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Isopycnic characterization of lipopolysaccharides from Pasteurella multocida
ANALYTICAL
lsopycnic
BIOCHEMISTRY
85,
265-270 (1978)
Characterization Pasteurella
of Lipopolysaccharides multocida
MARSHALLPHILLIPSANDPAUL
from
l A. REBERS
National Animal Disease Center, North Central Region, Agricultural P. 0. Box 70, Ames, Iowa 50010
Research Service,
Received July 5, 1977; accepted October 3, 1977 In a novel application of an established procedure, isopycnic density gradient centrifugation procedures were used to analyze material obtained from the Westphal phenol extraction procedure of Pasteurella multocida cells. The initial phenol phase contained most of the lipopolysaccharides (LPS) and the major component had a buoyant density of 1.38 g/ml in CsCl density gradients. Repartitioning the phenol phase with an equal volume of water produced a second aqueous phase which contained most of the LPS. This LPS appeared as a single symmetrical band with a buoyant density of 1.40 g/ml. Buoyant density patterns obtained with schlieren optics in CsCl density gradients were useful in characterizing LPSs from P. multocida.
In order to establish the chemical basis of immunological specificity of the lipopolysaccharides (LPS) from Pasteurella multocida, it was necessary to establish the purity of the preparations. The most widely accepted procedure for the isolation of LPSs from gram negative bacteria is the phenol-water extraction originated by Westphal et al. (1). Most LPSs obtained by the Westphal procedure are heterogeneous (2). Although applications of analytical ultracentrifugation for the identification of protein-polysaccharides in CsCl isopycnic gradients have been described (3, 4) there is a paucity of published reports describing schlieren optical patterns of LPSs in isopycnic density gradients. Sucrose density gradients have been used to characterize LPSs from Thiobacillusferrooxiduns (5), and CsCl density gradients have been used to characterize LPSs from Escherichiu coli (6). Our investigations on the use of CsCl density gradients for characterizing LPS are presented. With the buoyant density patterns as a criterion, a slight modification of the usual Westphal procedure was found to improve the purity of the LPSs obtained from P. multocidu. I Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the U. S. Department of Agriculture and does not imply its approval to the exclusion of other products that may be suitable. 265
0003-2697/78/0851-0265$02.00/O Copyright 0 1978 by Academic Rcss, Inc. All rights of reproduction in any form reserved.
266
PHILLIPS
MATERIALS
AND REBERS
AND METHODS
Cells. P. multocida cells strain X-73F (ATCC 11039) were grown as described by Rebers et al. (7). Phenol extraction. P. multocida formaldehyde-treated cells (40 g wet weight of packed cells) were washed two times with 200 ml of 150 mM NaCl, 20 mM sodium phosphate at pH 7.2 (PBS), and treated with 200 ml of Hz0 and 200 ml of glass-distilled phenol2 at 65-70°C for 10 min according to the method of Westphal and Jann (8). After centrifugation at 4°C the interface and phenol layer were extracted a second time with 200 ml of H,O at 6%70°C for 10 min. Zsopycnic ultracentrifugation. The procedures as described by Ifft (9) were used for the buoyant density determinations. Optical grade CsCl (Calbiochem Corp., Los Angeles, California) was added to solutions of LPS in 10 mM sodium phosphate at pH 6.8. Equilibrium was obtained after 20 hr at 36,000 r-pm in a Model E ultracentrifuge (Beckman Instruments, Fullerton, California). The refractive index of the CsCl-LPS solution was determined before and after the run. The density of CsCl as determined by the refractive index was assumed to be the isoconcentration point, re, for the density calculations. The distributive factors, p, were obtained from Vinograd and Hearst (10). All runs were made at 25°C and the plates were read on a Gaertner comparator (Gaertner Corp., Chicago, Illinois). Serology. Each fraction was analyzed by gel diffusion in agar containing 1.5 M NaCl with antisera from turkeys which had been given multiple iv inoculations of formaldehyde-treated P. multocida cells. The relative concentration of the antigen in the various fractions was estimated by determination of the highest dilution which was capable of giving a visible precipitin line in gel diffusion, and also by the radial immunodiffusion procedure for quantitation of antigens of Mancini et al. (11). Chemical analyses. Analyses for fatty acids, sugars, and amino acids were made by gas-liquid chromatography at the Analytical Biochemistry Laboratories, Inc., Columbia, Missouri. Fatty acids were estimated as their methyl esters and sugars as the trimethylsilyl derivatives according to the procedure of Wang et al. (12). RESULTS AND DISCUSSION
A flow diagram describing the fractions obtained by the Westphal extraction is shown in Fig. 1. Analyses of the fractions A-l, A-2, P-l, and P-2 by radial immunodiffusion indicated that the ratios of antigen, presumably LPS, in each were as follows: A-2/A- 1, 1.4; A-2/P- 1, 1 .O; and * Since even glass-distilled phenol gave an acid reaction when mixed with water and brom cresol green (yellow color), a small amount of sodium bicarbonate was added to reduce carry-over of volatile acids. This distillate gave a green color with the indicator.
ISOPYCNIC
CHARACTERIZATION
OF LPS
267
FIG. 1. Flow diagram of phenol-water extraction ofPasteurella multocida cells. Fractions are designated P-l, P-2, A- 1, and A-2. For details of extraction see Materials and Methods.
A-2/P-2,9.0. Hence most of the antigen was in A-2 or P-l. As expected in a Westphal extraction, most of the nucleic acids were recovered in A-l. Since this strain of P. multocida is a highly virulent organism with a large capsule, it was not expected that most of the LPS would be partitioned in the first phenol layer. However, after a second extraction of the phenol layer with water, most of the LPS was now in the aqueous layer. Because A-2 not only had more antigen but also had a higher activity in terms of gel diffision, the procedure was modified by keeping fractions A-l and A-2 separate. Fraction P-l was examined in a CsCl density gradient (Fig. 2a). A major band was observed at a density of 1.378 g/ml, and lesser amounts of smaller components were banded at higher densities. Buoyant density determinations were made on a number of P-l fractions, and the major density band was determined to be 1.381 + 0.005 g/ml. The second aqueous fraction A-2 was examined in a CsCl density gradient. A single symmetrical peak was observed with a density of 1.406 g/ml indicating homogeneity by this criterion (Fig. 2~). Buoyant density profiles were obtained from a series of four dilutions of the A-2 fraction in CsCl density gradients. The pattern in Fig. 2c was representative of the fraction at all concentrations, and the density of the LPS in A-2 was determined to be 1.403 & 0.005 g/ml. Fractions A-l were examined in similar CsCl gradients, and the nucleic acids from the extraction were banded at the bottom of the cell. The LPSs in this fraction were entrained with the nucleic acids and no bands were observed in the 1.38-1.40 g/ml density region. In order to verify that the LPSs in the fractions A-2 and P-l did have different buoyant densities, a mixture in CsCl was prepared consisting of
268
PHILLIPS
AND REBERS
FIG. 2. Equilibrium schlieren photographs of 0.2% LPS in CsCI, 10 mM sodium phosphate buffer, pH 6.8, at 36,000 rpm and 25°C: (a) fraction P-l, pe = 1.3% g/ml; (b) mixture of fraction A-2 and P-l, pe = 1.400 g/ml; and (c) fraction A-2, pe = 1.415 g/ml. Single-sector, 2”, Kel-F centerpiece.
equal amounts of both. Two major bands were observed buoying at 1.404 and 1.387 g/ml corresponding to the individual bands from the respective A-2 and P-l fractions (Fig. 2b). The smaller bands from the P-l fraction were also observed. The following data suggest that we are dealing with a typical LPS. The chemical composition of some of the components of fraction A-2 as indicated by gas-liquid chromatographic analyses is given in Table 1. The lauric and myristic acid contents and the glucose, galactose, and D-glycero-L-mannoheptose are as expected for LPSs. A previously described standard Westphal preparation from the X-73F strain of P. multocida in which A-l and A-2 were combined, treated with RNase and DNase, and then purified by sedimentation at 165,000 g contained 2.8% total phosphorus, 2.9% total nitrogen, 7.7% hexose, and 13.6% heptose (13). This preparation killed mice and produced a local Shwartzman reaction in rabbits. Since the complete chemical analysis of the constituents of LPS in A-2 was not available, it was not possible to calculate the predicted buoyant density. If the ratios of the amounts of lipid, carbohydrate, and protein as expressed in Table 1 are assumed to be representative of the LPS and contribute’to the density accordingly, then an observed density of 1.40 g/ml would be expected. Buoyant densities of 1.40- 1.44 g/ml in CsCl have been reported for LPS from E. coli (6). The mixing experiments indicate that the LPSs in the P-l and the A-2 buoy at different respective densities and are not readily interconverted in CsCl solutions. Since the monodisperse LPS in A-2 is recovered from the P-l fraction, explanations for the change in buoyant density were
ISOPYCNIC
CHARACTERIZATION TABLE
CHEMICAL
ANALYSIS
269
OF LPS
1
OF LIPOPOLYSACCHARIDE
IN FRACTION
A-2 Percentage w/w
I. Fatty acid analysis Lauric-C,z Myristic-C,, Palmitic-C,, Palmitoleic-C,,l Stearic-C,s Oleic-C,J
2.24 9.66 1.21 0.56 0.04 0.09 Total
II. Sugar analysis Galactose Glucose D-Glycero-L-mannoheptose
14.97 9.08 22.98 Total
III. Amino acid analysis 17 Amino acids
13.80%
Total (protein)
47.03% 4.61%
considered. The carbohydrate content would not be expected to change. There is the possibility that the amount of protein or lipid has been reduced and results in an increase of density from 1.38 to 1.40 g/ml. Although acid-free phenol was utilized for the extractions to reduce hydrolysis, labile linkages may still be hydrolyzed (14). The amount of hydrolysis was estimated by dividing one of the P-l fractions into two equal parts for the second extraction. The first part was extracted with water at 22°C and the other part with water at 65°C. The difference in the buoyant densities between the A-2 LPSs was only 0.003 g/ml. Since the buoyant densities of the two LPSs were nearly identical even though the temperatures were 65 and 22°C we concluded that relatively little degradation occurred. Although the different densities can be accounted for by differences in chemical composition, other changes such as hydration, net charge, ionic constituents, and hydrophobic bonding must be considered (15). Although the distribution of molecules in the A-2 LPS has yet to be determined by other procedures, the symmetrically shaped schlieren buoyant density patterns indicate a limited molecular weight distribution. The technique of equilibrium buoyant density determinations in CsCl may be used advantageously to characterize LPS fractions and these applications of the analytical ultracentrifuge are a useful adjunct to the Westphal extraction procedures.
270
PHILLIPS
AND
REBERS
ACKNOWLEDGMENTS The authors wish to acknowledge the technical assistance of Robert E. Patterson, Kim Brogden, and Bill Brown.
REFERENCES 1. Westphal, O., Luderitz, O., and Bister, F. (1952) Z. Nutueorsch 7B, 148-151. 2. Shands, J. W. (1971)in Microbial Toxins (Weinbaum, G., Kadis, S., and Ajl, S. S., eds.), Vol. IV, pp. 127-144, Academic Press, New York. 3. Dunstone, J. R. (1969) Separation Sci. 4, 267-285. 4. Creeth, J. M., and Denborough, M. A. (1970) Biochem. J. 117, 879-891. 5. Hirt, W. E., and Vestal, J. R. (1975) J. Bacterial. 123, 642-650. 6. Morrison, D. C., and Leive, L. (1975) J. Biol. Chem. 250, 2911-2919. 7. Rebers, Pi, Heddleston, K., and Rhoades, K. (1967) J. Bacterial. 93, 7-14. 8. Westphal, O., and Jann, K. (1965) in Methods in Carbohydrate Chemistry (Whistler, R. L., ed.), Vol. V, pp. 83-91, Academic Press, New York. 9. Ifft, J. B. (1969) in A Laboratory Manual of Analytical Methods of Protein Chemistry (Alexander, P., and Lundgren, H. P., eds.), Vol. 5, pp. 151-223, Pergamon Press, New York. 10. Vinograd, J., and Hearst, J. E. (1962), Fortschr. Chem. Org. Naturst. 20, 372-422. 11. Mancini, G., Carbonara, A. O., and Heremans, J. F. (1965) Zmtnunochemistty 2, 235-254. 12. Wang, W. S., Korczynski, M. S., and Lundgren, D. G. (1970)5. Bacterial. 104,556-565. 13. Rebers, P., and Heddleston, K. (1974) Amer. J. Vet. Res. 35, 555-560. 14. Tsang, J. C., Wang, C. S., and Alaupovic, P. (1974) J. Bncteriol. 117, 786-795. 15. Ifft, J. B. (1976) in Methods of Protein Separation (Catsimpoolas, N., ed.), Vol. I, pp. 193-237, Plenum, New York.
lsopycnic
BIOCHEMISTRY
85,
265-270 (1978)
Characterization Pasteurella
of Lipopolysaccharides multocida
MARSHALLPHILLIPSANDPAUL
from
l A. REBERS
National Animal Disease Center, North Central Region, Agricultural P. 0. Box 70, Ames, Iowa 50010
Research Service,
Received July 5, 1977; accepted October 3, 1977 In a novel application of an established procedure, isopycnic density gradient centrifugation procedures were used to analyze material obtained from the Westphal phenol extraction procedure of Pasteurella multocida cells. The initial phenol phase contained most of the lipopolysaccharides (LPS) and the major component had a buoyant density of 1.38 g/ml in CsCl density gradients. Repartitioning the phenol phase with an equal volume of water produced a second aqueous phase which contained most of the LPS. This LPS appeared as a single symmetrical band with a buoyant density of 1.40 g/ml. Buoyant density patterns obtained with schlieren optics in CsCl density gradients were useful in characterizing LPSs from P. multocida.
In order to establish the chemical basis of immunological specificity of the lipopolysaccharides (LPS) from Pasteurella multocida, it was necessary to establish the purity of the preparations. The most widely accepted procedure for the isolation of LPSs from gram negative bacteria is the phenol-water extraction originated by Westphal et al. (1). Most LPSs obtained by the Westphal procedure are heterogeneous (2). Although applications of analytical ultracentrifugation for the identification of protein-polysaccharides in CsCl isopycnic gradients have been described (3, 4) there is a paucity of published reports describing schlieren optical patterns of LPSs in isopycnic density gradients. Sucrose density gradients have been used to characterize LPSs from Thiobacillusferrooxiduns (5), and CsCl density gradients have been used to characterize LPSs from Escherichiu coli (6). Our investigations on the use of CsCl density gradients for characterizing LPS are presented. With the buoyant density patterns as a criterion, a slight modification of the usual Westphal procedure was found to improve the purity of the LPSs obtained from P. multocidu. I Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the U. S. Department of Agriculture and does not imply its approval to the exclusion of other products that may be suitable. 265
0003-2697/78/0851-0265$02.00/O Copyright 0 1978 by Academic Rcss, Inc. All rights of reproduction in any form reserved.
266
PHILLIPS
MATERIALS
AND REBERS
AND METHODS
Cells. P. multocida cells strain X-73F (ATCC 11039) were grown as described by Rebers et al. (7). Phenol extraction. P. multocida formaldehyde-treated cells (40 g wet weight of packed cells) were washed two times with 200 ml of 150 mM NaCl, 20 mM sodium phosphate at pH 7.2 (PBS), and treated with 200 ml of Hz0 and 200 ml of glass-distilled phenol2 at 65-70°C for 10 min according to the method of Westphal and Jann (8). After centrifugation at 4°C the interface and phenol layer were extracted a second time with 200 ml of H,O at 6%70°C for 10 min. Zsopycnic ultracentrifugation. The procedures as described by Ifft (9) were used for the buoyant density determinations. Optical grade CsCl (Calbiochem Corp., Los Angeles, California) was added to solutions of LPS in 10 mM sodium phosphate at pH 6.8. Equilibrium was obtained after 20 hr at 36,000 r-pm in a Model E ultracentrifuge (Beckman Instruments, Fullerton, California). The refractive index of the CsCl-LPS solution was determined before and after the run. The density of CsCl as determined by the refractive index was assumed to be the isoconcentration point, re, for the density calculations. The distributive factors, p, were obtained from Vinograd and Hearst (10). All runs were made at 25°C and the plates were read on a Gaertner comparator (Gaertner Corp., Chicago, Illinois). Serology. Each fraction was analyzed by gel diffusion in agar containing 1.5 M NaCl with antisera from turkeys which had been given multiple iv inoculations of formaldehyde-treated P. multocida cells. The relative concentration of the antigen in the various fractions was estimated by determination of the highest dilution which was capable of giving a visible precipitin line in gel diffusion, and also by the radial immunodiffusion procedure for quantitation of antigens of Mancini et al. (11). Chemical analyses. Analyses for fatty acids, sugars, and amino acids were made by gas-liquid chromatography at the Analytical Biochemistry Laboratories, Inc., Columbia, Missouri. Fatty acids were estimated as their methyl esters and sugars as the trimethylsilyl derivatives according to the procedure of Wang et al. (12). RESULTS AND DISCUSSION
A flow diagram describing the fractions obtained by the Westphal extraction is shown in Fig. 1. Analyses of the fractions A-l, A-2, P-l, and P-2 by radial immunodiffusion indicated that the ratios of antigen, presumably LPS, in each were as follows: A-2/A- 1, 1.4; A-2/P- 1, 1 .O; and * Since even glass-distilled phenol gave an acid reaction when mixed with water and brom cresol green (yellow color), a small amount of sodium bicarbonate was added to reduce carry-over of volatile acids. This distillate gave a green color with the indicator.
ISOPYCNIC
CHARACTERIZATION
OF LPS
267
FIG. 1. Flow diagram of phenol-water extraction ofPasteurella multocida cells. Fractions are designated P-l, P-2, A- 1, and A-2. For details of extraction see Materials and Methods.
A-2/P-2,9.0. Hence most of the antigen was in A-2 or P-l. As expected in a Westphal extraction, most of the nucleic acids were recovered in A-l. Since this strain of P. multocida is a highly virulent organism with a large capsule, it was not expected that most of the LPS would be partitioned in the first phenol layer. However, after a second extraction of the phenol layer with water, most of the LPS was now in the aqueous layer. Because A-2 not only had more antigen but also had a higher activity in terms of gel diffision, the procedure was modified by keeping fractions A-l and A-2 separate. Fraction P-l was examined in a CsCl density gradient (Fig. 2a). A major band was observed at a density of 1.378 g/ml, and lesser amounts of smaller components were banded at higher densities. Buoyant density determinations were made on a number of P-l fractions, and the major density band was determined to be 1.381 + 0.005 g/ml. The second aqueous fraction A-2 was examined in a CsCl density gradient. A single symmetrical peak was observed with a density of 1.406 g/ml indicating homogeneity by this criterion (Fig. 2~). Buoyant density profiles were obtained from a series of four dilutions of the A-2 fraction in CsCl density gradients. The pattern in Fig. 2c was representative of the fraction at all concentrations, and the density of the LPS in A-2 was determined to be 1.403 & 0.005 g/ml. Fractions A-l were examined in similar CsCl gradients, and the nucleic acids from the extraction were banded at the bottom of the cell. The LPSs in this fraction were entrained with the nucleic acids and no bands were observed in the 1.38-1.40 g/ml density region. In order to verify that the LPSs in the fractions A-2 and P-l did have different buoyant densities, a mixture in CsCl was prepared consisting of
268
PHILLIPS
AND REBERS
FIG. 2. Equilibrium schlieren photographs of 0.2% LPS in CsCI, 10 mM sodium phosphate buffer, pH 6.8, at 36,000 rpm and 25°C: (a) fraction P-l, pe = 1.3% g/ml; (b) mixture of fraction A-2 and P-l, pe = 1.400 g/ml; and (c) fraction A-2, pe = 1.415 g/ml. Single-sector, 2”, Kel-F centerpiece.
equal amounts of both. Two major bands were observed buoying at 1.404 and 1.387 g/ml corresponding to the individual bands from the respective A-2 and P-l fractions (Fig. 2b). The smaller bands from the P-l fraction were also observed. The following data suggest that we are dealing with a typical LPS. The chemical composition of some of the components of fraction A-2 as indicated by gas-liquid chromatographic analyses is given in Table 1. The lauric and myristic acid contents and the glucose, galactose, and D-glycero-L-mannoheptose are as expected for LPSs. A previously described standard Westphal preparation from the X-73F strain of P. multocida in which A-l and A-2 were combined, treated with RNase and DNase, and then purified by sedimentation at 165,000 g contained 2.8% total phosphorus, 2.9% total nitrogen, 7.7% hexose, and 13.6% heptose (13). This preparation killed mice and produced a local Shwartzman reaction in rabbits. Since the complete chemical analysis of the constituents of LPS in A-2 was not available, it was not possible to calculate the predicted buoyant density. If the ratios of the amounts of lipid, carbohydrate, and protein as expressed in Table 1 are assumed to be representative of the LPS and contribute’to the density accordingly, then an observed density of 1.40 g/ml would be expected. Buoyant densities of 1.40- 1.44 g/ml in CsCl have been reported for LPS from E. coli (6). The mixing experiments indicate that the LPSs in the P-l and the A-2 buoy at different respective densities and are not readily interconverted in CsCl solutions. Since the monodisperse LPS in A-2 is recovered from the P-l fraction, explanations for the change in buoyant density were
ISOPYCNIC
CHARACTERIZATION TABLE
CHEMICAL
ANALYSIS
269
OF LPS
1
OF LIPOPOLYSACCHARIDE
IN FRACTION
A-2 Percentage w/w
I. Fatty acid analysis Lauric-C,z Myristic-C,, Palmitic-C,, Palmitoleic-C,,l Stearic-C,s Oleic-C,J
2.24 9.66 1.21 0.56 0.04 0.09 Total
II. Sugar analysis Galactose Glucose D-Glycero-L-mannoheptose
14.97 9.08 22.98 Total
III. Amino acid analysis 17 Amino acids
13.80%
Total (protein)
47.03% 4.61%
considered. The carbohydrate content would not be expected to change. There is the possibility that the amount of protein or lipid has been reduced and results in an increase of density from 1.38 to 1.40 g/ml. Although acid-free phenol was utilized for the extractions to reduce hydrolysis, labile linkages may still be hydrolyzed (14). The amount of hydrolysis was estimated by dividing one of the P-l fractions into two equal parts for the second extraction. The first part was extracted with water at 22°C and the other part with water at 65°C. The difference in the buoyant densities between the A-2 LPSs was only 0.003 g/ml. Since the buoyant densities of the two LPSs were nearly identical even though the temperatures were 65 and 22°C we concluded that relatively little degradation occurred. Although the different densities can be accounted for by differences in chemical composition, other changes such as hydration, net charge, ionic constituents, and hydrophobic bonding must be considered (15). Although the distribution of molecules in the A-2 LPS has yet to be determined by other procedures, the symmetrically shaped schlieren buoyant density patterns indicate a limited molecular weight distribution. The technique of equilibrium buoyant density determinations in CsCl may be used advantageously to characterize LPS fractions and these applications of the analytical ultracentrifuge are a useful adjunct to the Westphal extraction procedures.
270
PHILLIPS
AND
REBERS
ACKNOWLEDGMENTS The authors wish to acknowledge the technical assistance of Robert E. Patterson, Kim Brogden, and Bill Brown.
REFERENCES 1. Westphal, O., Luderitz, O., and Bister, F. (1952) Z. Nutueorsch 7B, 148-151. 2. Shands, J. W. (1971)in Microbial Toxins (Weinbaum, G., Kadis, S., and Ajl, S. S., eds.), Vol. IV, pp. 127-144, Academic Press, New York. 3. Dunstone, J. R. (1969) Separation Sci. 4, 267-285. 4. Creeth, J. M., and Denborough, M. A. (1970) Biochem. J. 117, 879-891. 5. Hirt, W. E., and Vestal, J. R. (1975) J. Bacterial. 123, 642-650. 6. Morrison, D. C., and Leive, L. (1975) J. Biol. Chem. 250, 2911-2919. 7. Rebers, Pi, Heddleston, K., and Rhoades, K. (1967) J. Bacterial. 93, 7-14. 8. Westphal, O., and Jann, K. (1965) in Methods in Carbohydrate Chemistry (Whistler, R. L., ed.), Vol. V, pp. 83-91, Academic Press, New York. 9. Ifft, J. B. (1969) in A Laboratory Manual of Analytical Methods of Protein Chemistry (Alexander, P., and Lundgren, H. P., eds.), Vol. 5, pp. 151-223, Pergamon Press, New York. 10. Vinograd, J., and Hearst, J. E. (1962), Fortschr. Chem. Org. Naturst. 20, 372-422. 11. Mancini, G., Carbonara, A. O., and Heremans, J. F. (1965) Zmtnunochemistty 2, 235-254. 12. Wang, W. S., Korczynski, M. S., and Lundgren, D. G. (1970)5. Bacterial. 104,556-565. 13. Rebers, P., and Heddleston, K. (1974) Amer. J. Vet. Res. 35, 555-560. 14. Tsang, J. C., Wang, C. S., and Alaupovic, P. (1974) J. Bncteriol. 117, 786-795. 15. Ifft, J. B. (1976) in Methods of Protein Separation (Catsimpoolas, N., ed.), Vol. I, pp. 193-237, Plenum, New York.