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Some properties of pigeon and other avian serum proteins
ht. 3.
419-422.
Biochem.,
1971,
SOME
PROPERTIES
2,
[Scientechnica
OF PIGEON SERUM
W. BAXENDALE,
(Publishers) Ltd.]
4’9
AND OTHER
AVIAN
PROTEINS
J. S. COURTENAY,
A. W. PHILLIPS,
AND
H. ZOLA
The Wellcome Research Laboratories (Biological Division), Langley Court, Beckenham, Kent BR3 3BS (Receivedx7 Oct., 1970) ABSTRACT I. The serum proteins of 6 avian sera are compared using electrophoretic gel filtration, and ultracentrifugation. 2. Antigenic similarities are demonstrated by immuno-electrophoresis.
antisera are used extensively in these laboratories for the detection of the group specific antigen of the avian leucosis and sarcoma group of viruses (Baxendale, 1971). We have set out to purify the antibody globulin fraction from pigeon serum in order to prepare fluorescent-labelled antibody to pigeon antibody protein. During a preliminary examination the electrophoretic pattern of pigeon serum proteins, particularly in the albumin-pre-albumin region, was observed to differ from the patterns of mammalian sera and chicken serum. Other avian sera were therefore examined to see how they compared with pigeon serum. Although the immunoglobulins of some avian species, particularly the chicken, have been the subject of a number of fairly detailed physicochemical studies (Leslie and Clem, 1964; Tenenhouse and Deutsch, 1966; Gallagher and Voss, rg6g; Hersh, Kubo, Leslie, and Benedict, 1969; Kubo and Benedict, 1969; Leslie and Benedict, 1969) a more general comparative study of avian sera appears to be lacking. The present communication sets out some general features of the serum proteins of 6 avian species. The species examined are: pigeon (Columba palumbus), domestic chicken (Gallus domesticus), turkey (Meleagris galloparo) , pheasant (Phasianus colchicus), sparrow (Passer domesticus), and duck (Anus platyrhynchos) . PIGEON
techniques,
EXPERIMENTAL Avian blood was allowed to clot at 37” C. for 30 minutes and the serum drawn off. Antisera were prepared in rabbits by intramuscular injection of 0.5 ml. of serum with 0.5 ml. of Freund’s complete adjuvant at monthly intervals, bleedings being taken at intervals and tested for precipitating activity by gel diffusion. Electrophoresis was performed in Verona1 buffer at pH 8.6, Z=o*o5, using Sepraphore III cellulose acetate membranes (Gelman Instrument Co., Ann Arbor, Michigan) and a voltage of 250 V. After I hour the proteins were stained with Ponceau S (G. T. Gun, London), dried, and cleared using IO per cent acetic acid in methanol. Cleared cellulose acetate membranes were scanned using an Analytrol photometer (Beckman Instruments Ltd., Palo Alto. California. U.S.A.). Acrylamide gel electrophdresis was carried out at PH 8.3 using the buffer system of Davis ( 1964) and gels made from a mixture containing 7.5 per cent total acrylamide. The gels were stained with Coomasie Blue using the t&hnique of Chrambach, Reisfield, Wyckoff, and Zaccari (1967). Gel immuno-electrophoresis was performed by the method of Scheidegger (1955) modified by using 8 x 8-cm. plates covered with a ml. of 2 ner r~ cent- agarose (i’Industrie Biologique Franwise S.A.) in pH 8.9 Tris-EDTA buffer (10.08 g. tris(hydroxymethyl)-aminomethane (Tris), + ethylenediaminetetra-acetic acid (disodiu: z&T; 0.79 g. boric acid per 1.). Electrophoresis was carried out at 150 V. for 70 minutes. After the addition of antiserum and incubation at 17’ C. for 3 hours, 8 per cent w/v polyethylene gly&l, 20 M (British Drue Houses. Poole. Dot-set). was added to the trough; to increke sensitivity (&&a, I 969). The plates were then incubated for a further 20 hours before being examined. .3
420
BAXENDALE
Columns of Sephadex G200 (Pharmacia Ltd., Uppsala, Sweden) were run in 0.2 34 Tris-HCI buffer containing 0.2 M NaCI. Sedimentation velocity experiments were performed at 50,740 r.p.m. using a Spinco mode1 E analytical ultracentrifuge.
ET AL.
Int. J. Biochem.
mammalian */-globulins. Also the electrophoretic mobilities of the albumin bands var) considerabfy from species to species. Pigeon and sparrow sera both show a slow albumin and a pre-albumin band, the latter being
Fro. I.- -Zone electrophoresis of avian sera and horse serum on cellulose acetate membr rane. From left : turkey, chicken, horse, duck, pigeon, horse, sparrow, pheasant.
Fro. z--Pigeon serum showing pre-albumin components {on right) : electrophoresis on celiulose acetate membrane. The photometer scan is superimposed on the electrophoretic pattern. RESULTS ZONE ELECTROPHORESIS Electrophoresis of the 6 avian sera and horse serum on cellulose acetate membranes gave the patterns shown in Fig. I. From this it can be seen that the avian y-globulins have on average a higher negative charge than
rather more pronounced in the sparrow serum. However, this difference is probably not significant as the amount of pre-albumin was found to vary considerably from sample to sample within a species. Fig. 2 shows the pattern obtained from another batch of pigeon serum relatively rich in pre-albumin,
1971, 2
AVIAN SERUMPROTEINS
the superimposed photometer scan indicating that the pre-albumin region is complex and contains at least two protein bands. The electrophoretic patterns of avian sera in
42’
shown in Fig. 4. On immuno-electrophoresis peak I showed components in the yhl- and a,-macroglobulin regions, peaks II and III showed proteins with the full
is
FIG. 3.--Immuno-electrophoresis of avian sera. The troughs contain a rabbit antiserum to pigeon serum. acrylamide gel are broadly similar to those on cellulose acetate. Besides the differences mentioned, there are. as can be seen in Fig. I, many other variations among the avian sera as well as differences between avian sera and horse serum, but the complexity of the patterns renders more detailed interpretation difficult without fractionation of the sera. ANTIVENIN COMPARISON Rabbit antiserum to pigeon whole serum gave several bands with all the other avian sera tested (Fig. 3). Rabbit anti-chicken serum similarI>- cross-reacted with all the avian sera tested. With both these antisera cross-reaction was extensive but incomplete. MOLECULAR WEIGHT DISTRIBCTION OF _4VIAN SERI’M PROTEINS The elution pattern obtained when pigeon serum \zas fractionated on Sephadex G2oo
FIG. a.-Elution
of proteins of pigeon serum G200. Ordinate, extinction: abscissa,elution volume.
from
Sephadex
mobility range from y through to prealbumin, whilst peak IV gave a single band in the pre-albumin region. Chicken and turke) sera also showed components which were retarded on Sephadex G200. Horse serum gave a relatively small peak in the same area. A I : 4 vjv dilution of pigeon serum with isotonic saline was examined in the analytical
BAXENDALE ET .4L.
422
ultracentrifuge. The pattern was qualitatively similar to that of horse serum, and consisted of components with sedimentation coefficients of 15.6 s, 7-1 s, and the main peak at 3.6 5. By comparison with horse serum run at the same concentration it could be ascertained that the heavy component was approximately equivalent to the normal mammalian macroglobulin ( I g s) component and the main peak was equivalent to the 4 s of albumin. The second component sedimented faster than the corresponding mammalian globulin which has a sedimentation coefficient of about 6.5 s at the concentrations used. The other avian sera gave similar results on ultracentrifugation. DISCUSSION The albumin bands in the different avian sera show considerable differences in electrophoretic mobility, pigeon albumin being particularly slow, whilst a strong complex pre-albumin region is also present in some samples of pigeon serum. The name ‘ prealbumin ’ is used here to indicate the electrophoretic mobility of the protein; it is not known whether this protein bears any other resemblance to mammalian pre-albumin. The pre-albumin components are either very low in molecular weight (around ro,ooo) or are retarded on Sephadex gels by adsorption. .Antisera to one species of avian serum cross-react extensively with sera from other species, although not all the proteins crossreact. ACKNOWLEDGEMENTS We are indebted to Mr. D. Veal, Miss Lizbeth Sayer, Miss Isabelle Trim, and Miss Susan Richard for skilled technical assistance.
REFERENCES BAXENDALE.W. (rg71), in preparation. CESKA,M. (196g), ’ Antigen-antibody interaction in the presence of water-soluble nonionic resins ‘, Biochim. biopbvs. .lcta. 177, 625628.
CHRAMBACH, A., REISPIELD,R. A., WYCKOFF. bl., and ZACCARI, J. (1g67), ‘ A procedure for rapid and sensitive staining of protein fractionated by polyacrylamide gel electrophoresis ‘, dnalyt. Biochcm., ao,
DAVIS, B. J.
I 50-t 54.
(rg64), ‘ Disc electrophoresis, II ‘, Ann. N. r. Acad. &i., sax, 404-427. GALLAGHER,J. S., and Voss, E. W. ( 1969). ‘ Molecular weight of a purified chicken antibody ‘, Immunochem., 6, lgg-206. HERJH, R. T., Ktrao, R. T., LESLIE, G. A.. and BENEDICT,A. A. (rg6g), ‘ Molecular weight of chicken, pheasant and auail IgG 1 UI ‘. Immunochem.. 6, 762~$5.
KUBO, R. T., and BENEDICT, A. A. ( 1969). ‘ Comparison of various avian and mammalian IgG for salt induced aggregation ‘. 3. Immun.. I-,
1022-1028.
LESLIE, G. A., and BENEDICT, A. .4. ( rg6gi. ‘ Structural and antigenic relationships between avian immunoglobulins ‘, 3. Zmmun.. 1% I 3561365.
LESLIE, G. A., and CLEM, L. W. (1964). ‘ Phylogeny of immunoglobulin structure and function. III ‘, 3. .5X/J.Med., 130, 1337-1352. SCHEIDEGGER, J. J. (rg55), ‘ Une micro-methode de l’immune electrophortse ‘, Znt. Archs .-I&TQ appl.
Immun., 7, 103-r 10.
TENENHOUSE,H. S., and DEUTSCH,H. F. ( rg66i, ’ Some physical-chemical properties of chicken y-globulinsand theirpepsin and papain digestion products ‘, Immunochem., 3, 1t-20.
Key Word Index: Avian sera, serum proteins. avian pre-albumin, electrophoresis, gel filtration. ultracentrifuge, immuno-electrophoresis, Columba palumbus, Phasianus rhynchos.
Gallus domesticus, Meleagris galloparo, colchicus, Passer domesticus. Anas pla&-
419-422.
Biochem.,
1971,
SOME
PROPERTIES
2,
[Scientechnica
OF PIGEON SERUM
W. BAXENDALE,
(Publishers) Ltd.]
4’9
AND OTHER
AVIAN
PROTEINS
J. S. COURTENAY,
A. W. PHILLIPS,
AND
H. ZOLA
The Wellcome Research Laboratories (Biological Division), Langley Court, Beckenham, Kent BR3 3BS (Receivedx7 Oct., 1970) ABSTRACT I. The serum proteins of 6 avian sera are compared using electrophoretic gel filtration, and ultracentrifugation. 2. Antigenic similarities are demonstrated by immuno-electrophoresis.
antisera are used extensively in these laboratories for the detection of the group specific antigen of the avian leucosis and sarcoma group of viruses (Baxendale, 1971). We have set out to purify the antibody globulin fraction from pigeon serum in order to prepare fluorescent-labelled antibody to pigeon antibody protein. During a preliminary examination the electrophoretic pattern of pigeon serum proteins, particularly in the albumin-pre-albumin region, was observed to differ from the patterns of mammalian sera and chicken serum. Other avian sera were therefore examined to see how they compared with pigeon serum. Although the immunoglobulins of some avian species, particularly the chicken, have been the subject of a number of fairly detailed physicochemical studies (Leslie and Clem, 1964; Tenenhouse and Deutsch, 1966; Gallagher and Voss, rg6g; Hersh, Kubo, Leslie, and Benedict, 1969; Kubo and Benedict, 1969; Leslie and Benedict, 1969) a more general comparative study of avian sera appears to be lacking. The present communication sets out some general features of the serum proteins of 6 avian species. The species examined are: pigeon (Columba palumbus), domestic chicken (Gallus domesticus), turkey (Meleagris galloparo) , pheasant (Phasianus colchicus), sparrow (Passer domesticus), and duck (Anus platyrhynchos) . PIGEON
techniques,
EXPERIMENTAL Avian blood was allowed to clot at 37” C. for 30 minutes and the serum drawn off. Antisera were prepared in rabbits by intramuscular injection of 0.5 ml. of serum with 0.5 ml. of Freund’s complete adjuvant at monthly intervals, bleedings being taken at intervals and tested for precipitating activity by gel diffusion. Electrophoresis was performed in Verona1 buffer at pH 8.6, Z=o*o5, using Sepraphore III cellulose acetate membranes (Gelman Instrument Co., Ann Arbor, Michigan) and a voltage of 250 V. After I hour the proteins were stained with Ponceau S (G. T. Gun, London), dried, and cleared using IO per cent acetic acid in methanol. Cleared cellulose acetate membranes were scanned using an Analytrol photometer (Beckman Instruments Ltd., Palo Alto. California. U.S.A.). Acrylamide gel electrophdresis was carried out at PH 8.3 using the buffer system of Davis ( 1964) and gels made from a mixture containing 7.5 per cent total acrylamide. The gels were stained with Coomasie Blue using the t&hnique of Chrambach, Reisfield, Wyckoff, and Zaccari (1967). Gel immuno-electrophoresis was performed by the method of Scheidegger (1955) modified by using 8 x 8-cm. plates covered with a ml. of 2 ner r~ cent- agarose (i’Industrie Biologique Franwise S.A.) in pH 8.9 Tris-EDTA buffer (10.08 g. tris(hydroxymethyl)-aminomethane (Tris), + ethylenediaminetetra-acetic acid (disodiu: z&T; 0.79 g. boric acid per 1.). Electrophoresis was carried out at 150 V. for 70 minutes. After the addition of antiserum and incubation at 17’ C. for 3 hours, 8 per cent w/v polyethylene gly&l, 20 M (British Drue Houses. Poole. Dot-set). was added to the trough; to increke sensitivity (&&a, I 969). The plates were then incubated for a further 20 hours before being examined. .3
420
BAXENDALE
Columns of Sephadex G200 (Pharmacia Ltd., Uppsala, Sweden) were run in 0.2 34 Tris-HCI buffer containing 0.2 M NaCI. Sedimentation velocity experiments were performed at 50,740 r.p.m. using a Spinco mode1 E analytical ultracentrifuge.
ET AL.
Int. J. Biochem.
mammalian */-globulins. Also the electrophoretic mobilities of the albumin bands var) considerabfy from species to species. Pigeon and sparrow sera both show a slow albumin and a pre-albumin band, the latter being
Fro. I.- -Zone electrophoresis of avian sera and horse serum on cellulose acetate membr rane. From left : turkey, chicken, horse, duck, pigeon, horse, sparrow, pheasant.
Fro. z--Pigeon serum showing pre-albumin components {on right) : electrophoresis on celiulose acetate membrane. The photometer scan is superimposed on the electrophoretic pattern. RESULTS ZONE ELECTROPHORESIS Electrophoresis of the 6 avian sera and horse serum on cellulose acetate membranes gave the patterns shown in Fig. I. From this it can be seen that the avian y-globulins have on average a higher negative charge than
rather more pronounced in the sparrow serum. However, this difference is probably not significant as the amount of pre-albumin was found to vary considerably from sample to sample within a species. Fig. 2 shows the pattern obtained from another batch of pigeon serum relatively rich in pre-albumin,
1971, 2
AVIAN SERUMPROTEINS
the superimposed photometer scan indicating that the pre-albumin region is complex and contains at least two protein bands. The electrophoretic patterns of avian sera in
42’
shown in Fig. 4. On immuno-electrophoresis peak I showed components in the yhl- and a,-macroglobulin regions, peaks II and III showed proteins with the full
is
FIG. 3.--Immuno-electrophoresis of avian sera. The troughs contain a rabbit antiserum to pigeon serum. acrylamide gel are broadly similar to those on cellulose acetate. Besides the differences mentioned, there are. as can be seen in Fig. I, many other variations among the avian sera as well as differences between avian sera and horse serum, but the complexity of the patterns renders more detailed interpretation difficult without fractionation of the sera. ANTIVENIN COMPARISON Rabbit antiserum to pigeon whole serum gave several bands with all the other avian sera tested (Fig. 3). Rabbit anti-chicken serum similarI>- cross-reacted with all the avian sera tested. With both these antisera cross-reaction was extensive but incomplete. MOLECULAR WEIGHT DISTRIBCTION OF _4VIAN SERI’M PROTEINS The elution pattern obtained when pigeon serum \zas fractionated on Sephadex G2oo
FIG. a.-Elution
of proteins of pigeon serum G200. Ordinate, extinction: abscissa,elution volume.
from
Sephadex
mobility range from y through to prealbumin, whilst peak IV gave a single band in the pre-albumin region. Chicken and turke) sera also showed components which were retarded on Sephadex G200. Horse serum gave a relatively small peak in the same area. A I : 4 vjv dilution of pigeon serum with isotonic saline was examined in the analytical
BAXENDALE ET .4L.
422
ultracentrifuge. The pattern was qualitatively similar to that of horse serum, and consisted of components with sedimentation coefficients of 15.6 s, 7-1 s, and the main peak at 3.6 5. By comparison with horse serum run at the same concentration it could be ascertained that the heavy component was approximately equivalent to the normal mammalian macroglobulin ( I g s) component and the main peak was equivalent to the 4 s of albumin. The second component sedimented faster than the corresponding mammalian globulin which has a sedimentation coefficient of about 6.5 s at the concentrations used. The other avian sera gave similar results on ultracentrifugation. DISCUSSION The albumin bands in the different avian sera show considerable differences in electrophoretic mobility, pigeon albumin being particularly slow, whilst a strong complex pre-albumin region is also present in some samples of pigeon serum. The name ‘ prealbumin ’ is used here to indicate the electrophoretic mobility of the protein; it is not known whether this protein bears any other resemblance to mammalian pre-albumin. The pre-albumin components are either very low in molecular weight (around ro,ooo) or are retarded on Sephadex gels by adsorption. .Antisera to one species of avian serum cross-react extensively with sera from other species, although not all the proteins crossreact. ACKNOWLEDGEMENTS We are indebted to Mr. D. Veal, Miss Lizbeth Sayer, Miss Isabelle Trim, and Miss Susan Richard for skilled technical assistance.
REFERENCES BAXENDALE.W. (rg71), in preparation. CESKA,M. (196g), ’ Antigen-antibody interaction in the presence of water-soluble nonionic resins ‘, Biochim. biopbvs. .lcta. 177, 625628.
CHRAMBACH, A., REISPIELD,R. A., WYCKOFF. bl., and ZACCARI, J. (1g67), ‘ A procedure for rapid and sensitive staining of protein fractionated by polyacrylamide gel electrophoresis ‘, dnalyt. Biochcm., ao,
DAVIS, B. J.
I 50-t 54.
(rg64), ‘ Disc electrophoresis, II ‘, Ann. N. r. Acad. &i., sax, 404-427. GALLAGHER,J. S., and Voss, E. W. ( 1969). ‘ Molecular weight of a purified chicken antibody ‘, Immunochem., 6, lgg-206. HERJH, R. T., Ktrao, R. T., LESLIE, G. A.. and BENEDICT,A. A. (rg6g), ‘ Molecular weight of chicken, pheasant and auail IgG 1 UI ‘. Immunochem.. 6, 762~$5.
KUBO, R. T., and BENEDICT, A. A. ( 1969). ‘ Comparison of various avian and mammalian IgG for salt induced aggregation ‘. 3. Immun.. I-,
1022-1028.
LESLIE, G. A., and BENEDICT, A. .4. ( rg6gi. ‘ Structural and antigenic relationships between avian immunoglobulins ‘, 3. Zmmun.. 1% I 3561365.
LESLIE, G. A., and CLEM, L. W. (1964). ‘ Phylogeny of immunoglobulin structure and function. III ‘, 3. .5X/J.Med., 130, 1337-1352. SCHEIDEGGER, J. J. (rg55), ‘ Une micro-methode de l’immune electrophortse ‘, Znt. Archs .-I&TQ appl.
Immun., 7, 103-r 10.
TENENHOUSE,H. S., and DEUTSCH,H. F. ( rg66i, ’ Some physical-chemical properties of chicken y-globulinsand theirpepsin and papain digestion products ‘, Immunochem., 3, 1t-20.
Key Word Index: Avian sera, serum proteins. avian pre-albumin, electrophoresis, gel filtration. ultracentrifuge, immuno-electrophoresis, Columba palumbus, Phasianus rhynchos.
Gallus domesticus, Meleagris galloparo, colchicus, Passer domesticus. Anas pla&-