The Ultracentriige, University Press, Oxford (1940). Yphantis D. S., Biochemistry $3 (1964). Eisen H. N., Methods in MedicalResearch, Vol. 10, p. 94. Year Book Medical Publishers, Chicago (1964). Robbins J. B., Haemovich J. and Sela M., Immunochemistry 4,11 (1967). Voss E. W. and Eisen H. N., In preparation. Brand E., Ann. N.Y. Acod. Sci. 47,187 (1946). McFarlane S. S., Nature, Lond. 18253 (1958). Ornstein L., Ann. N.Y. Acud. Sci. 121,321 (1964). Davis B. J., Ann. N.Y. Acod. Sci. 121,404 (1964). Edelstein S. J. and Schachman H. K.,J. biol. Chem. 306,2 (1966). Lundgren H. P. and Ward W. H., Amino Acids and Proteins (Edited by Greenberg D. M.), Thomas, Springfield, Ill. (1951). Schachman H. K., Methods in Enzymology, Vol. 4, p. 32. Academic Press, New York (1957). Gallagher J. S. and Voss E. W. Unpublished results. McGuigan J. E. and Eisen H. N., Biochemistry 7, 1919 (1968). McGuigan J. E..and Eisen H. N., Biochemistry 7, 1929 (1968). Orlans E., Rose M. E. and Marrack J. R., Immunology 4,262 (1961).