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Immunochemical relationships of proteins of avian egg whites
ARCHIVES
OF BIOCHEMISTRY
immunochemical
AND
the Department
108, 117-124
Relationships
HERb’IAN From
BIOPHYSICS
of Proteins
T. MILLER2
of Food Science
(1964)
ROBERT
AND
and !l’echnology, Received
University
of Avian
Egg Whites’
E. FEENEY of California,
Davis,
California
April 20, 1964
The immunochemical interactions of the egg white proteins of different avian species were studied with homologous and heterologous antisera. Cross reactions were determined between egg whites of closely and distantly related species by using rabbit antisera to the egg whites of the closely and distantly related species, as well as rabbit antisera to the egg whites of intermediate species. The species studied included several galliformes, two anseriformes, a columbiforme, and several ratites. The interrelationships also were studied by using antisera to the purified conalbumins of two of the more distantly related species, chicken (a galliform), and cassowary (a ratite). The conalbumins gave the most extensive cross reactions of any of the major egg-white components, but a minor constituent gave a more extensive cross reaction than conalbumin. INTRODUCTION
The proteins of egg whites of avian species show extensive diversity between and within taxonomic groupings (l-6). Major differences have been found in the quantities of the individual proteins, as well as in their physical and chemical properties and biochemical activities. These differences make the avian egg-white proteins attractive systems for studies in molecular evolution. The immunochemical relationships of eggwhite proteins have been studied by many investigators. Landsteiner et al. (7), in earlier classical studies, found cross reactions among several ovalbumins. Deutsch and co-workers (8-10) showed that many components seen in free-boundary electrophoresis crossreacted to varying degrees in the turkey, pheasant, duck, and chicken. Kaminski (ll-13), in studying
chicken
egg-white
proteins
by
immunoelectroDhoresis, found as many as nine globulins in some preparations. Recent studies in our laboratory showed that there are major differences between the electrophoretic characteristics of the conalbumins (ovotransferrins) of the ratites and those of many of the galliformes (5). A further comparative study of the properties of the egg white proteins of a selected group of avian species therefore has been made, using immunodiffusion and immunoelectrophoresis. MATERIALS
AND
METHODS
Procurement of eggs. The eggs of the chicken (Gallus gallus var. domesticus), turkey (Meleagres gallopavo), Japanese quail (Coturniz coturniz
japonica) ,3 pigeon (Columba lioia),
and dove
(Zenaidura macroura) were obtained from the Poultry Department and the School of Veterinary Medicine of the University of California, Davis, California. The golden pheasant (Chrysolophus
1 Supported in part by grant AI-03484 from the United States Public Health Service. Presented in part before the 146th National Meeting of the American Chemical Society, Denver, Colorado, January 20-23, 1964. 2 Postdoctoral Fellow of the United States Public Health Service during the year 1963%1964.
3 The eggs of the Japanese quail (Coturniz coturnir japonica) were obtained from Dr. Hans Abplanalp, Department of Poultry Husbandry. Further descriptions of, and genetic studies on, these eggs will be published in more detail elsewhere in cooperation with Dr. Abplanalp. 117
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MILLER
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pictus) eggs were obtained from the State Game Farm, Yountville, California. Eggs of the following birds were obtained from the San Diego Zoo, San Diego, California: cassowary (Casuarius aruensis), rhea (Rhea americana), emu (Dromiceius n. hollundiue), ostrich (Struthio camelus), tinamou (Eudromiu eleguns), and green Java pea fowl (Pave muticus). Duck eggs (Anus plutyrhynchos) were obtained locally. The storage of the eggs and the separating and blending of the egg whites were done as previously described (3, 14). PuriJed proteins. Chicken ovalbumin, conalbumin, and lysozyme were all crystalline preparations made by methods involving chromatography on ion exchange cellulose as previously described (5, 15). The highly purified conalbumins of quail and cassowary were prepared as recently described (5). All chemical modifications of conalbumin were done with the iron complexes because of the much greater stability of the complex as compared to that of iron-free protein (16). Acetic anhydride in sodium acetate was used for acetylation so as to introduce approximately 20 acetyl groups (5,6). Iodination with Iz and KI was used to introduce lo-15 iodine groups (6, 16). In carbamylation, KNCO reacted with approximately 20 amino groups (6). Reaction with glucose was achieved by incubation of a solution containing 2% iron conalbumin and 5y0 glucose in 0.1 M NaHCOo at pH 9.0 and 50°C for 24 hours (17). Reaction with 5-dimethyl amino-1-naphthalene sulfonyl chloride was in 0.1 M NaHC03 at pH 9.0 for 1 hour at 20”-25” (18). Antisera. Antigens were freshly prepared solu-
FIG. 1. Comparisons egg white. Anti-chicken
FEENEY tions of the purified proteins in 1% NaCl or the individual egg whites diluted with an equal volume of 1% NaCl. Antiserums were prepared by Antibodies Incorporated, Davis, California, by injecting rabbits with the appropriate antigen mixed with Freunds adjuvant. The titers and specificities of all antisera were checked carefully. Anti-ovalbumin sera from two different commercial sources gave precipitin lines for conalbumin and for component 18 on immunoelectrophoresis of whole egg white. These antisera had been prepared against five times crystallized only a single precipitin ovalbumin. However, line was obtained with antisera prepared against ovalbumin which had been crystallized and chromatographed in our laboratory (15). Electrophoresis and diffusion. Micro-immunoelectrophoresis was done according to the method of Scheidegger (19). Longer slides were used. Barbital buffer of pH 8.2 and y/2 = 0.05 was used except when higher ionic strengths were tested for comparisons. Agar was either Ionagar (Consolidated Lab. Inc., Chicago Heights, Illinois) or Agarose (Marine Colloids, Inc., New York). Immunodiffusion was done according to the method of Ouchterlony (20). Direct transmitted light was used to photograph agar slides. Starch-gel electrophoresis was done as described by Poulik (21) as adapted in our laboratory (5,14), and the gels were stained and photographed. RESULTS
CHICKEN
EGG WHITE
Figure 1 compares the electrophoretic pattern of the stained starch gel of chicken
of immunoelectrophoretic and starch-gel patterns of whole chicken egg-white serum used for development of immunoelectrophoresis.
IMMUNOCHEMISTRY
OF EGG
egg white with the immunoelectrophoretic pattern of chicken egg white developed against anti-chicken egg-white serum. The major components easily recognizable are ovalbumin, conalbumin, and lysozyme. The patterns of these in starch gel have been described recently (14). Their positions in the immunoelectropherograms were identified by experiments with the individual crystalline proteins. Ovomucoid does not give easily apparent patterns in starch gel by this staining procedure (14) and also was not identified in these immunoelectropherograms. Both patterns show several of the more poorly characterized globulins, including one component which separates well in starch-gel electrophoresis and which has been described as component 18 or line 18 by Lush (22) and Feeney et al. (14). Differences between the relative migrations of the various components in the starch-gel electropherograms and in the agargel immunoelectropherograms are readily apparent (Fig. 1). For example, conalbumin migrates faster anodically in starch than does component 18, but in agar conalbumin migrates toward the cathode. Such differences may be attributable to differences in electroendosmosis and molecular filtration. The relative migrations also were influenced by the type of agar used. In general, fewer electroendosmotic effects were observed with a more purified agar, such as Agarose or Ionagar. Kaminski and co-workers, who used agar, stated that lysozyme does not electrophorese. Using Ionagar and barbital buffer of y/2 0.1, we have electrophoresed lysozyme. When we used purified lysozyme from our laboratory and subjected it to electrophoresis in agar, the path of this protein through the agar was visible. In almost all runs of immunoelectrophoresis of whole egg whites that contained measurable quantities of lysozyme, a “tongue,” or smear, which moved cathodically from the origin was evident, but when a more purified agar (Agarose) was used, it was not possible to observe the lysozyme without development with antiserum. However, the position of lysozyme in agarose was always evident when 0.5-l .O% solutions of crystalline lysozyme was subjected to electrophoresis
WHITE
PROTEINS
119
and the precipitin bands developed against antiserum to crystalline lysozyme. INTERRELATIONSHIPS OF DIFFERENT
AMONG EGG WHITES AVIAN SPECIES
The majority of the comparative studies were performed with antisera to cassowary and chicken egg whites and antisera to cassowary and chicken conalbumins. These species were selected because these particular species have conalbumins which showed large differences in physical properties (5), and these species were among the more distantly related of those studied. In addition, quantities of the egg whites and highly purified preparations of the conalbumins were available (5). Antisera to duck and Japanese quail egg whites were employed to substantiate the relative reactivity of these intermediately reacting species as determined with antisera to cassowary and chicken egg whites. The egg-white proteins of several avian species were compared by means of immunoelectrophoresis employing both chicken and cassowary egg white antisera simultaneously as shown in Fig. 2. It is evident that there is a good cross reaction between the chicken and golden pheasant egg-white proteins and between the proteins of the rhea, emu, and cassowary. All of these egg whites show a precipitin band for component 18 even where no other bands are present. In other experiments, the goose egg-white proterns reacted similar to those of the duck. The major egg-white proteins of the turkey reacted very strongly with anti-chicken eggwhite serum, but no precipitin band was observed for component 18; nor was there any reaction against anti-cassowary egg-white serum. The pigeon egg white showed no cross reactions of the major proteins with either antiserum. As will be discussed below, the egg whites of the turkey and pigeon lack component 18. Figure 3 compares egg whites of four of the ratites against anti-cassowary egg white serum in both troughs in order to delineate more sharply the patterns of this group. Extensive cross reactions were obtained. From these, and from a series of similar experiments, the phylogenetic relationships of these four ratites were in the following order: cassowary, emu, rhea, and
120
MILLER
-AND
FEENEY
FIG. 2. Immunoelectrophoresis of various avian egg whites. Cassowary egg-white antiserum (top trough) and chicken egg-white antiserum (bottom trough) were used for development. (A) rhea, (B) duck, (C) dove, (1~) peafowl, (E) cassowary, (F) emu, (G) golden pheasant, (H) chicken.
ostrich. The tinamou also showed cross reactivity with anti-cassowary serum, and probably can be added after the ostrich. CROSS REACTIVITIES WITH THE CONALBUMINS
Conalbumin was investigated in more detail because, (a) it appeared to be the most antigenic of the major components; (b) it could be prepared in crystalline form or in high purity from several species; and (c) in one group, the ratites, it showed multiple forms and large differences in electrophoretic mobilities as determined in both movingboundary and starch-gel electrophoresis (5). Immunoelectropherograms were made of the whites of eight representative species against both anti-chicken conalbumin serum and anti-cassowary conalbumin serum. The conalbumin of the cassowary, rhea, ostrich, and tinamou all reacted with anti-cassowary conalbumin serum but not with anti-chicken conalbumin serum. The turkey, dove, golden pheasant, and peafowl all reacted with antichicken conalbumin serum but not with anti-cassowary conalbumin serum. Quail white showed a strong reaction against antichicken conalbumin serum and occasionally a very weak reaction against anti-cassowary conalbumin serum. Duck white always gave weak reactions with both anti-chicken and
anti-cassowary conalbumin serums. As a representative of an intermediate group, antiserum prepared against duck egg white confirmed the results obtained with the antisera to chicken and cassowary conalbumins giving weak reactions with both chicken and cassowary conalbumin. The cross reactions in the ratite group were studied in more extensive experiments employing immunodiffusion of the egg whites and cassowary conalbumin against anticassowary conalbumin serum in Ouchterlony plates (7). The results of three typical experiments are shown in Fig. 4. Some of the reacting samples showed spurs and therefore reactions of partial identity. The precipitin bands formed with the tinamou and ostrich egg whites reacted very weakly, and in most cases the bands redissolved within 24 hours. The weak reactions of duck and quail against anti-cassowary serums on immunoelectrophoresis were not observed in Ouchterlony plates. Acetylated cassowary conalbumin was compared in an attempt to determine whether the difference in charge on the conalbumins in the ratite group were the main reasons for the differences in immunochemical reactivity. In Fig. 4A it can be seen that acetylation has removed some antigenic determinants from cassowary COIIalbumin. Rhea conalbumin from whole egg
IMMUNOCHEMISTRY
OF EGG WHITE
FIG. 3. Immunoelectrophoresis of ratite egg whites. Cassowary egg white used for development in both top and bottom troughs. (A) Cassowary, (B) rhea, (C) emu, and (D) ostrich followed by immunodiffusion against cassowary egg-white antiserum in both troughs.
white and acetylated cassowary conalbumin each showed antigenic determinants not present in the other, and both lacked determinants present in untreated protein. Emu conalbumin showed antigenic determinants not present in acetylated cassowary conalbumin. Acetylated cassowary conalbumin (precipitate) was the fraction which precipitated during the chemical reaction. After it was dissolved, it reacted as did the soluble fraction of acetylated cassowary conalbumin. Chicken conalbumin was modified chemically by a variety of procedures and the effects of these modifications were studied by immunoelectrophoresis and immunodiffusion. Chicken conalbumin was modified in the following ways : acetylation, iodination,
PROTEINS
121
FIG. 4. Immunodiffusion against anti-cassowary conalbumin serum. The antigens were: (.4): 1, cassowary conalbumin; 2, acetylated cassowary conalbumin; 3, rhea egg white; 4, duck egg white; 5, ostrich; and 6, emu egg white. (B): 1, quail conalbumin; 2, acetylated cassowary conalbumin; 3, emu egg white; 4, golden pheasant egg white; 5, tinamou egg white; and 6, cassowary conalbumin. (C) : 1, acetylated cassowary conalbumin (ppt.); 2, acetylated cassowary conalbumin; 3, duck egg white; 4, emu egg white; 5, quail conalbumin; and 6, cassowary conalbumin.
carbamylation, reaction with glucose, and reaction with 5-dimethylamino naphthalene sulfonyl chloride. With the exception of one of the 5-dimethylamino naphthalene sulfonyl chloride derivatives which showed two distinct areas, all derivatives showed one broad arc and increased mobility toward the anode. Comparison of the same or similar samples in starch-gel electrophoresis showed similar increases in relative mobilities. This would be expected from such modifications which produce more acidic proteins (6, 16, 17, 19). Many of the amino and tyrosyl groups were therefore probably modified without reduc ing greatly the interactions with anticonalbumin serum.
122
MILLER
AND
FEENEY
conalbumin near the arcs of component 18, as seen in the pheasants and peafowl, were barely discernible. The extensive cross reac. tions of the egg whites of three ratites with chicken egg white antiserum in Fig. 5 were particularly noteworthy. Direct confirmation of the identity of cornponent 18 as seen in starch gel was obtained by comparisons with egg whites which did not show component 18 on starch-gel electrophoresis. This was available in two different ways, One was by the use of turkey and pigeon whites, and the other by the use of genetic strains of Japanese quail whose whites gave starch-gel patterns differing only by the presence or absence of component 18 (Fig. 6). It is evident that component 18 was absent from Quail II, the pigeon, and the turkey. The eggs of insufficient numbers of turkeys and pigeons were examined to determine whether component 18 generally is absent from their egg whites. As judged from the relative intensities of staining in starch gel, the duck and the dove appeared to have elevated amounts of this constituent. The precipitin line designated as component 18 in imnmnoelectrophoresis was present on immunoelectrophoresis of Quail I but absent FIN. 5. Immunoelectrophoretic patterns of egg whites after prolonged diffusion. Anti-chicken from Quail II. The use of these quail strains egg white serum used for development. Immunotherefore confirmed the position of comdiffusion was allowed to proceed for 5 days (component 18 in agar immunoelectropherograms. ponent 18 is the only precipitin band remaining) : Conalbumin and component 18 are easily (A) cassowary and ostrich, (B) rhea and duck, confused in agar immunoelectropherograms (C) peafowl and golden-Amherst pheasant genetic because they may have similar mobilities. cross, (1)) golden pheasant and dove. The existence of component 18 in chicken serum was examined in two different samples CROSS REACTIONS WITH COMPONENT 18 by immunoelectrophoresis against antichicken egg-white serum. n-0 arcs similar to Component 18 (Fig. 1) not only gave the most extensive cross reactions of all the those for component 18 were found. The partial purification of component 18 egg-white proteins but also apparently was was accomplished. In a typical run a liter the most antigenic of all minor components. The extensive cross reaction was best ob- of egg white was adjusted to pH 4.44.6, and served in immunoelectropherograms of the resulting precipitate was separated by several egg whites of the more distantly re- centrifugation and discarded. Component 18 at pH lated species studied, which were allowed to was then precipitated quantitatively 6.8-7.0 by 35 % saturation with (NH&SO*. continue diffusion for 4-6 days against antichicken egg-white serum. After this time all The precipitate was dissolved in water and dialyzed extensively against water. The the precipitin arcs due to the major constituents dissolved probably because of excess soluble fraction after dialysis and lyophilization produced approximately 5 gm of proantigen; however, the arcs due to component tein. This protein was dissolved in phosphate 18 showed more clearly than at the beginning (Fig. 5). The usually much heavier arcs of buffer and was mixed with 20 gm carboxy-
IMMUNOCHEMISTRY
FIG. 6. Starch-gel electrophoretic
OF EGG WHITE
the cellulose at this pH was mostly ovalbumin, ovomucoid, and component 18. This portion of the egg-white protein was then subjected to two successive experiments of
chromatography on G-200 Sephadex in 0.05 M phosphate at pH 6.5 in a column 68 cm long by 3 cm wide. The resulting first peak showed only component 18 by starch-gel electrophoretic analysis. DISCUSSION
Our primary interest was to study the interactions of the constituents which elicit good antibody production and which give interactions
123
patterns of whole egg whites of several avian species
methyl cellulose in 0.01 M sodium phosphate at pH 5.2. The protein that did not adsorb to
well-defined
PROTEINS
in immunoelectro-
phoresis and immunodiffusion. An important aspect of this study was the use of antisera to the egg whites of two distantly related species. The two constituents which showed the greatest degree of cross reaction in this study were the conalbumins and the protein designated as component 18 by Lush (22), and which was further described by Feeney et al. (14) as a relatively high molecular weight substance. Deutsch (9), in early reports on the immunochemistry of egg white proteins, stated that, although the four major components account for 92% of the protein egg white, there was an unexpectedly small percentage of the total antibody directed against these proteins. Most of the
antibody response was to minor and poorly defined antigenic components of egg white. Further,
in experiments
in Deutsch’s
labora-
tory (8-10) it was noted that several of these minor components always was associated with conalbumin, and one of these minor components may have been the material we presently term component 18. The relatively extensive cross reactivity and the apparently high antigenicity of component 18 present a challenge for further studies of its chemical, physical, and immunological properties. In the present limited comparisons, the cross reactions of the various proteins grouped the species into those reacting primarily with the anti-chicken egg white serum, those reacting primarily with the anti-cassowary egg white serum and those reacting weakly with both serums. According to hypothetical taxonomic relationships (23), the ducks are lower than the chicken on the taxonomic scale and one might expect duck egg white to react slightly with anti-chicken egg white serum, but one perhaps would not predict that cassowary egg white would react with anti-chicken egg white serum. Pigeons and doves are closely related taxonomically according to Welty (23), but the cross reactions found in this study would indicate wider separations. Emu egg-white proteins reacted almost as well as did those of cassowary against anti-cassowary egg white serum, but, while the tinamou is only slightly above the
124
MILLER
AND
ratites, it only gave a very slight cross reaction with anti-cassowary egg white serum. The position of tinamou is thus still an anomaly (5). ACKNOWLEDGMENT The authors appreciate the technical assistance of Mr. Merrill E. Gershwin in certain phases of this study and are indebted to Dr. Alan Wilson, Brandeis University, for allowing us to see his tabulated data on immunological comparisons of different avian egg whites reacted against antichicken ovalbumin serum. REFERENCES 1. MCCABE, R. A., AND DEUTSCH, H. F., The Auk 69, 1 (1952). 2. RHODES, M. B., BENNETT, N., AND FEENEY, R. E., J. Biol. Chem. 235, 1686 (1960). 3. FEENEY, R. E., ANDERSON, J. S., AZARI, P. R., BENNETT, N., AND RHODES, M. B., J. Biol. Chem. 235, 2307 (1960). 4. SIBLEY, C. G., Ibis. 102, 215 (1960). 5. CLARK, J. R., OSUGA, D. T., AND FEENEY, R. E., J. Biol. Chem. 238,362l (1963). 6. STEVENS, F. C., ANII FEENEY, R. E., Biochemistry 2, 1346 (1963). 7. LANDSTEINER, K., LONGSWORTH, L. G., AND VAN DER SCHEER, J., Science 88, 83 (1938).
FEENEY 8. WETTER, L. R., COHN, M., AND DETTSCH, H. E., J. Immunol. 69, 109 (1952). 9. DEUTSCH, H. R.., Federation Proc. 12, 729 (1953). 10. WETTER, L. R., COHN, M., AND DEUTSCH, H. R., J. Immunol. 70, 507 (1953). 11. KAMINSKI, ;LI., Nature 178, 981 (1956). 12. KAMINSKI, XI., Ann. Z’lnst. Pasteltr 92, 802 (1957). 13. KAMINSKI, M., Immunology 6, 322 (1962). 14. FEENEY, R. E., ABPLANALP, H., CLARY, J. J., EDWARDS, D. L., AND CLARK, J. R., J. Biol. Chem. 238, 1732 (1963). 15. RHODES, M. B., AZARI, P. R., AND FEENEY, R. E., J. BioZ. Chen+ 230, 399 (1953). 16. AZARI, P. R., AND FEENEY, R. E., Arch. Biothem. Biophys. 92, 44 (1961). 17. FEENEY, R. E., CLARK, J. R., AND CLARY, J. J., Kature 201, 192 (1964). 18. WEBER, G., Biochem. J. 51, 155 (1952). 19. SCHEIDEGGEH, J. J., Intern. Arch. Allergy Appl. Immunol. 7, 103 (1955). 20. OUCHTERLONY, O., Acta Pathol. Microbial. Stand. 26, 516 (1949). 21. POULIK, M. D., Nature 130, 1477 (1957). 22. LUSH, I. E., K’ature 189, 931 (1961). 23. WELTY, J. C., 1962. !/‘h,e Life of Birds, W. B. Saunders & Co., Philadelphia, Pennsylvania.
OF BIOCHEMISTRY
immunochemical
AND
the Department
108, 117-124
Relationships
HERb’IAN From
BIOPHYSICS
of Proteins
T. MILLER2
of Food Science
(1964)
ROBERT
AND
and !l’echnology, Received
University
of Avian
Egg Whites’
E. FEENEY of California,
Davis,
California
April 20, 1964
The immunochemical interactions of the egg white proteins of different avian species were studied with homologous and heterologous antisera. Cross reactions were determined between egg whites of closely and distantly related species by using rabbit antisera to the egg whites of the closely and distantly related species, as well as rabbit antisera to the egg whites of intermediate species. The species studied included several galliformes, two anseriformes, a columbiforme, and several ratites. The interrelationships also were studied by using antisera to the purified conalbumins of two of the more distantly related species, chicken (a galliform), and cassowary (a ratite). The conalbumins gave the most extensive cross reactions of any of the major egg-white components, but a minor constituent gave a more extensive cross reaction than conalbumin. INTRODUCTION
The proteins of egg whites of avian species show extensive diversity between and within taxonomic groupings (l-6). Major differences have been found in the quantities of the individual proteins, as well as in their physical and chemical properties and biochemical activities. These differences make the avian egg-white proteins attractive systems for studies in molecular evolution. The immunochemical relationships of eggwhite proteins have been studied by many investigators. Landsteiner et al. (7), in earlier classical studies, found cross reactions among several ovalbumins. Deutsch and co-workers (8-10) showed that many components seen in free-boundary electrophoresis crossreacted to varying degrees in the turkey, pheasant, duck, and chicken. Kaminski (ll-13), in studying
chicken
egg-white
proteins
by
immunoelectroDhoresis, found as many as nine globulins in some preparations. Recent studies in our laboratory showed that there are major differences between the electrophoretic characteristics of the conalbumins (ovotransferrins) of the ratites and those of many of the galliformes (5). A further comparative study of the properties of the egg white proteins of a selected group of avian species therefore has been made, using immunodiffusion and immunoelectrophoresis. MATERIALS
AND
METHODS
Procurement of eggs. The eggs of the chicken (Gallus gallus var. domesticus), turkey (Meleagres gallopavo), Japanese quail (Coturniz coturniz
japonica) ,3 pigeon (Columba lioia),
and dove
(Zenaidura macroura) were obtained from the Poultry Department and the School of Veterinary Medicine of the University of California, Davis, California. The golden pheasant (Chrysolophus
1 Supported in part by grant AI-03484 from the United States Public Health Service. Presented in part before the 146th National Meeting of the American Chemical Society, Denver, Colorado, January 20-23, 1964. 2 Postdoctoral Fellow of the United States Public Health Service during the year 1963%1964.
3 The eggs of the Japanese quail (Coturniz coturnir japonica) were obtained from Dr. Hans Abplanalp, Department of Poultry Husbandry. Further descriptions of, and genetic studies on, these eggs will be published in more detail elsewhere in cooperation with Dr. Abplanalp. 117
118
MILLER
AND
pictus) eggs were obtained from the State Game Farm, Yountville, California. Eggs of the following birds were obtained from the San Diego Zoo, San Diego, California: cassowary (Casuarius aruensis), rhea (Rhea americana), emu (Dromiceius n. hollundiue), ostrich (Struthio camelus), tinamou (Eudromiu eleguns), and green Java pea fowl (Pave muticus). Duck eggs (Anus plutyrhynchos) were obtained locally. The storage of the eggs and the separating and blending of the egg whites were done as previously described (3, 14). PuriJed proteins. Chicken ovalbumin, conalbumin, and lysozyme were all crystalline preparations made by methods involving chromatography on ion exchange cellulose as previously described (5, 15). The highly purified conalbumins of quail and cassowary were prepared as recently described (5). All chemical modifications of conalbumin were done with the iron complexes because of the much greater stability of the complex as compared to that of iron-free protein (16). Acetic anhydride in sodium acetate was used for acetylation so as to introduce approximately 20 acetyl groups (5,6). Iodination with Iz and KI was used to introduce lo-15 iodine groups (6, 16). In carbamylation, KNCO reacted with approximately 20 amino groups (6). Reaction with glucose was achieved by incubation of a solution containing 2% iron conalbumin and 5y0 glucose in 0.1 M NaHCOo at pH 9.0 and 50°C for 24 hours (17). Reaction with 5-dimethyl amino-1-naphthalene sulfonyl chloride was in 0.1 M NaHC03 at pH 9.0 for 1 hour at 20”-25” (18). Antisera. Antigens were freshly prepared solu-
FIG. 1. Comparisons egg white. Anti-chicken
FEENEY tions of the purified proteins in 1% NaCl or the individual egg whites diluted with an equal volume of 1% NaCl. Antiserums were prepared by Antibodies Incorporated, Davis, California, by injecting rabbits with the appropriate antigen mixed with Freunds adjuvant. The titers and specificities of all antisera were checked carefully. Anti-ovalbumin sera from two different commercial sources gave precipitin lines for conalbumin and for component 18 on immunoelectrophoresis of whole egg white. These antisera had been prepared against five times crystallized only a single precipitin ovalbumin. However, line was obtained with antisera prepared against ovalbumin which had been crystallized and chromatographed in our laboratory (15). Electrophoresis and diffusion. Micro-immunoelectrophoresis was done according to the method of Scheidegger (19). Longer slides were used. Barbital buffer of pH 8.2 and y/2 = 0.05 was used except when higher ionic strengths were tested for comparisons. Agar was either Ionagar (Consolidated Lab. Inc., Chicago Heights, Illinois) or Agarose (Marine Colloids, Inc., New York). Immunodiffusion was done according to the method of Ouchterlony (20). Direct transmitted light was used to photograph agar slides. Starch-gel electrophoresis was done as described by Poulik (21) as adapted in our laboratory (5,14), and the gels were stained and photographed. RESULTS
CHICKEN
EGG WHITE
Figure 1 compares the electrophoretic pattern of the stained starch gel of chicken
of immunoelectrophoretic and starch-gel patterns of whole chicken egg-white serum used for development of immunoelectrophoresis.
IMMUNOCHEMISTRY
OF EGG
egg white with the immunoelectrophoretic pattern of chicken egg white developed against anti-chicken egg-white serum. The major components easily recognizable are ovalbumin, conalbumin, and lysozyme. The patterns of these in starch gel have been described recently (14). Their positions in the immunoelectropherograms were identified by experiments with the individual crystalline proteins. Ovomucoid does not give easily apparent patterns in starch gel by this staining procedure (14) and also was not identified in these immunoelectropherograms. Both patterns show several of the more poorly characterized globulins, including one component which separates well in starch-gel electrophoresis and which has been described as component 18 or line 18 by Lush (22) and Feeney et al. (14). Differences between the relative migrations of the various components in the starch-gel electropherograms and in the agargel immunoelectropherograms are readily apparent (Fig. 1). For example, conalbumin migrates faster anodically in starch than does component 18, but in agar conalbumin migrates toward the cathode. Such differences may be attributable to differences in electroendosmosis and molecular filtration. The relative migrations also were influenced by the type of agar used. In general, fewer electroendosmotic effects were observed with a more purified agar, such as Agarose or Ionagar. Kaminski and co-workers, who used agar, stated that lysozyme does not electrophorese. Using Ionagar and barbital buffer of y/2 0.1, we have electrophoresed lysozyme. When we used purified lysozyme from our laboratory and subjected it to electrophoresis in agar, the path of this protein through the agar was visible. In almost all runs of immunoelectrophoresis of whole egg whites that contained measurable quantities of lysozyme, a “tongue,” or smear, which moved cathodically from the origin was evident, but when a more purified agar (Agarose) was used, it was not possible to observe the lysozyme without development with antiserum. However, the position of lysozyme in agarose was always evident when 0.5-l .O% solutions of crystalline lysozyme was subjected to electrophoresis
WHITE
PROTEINS
119
and the precipitin bands developed against antiserum to crystalline lysozyme. INTERRELATIONSHIPS OF DIFFERENT
AMONG EGG WHITES AVIAN SPECIES
The majority of the comparative studies were performed with antisera to cassowary and chicken egg whites and antisera to cassowary and chicken conalbumins. These species were selected because these particular species have conalbumins which showed large differences in physical properties (5), and these species were among the more distantly related of those studied. In addition, quantities of the egg whites and highly purified preparations of the conalbumins were available (5). Antisera to duck and Japanese quail egg whites were employed to substantiate the relative reactivity of these intermediately reacting species as determined with antisera to cassowary and chicken egg whites. The egg-white proteins of several avian species were compared by means of immunoelectrophoresis employing both chicken and cassowary egg white antisera simultaneously as shown in Fig. 2. It is evident that there is a good cross reaction between the chicken and golden pheasant egg-white proteins and between the proteins of the rhea, emu, and cassowary. All of these egg whites show a precipitin band for component 18 even where no other bands are present. In other experiments, the goose egg-white proterns reacted similar to those of the duck. The major egg-white proteins of the turkey reacted very strongly with anti-chicken eggwhite serum, but no precipitin band was observed for component 18; nor was there any reaction against anti-cassowary egg-white serum. The pigeon egg white showed no cross reactions of the major proteins with either antiserum. As will be discussed below, the egg whites of the turkey and pigeon lack component 18. Figure 3 compares egg whites of four of the ratites against anti-cassowary egg white serum in both troughs in order to delineate more sharply the patterns of this group. Extensive cross reactions were obtained. From these, and from a series of similar experiments, the phylogenetic relationships of these four ratites were in the following order: cassowary, emu, rhea, and
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FIG. 2. Immunoelectrophoresis of various avian egg whites. Cassowary egg-white antiserum (top trough) and chicken egg-white antiserum (bottom trough) were used for development. (A) rhea, (B) duck, (C) dove, (1~) peafowl, (E) cassowary, (F) emu, (G) golden pheasant, (H) chicken.
ostrich. The tinamou also showed cross reactivity with anti-cassowary serum, and probably can be added after the ostrich. CROSS REACTIVITIES WITH THE CONALBUMINS
Conalbumin was investigated in more detail because, (a) it appeared to be the most antigenic of the major components; (b) it could be prepared in crystalline form or in high purity from several species; and (c) in one group, the ratites, it showed multiple forms and large differences in electrophoretic mobilities as determined in both movingboundary and starch-gel electrophoresis (5). Immunoelectropherograms were made of the whites of eight representative species against both anti-chicken conalbumin serum and anti-cassowary conalbumin serum. The conalbumin of the cassowary, rhea, ostrich, and tinamou all reacted with anti-cassowary conalbumin serum but not with anti-chicken conalbumin serum. The turkey, dove, golden pheasant, and peafowl all reacted with antichicken conalbumin serum but not with anti-cassowary conalbumin serum. Quail white showed a strong reaction against antichicken conalbumin serum and occasionally a very weak reaction against anti-cassowary conalbumin serum. Duck white always gave weak reactions with both anti-chicken and
anti-cassowary conalbumin serums. As a representative of an intermediate group, antiserum prepared against duck egg white confirmed the results obtained with the antisera to chicken and cassowary conalbumins giving weak reactions with both chicken and cassowary conalbumin. The cross reactions in the ratite group were studied in more extensive experiments employing immunodiffusion of the egg whites and cassowary conalbumin against anticassowary conalbumin serum in Ouchterlony plates (7). The results of three typical experiments are shown in Fig. 4. Some of the reacting samples showed spurs and therefore reactions of partial identity. The precipitin bands formed with the tinamou and ostrich egg whites reacted very weakly, and in most cases the bands redissolved within 24 hours. The weak reactions of duck and quail against anti-cassowary serums on immunoelectrophoresis were not observed in Ouchterlony plates. Acetylated cassowary conalbumin was compared in an attempt to determine whether the difference in charge on the conalbumins in the ratite group were the main reasons for the differences in immunochemical reactivity. In Fig. 4A it can be seen that acetylation has removed some antigenic determinants from cassowary COIIalbumin. Rhea conalbumin from whole egg
IMMUNOCHEMISTRY
OF EGG WHITE
FIG. 3. Immunoelectrophoresis of ratite egg whites. Cassowary egg white used for development in both top and bottom troughs. (A) Cassowary, (B) rhea, (C) emu, and (D) ostrich followed by immunodiffusion against cassowary egg-white antiserum in both troughs.
white and acetylated cassowary conalbumin each showed antigenic determinants not present in the other, and both lacked determinants present in untreated protein. Emu conalbumin showed antigenic determinants not present in acetylated cassowary conalbumin. Acetylated cassowary conalbumin (precipitate) was the fraction which precipitated during the chemical reaction. After it was dissolved, it reacted as did the soluble fraction of acetylated cassowary conalbumin. Chicken conalbumin was modified chemically by a variety of procedures and the effects of these modifications were studied by immunoelectrophoresis and immunodiffusion. Chicken conalbumin was modified in the following ways : acetylation, iodination,
PROTEINS
121
FIG. 4. Immunodiffusion against anti-cassowary conalbumin serum. The antigens were: (.4): 1, cassowary conalbumin; 2, acetylated cassowary conalbumin; 3, rhea egg white; 4, duck egg white; 5, ostrich; and 6, emu egg white. (B): 1, quail conalbumin; 2, acetylated cassowary conalbumin; 3, emu egg white; 4, golden pheasant egg white; 5, tinamou egg white; and 6, cassowary conalbumin. (C) : 1, acetylated cassowary conalbumin (ppt.); 2, acetylated cassowary conalbumin; 3, duck egg white; 4, emu egg white; 5, quail conalbumin; and 6, cassowary conalbumin.
carbamylation, reaction with glucose, and reaction with 5-dimethylamino naphthalene sulfonyl chloride. With the exception of one of the 5-dimethylamino naphthalene sulfonyl chloride derivatives which showed two distinct areas, all derivatives showed one broad arc and increased mobility toward the anode. Comparison of the same or similar samples in starch-gel electrophoresis showed similar increases in relative mobilities. This would be expected from such modifications which produce more acidic proteins (6, 16, 17, 19). Many of the amino and tyrosyl groups were therefore probably modified without reduc ing greatly the interactions with anticonalbumin serum.
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conalbumin near the arcs of component 18, as seen in the pheasants and peafowl, were barely discernible. The extensive cross reac. tions of the egg whites of three ratites with chicken egg white antiserum in Fig. 5 were particularly noteworthy. Direct confirmation of the identity of cornponent 18 as seen in starch gel was obtained by comparisons with egg whites which did not show component 18 on starch-gel electrophoresis. This was available in two different ways, One was by the use of turkey and pigeon whites, and the other by the use of genetic strains of Japanese quail whose whites gave starch-gel patterns differing only by the presence or absence of component 18 (Fig. 6). It is evident that component 18 was absent from Quail II, the pigeon, and the turkey. The eggs of insufficient numbers of turkeys and pigeons were examined to determine whether component 18 generally is absent from their egg whites. As judged from the relative intensities of staining in starch gel, the duck and the dove appeared to have elevated amounts of this constituent. The precipitin line designated as component 18 in imnmnoelectrophoresis was present on immunoelectrophoresis of Quail I but absent FIN. 5. Immunoelectrophoretic patterns of egg whites after prolonged diffusion. Anti-chicken from Quail II. The use of these quail strains egg white serum used for development. Immunotherefore confirmed the position of comdiffusion was allowed to proceed for 5 days (component 18 in agar immunoelectropherograms. ponent 18 is the only precipitin band remaining) : Conalbumin and component 18 are easily (A) cassowary and ostrich, (B) rhea and duck, confused in agar immunoelectropherograms (C) peafowl and golden-Amherst pheasant genetic because they may have similar mobilities. cross, (1)) golden pheasant and dove. The existence of component 18 in chicken serum was examined in two different samples CROSS REACTIONS WITH COMPONENT 18 by immunoelectrophoresis against antichicken egg-white serum. n-0 arcs similar to Component 18 (Fig. 1) not only gave the most extensive cross reactions of all the those for component 18 were found. The partial purification of component 18 egg-white proteins but also apparently was was accomplished. In a typical run a liter the most antigenic of all minor components. The extensive cross reaction was best ob- of egg white was adjusted to pH 4.44.6, and served in immunoelectropherograms of the resulting precipitate was separated by several egg whites of the more distantly re- centrifugation and discarded. Component 18 at pH lated species studied, which were allowed to was then precipitated quantitatively 6.8-7.0 by 35 % saturation with (NH&SO*. continue diffusion for 4-6 days against antichicken egg-white serum. After this time all The precipitate was dissolved in water and dialyzed extensively against water. The the precipitin arcs due to the major constituents dissolved probably because of excess soluble fraction after dialysis and lyophilization produced approximately 5 gm of proantigen; however, the arcs due to component tein. This protein was dissolved in phosphate 18 showed more clearly than at the beginning (Fig. 5). The usually much heavier arcs of buffer and was mixed with 20 gm carboxy-
IMMUNOCHEMISTRY
FIG. 6. Starch-gel electrophoretic
OF EGG WHITE
the cellulose at this pH was mostly ovalbumin, ovomucoid, and component 18. This portion of the egg-white protein was then subjected to two successive experiments of
chromatography on G-200 Sephadex in 0.05 M phosphate at pH 6.5 in a column 68 cm long by 3 cm wide. The resulting first peak showed only component 18 by starch-gel electrophoretic analysis. DISCUSSION
Our primary interest was to study the interactions of the constituents which elicit good antibody production and which give interactions
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patterns of whole egg whites of several avian species
methyl cellulose in 0.01 M sodium phosphate at pH 5.2. The protein that did not adsorb to
well-defined
PROTEINS
in immunoelectro-
phoresis and immunodiffusion. An important aspect of this study was the use of antisera to the egg whites of two distantly related species. The two constituents which showed the greatest degree of cross reaction in this study were the conalbumins and the protein designated as component 18 by Lush (22), and which was further described by Feeney et al. (14) as a relatively high molecular weight substance. Deutsch (9), in early reports on the immunochemistry of egg white proteins, stated that, although the four major components account for 92% of the protein egg white, there was an unexpectedly small percentage of the total antibody directed against these proteins. Most of the
antibody response was to minor and poorly defined antigenic components of egg white. Further,
in experiments
in Deutsch’s
labora-
tory (8-10) it was noted that several of these minor components always was associated with conalbumin, and one of these minor components may have been the material we presently term component 18. The relatively extensive cross reactivity and the apparently high antigenicity of component 18 present a challenge for further studies of its chemical, physical, and immunological properties. In the present limited comparisons, the cross reactions of the various proteins grouped the species into those reacting primarily with the anti-chicken egg white serum, those reacting primarily with the anti-cassowary egg white serum and those reacting weakly with both serums. According to hypothetical taxonomic relationships (23), the ducks are lower than the chicken on the taxonomic scale and one might expect duck egg white to react slightly with anti-chicken egg white serum, but one perhaps would not predict that cassowary egg white would react with anti-chicken egg white serum. Pigeons and doves are closely related taxonomically according to Welty (23), but the cross reactions found in this study would indicate wider separations. Emu egg-white proteins reacted almost as well as did those of cassowary against anti-cassowary egg white serum, but, while the tinamou is only slightly above the
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ratites, it only gave a very slight cross reaction with anti-cassowary egg white serum. The position of tinamou is thus still an anomaly (5). ACKNOWLEDGMENT The authors appreciate the technical assistance of Mr. Merrill E. Gershwin in certain phases of this study and are indebted to Dr. Alan Wilson, Brandeis University, for allowing us to see his tabulated data on immunological comparisons of different avian egg whites reacted against antichicken ovalbumin serum. REFERENCES 1. MCCABE, R. A., AND DEUTSCH, H. F., The Auk 69, 1 (1952). 2. RHODES, M. B., BENNETT, N., AND FEENEY, R. E., J. Biol. Chem. 235, 1686 (1960). 3. FEENEY, R. E., ANDERSON, J. S., AZARI, P. R., BENNETT, N., AND RHODES, M. B., J. Biol. Chem. 235, 2307 (1960). 4. SIBLEY, C. G., Ibis. 102, 215 (1960). 5. CLARK, J. R., OSUGA, D. T., AND FEENEY, R. E., J. Biol. Chem. 238,362l (1963). 6. STEVENS, F. C., ANII FEENEY, R. E., Biochemistry 2, 1346 (1963). 7. LANDSTEINER, K., LONGSWORTH, L. G., AND VAN DER SCHEER, J., Science 88, 83 (1938).
FEENEY 8. WETTER, L. R., COHN, M., AND DETTSCH, H. E., J. Immunol. 69, 109 (1952). 9. DEUTSCH, H. R.., Federation Proc. 12, 729 (1953). 10. WETTER, L. R., COHN, M., AND DEUTSCH, H. R., J. Immunol. 70, 507 (1953). 11. KAMINSKI, ;LI., Nature 178, 981 (1956). 12. KAMINSKI, XI., Ann. Z’lnst. Pasteltr 92, 802 (1957). 13. KAMINSKI, M., Immunology 6, 322 (1962). 14. FEENEY, R. E., ABPLANALP, H., CLARY, J. J., EDWARDS, D. L., AND CLARK, J. R., J. Biol. Chem. 238, 1732 (1963). 15. RHODES, M. B., AZARI, P. R., AND FEENEY, R. E., J. BioZ. Chen+ 230, 399 (1953). 16. AZARI, P. R., AND FEENEY, R. E., Arch. Biothem. Biophys. 92, 44 (1961). 17. FEENEY, R. E., CLARK, J. R., AND CLARY, J. J., Kature 201, 192 (1964). 18. WEBER, G., Biochem. J. 51, 155 (1952). 19. SCHEIDEGGEH, J. J., Intern. Arch. Allergy Appl. Immunol. 7, 103 (1955). 20. OUCHTERLONY, O., Acta Pathol. Microbial. Stand. 26, 516 (1949). 21. POULIK, M. D., Nature 130, 1477 (1957). 22. LUSH, I. E., K’ature 189, 931 (1961). 23. WELTY, J. C., 1962. !/‘h,e Life of Birds, W. B. Saunders & Co., Philadelphia, Pennsylvania.