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Electrophoretic and immunologic study of the hemoglobins of the Greek island populations of the lizard Agama stellio
Comp. Biochem. Physiol. Vol. 68B. pp. 359 to 360
0305-0491/81/0201-0359102.00/0
© Pergamon Press Ltd 1981. Printed in Great Britain
ELECTROPHORETIC A N D I M M U N O L O G I C STUDY OF THE HEMOGLOBINS OF THE GREEK ISLAND POPULATIONS OF THE LIZARD AGAMA STELLIO ANGELA XYDA and A. A. HARITOS Zoological Laboratory and Museum, University of Athens, Panepistimiopolis, Athens 621, Greece
(Received 13 March 1980) Abstract--l. Polyacrylamide gel electrophoresis of 98 hemolysates of the lizard Agama stellio from 10
Greek islands, revealed a constant electrophoretic pattern consisting of two slow hemoglobin bands. 2. Aging of hemolysates produced a diffuse electrophoretic profile that was restored to normal by reduction and alkylation. 3. Rabbit antisera against Agama stellio hemolysates from three islands gave a continuous precipitin line in Ouchterlony plates with hemolysates from several islands.
INTRODUCTION
trophoretic run was that described by Maurer (1971). Samples were reduced and alkylated by incubation for 10min with fl-mercaptoethanol and subsequently for 10 min with iodoacetamide at final concentrations of 3~o (v/v) and 4~ (w/v) respectively. Gels were stained for hemoglobin with the benzidine stain (Sunderman, 1964). Rabbit antisera were prepared against hemolysates of Agama stellio by the following procedure. Equal volumes of hemolysate and Freund's conplete adjuvant (Difco) were emulsified and 1 ml of this emulsion was distributed subcutaneously into the neck region. The same injection was given 15 days later in the foot-pads. Four anamnestic injections with 1 ml of hemolysate were given on the 34, 38, 42 and 46th day after the primary immunization. The rabbits were bled 1 week after the last injection. Antisera were kept -25°C. Immunodiffusion was carried out in 0.85?/0 Ionoagar No 2 (Difco) prepared in borate-saline (Campbell et al., 1970). Immunoprecipitation arcs were stained with 0.2~o acid fuchsin solution in methyl alcohol, water, acetic acid (5/4/1, v/v/v).
Electrophoresis of hemolysates from various lizard species has shown the existence of one to three hemoglobin bands (Guttman 1970a,b 1971 ; Sullivan, 1974). Most species show a constant electrophoretic profile for hemoglobin, although the number of individuals tested for each species was usually limited. Hemoglobin polymorphism as revealed by electrophoresis has been reported for several lizards (Dessauer & Fox, 1964; Guttman, 1970a, 1971; Webster et al., 1972), as well as for Agama stellio from Israel (Gorman & Shochat, 1972). Aging of hemolysates from reptiles has been reported to lead possibly by polymerization to diffuse electrophoretic patterns that could be reversed to normal by reduction and alkylation (Sullivan & Riggs, 1967; Reischl & Diefenbach, 1976). However in vitro polymerization occurs also in other vertebrate groups. Agama stellio in Greece is present mainly in the islands of the Aegean Sea, with only one subspecies (Agama s. stellio) (Daan, 1967; Ondrias, 1968). The present investigation was intended to initiate the study of hemoglobins of the Greek lizards as well as to identify any effect of the geographic isolation on the electrophoretic and antigenic properties of Agama stellio hemoglobins.
RESULTS AND DISCUSSION
MATERIALSAND METHODS Blood was obtained by cardiac puncture and heparin was added as anticoagulant. The erythrocytes were collected by centrifugation at 3000 rpm in a bench centrifuge and washed three times with 3 vol of 0.85~o saline. Packed red cells were lysed with three volumes of cold distilled water. The hemolysate was centrifuged at 27,000g for 20 min at 4°C in a RC2-B Sorvall preparative centrifuge and the supernatant was immediately electrophoresed or stored at - 2 5 ° C Polyacrylamide gel electrophoresis was performed using a 4~ spacer and 7.5~o separation gel prepared with Cyanogum 41 (B.D.H.), in Tris-HCl buffer, pH 8.9. Gels were formed in 7.5 x 0.5 cm glass tubes. Electrode chambers were filled with Tris-glycine buffer, pH 8.3. Each gel was loaded with 52 of hemolysate mixed with 252 of sucrose solution 40~o. The procedure for gel preparation and elec359
The electrophoretic patterns of 98 hemolysates of Agama stellio (50d', 48~) from the islands of Mikonos (20), Dilos (6), Naxos (10), Paros (3), Chios (24), Leros (2), Kalimnos (8), Kos (16), Rodos (5) and Kastellorizo (4) were examined. With fresh hemolysates two bands were stained for hemoglobin, the faster being broader (Fig. 1). No difference in the relative mobilities of the two bands or in their relative staining intensity was observed between individuals of the same island or of different islands. G o r m a n & Shochat (1972) reported variation of the electrophoretic pattern of hemoglobin of Agama stellio collected from Israel, consisting of one, two or three major bands. This variability was not found to be geographically limited. However information about the number of individuals tested, locations of collection and subspecies found was not given. Daan (1967) reported the presence of Agama s. stellio and Agama s. brachydactyla in Iarael while only the former subspecies was found in the Greek islands (Ondrias, 1968). Immunodiffusion experiments offered a further evidence for the identity of hemoglobins of Agama stellio island populations. Seven pools of hemolysates were
360
ANGELA XYDA and A. A. HARITOS m
m
may simply imply the deterioration of the reducing environment of the hemolysate upon aging. Obviously variability of hemoglobins of Agama stellio Greek island populations that can be revealed by other techniques or by a considerably greater sample of animals, can not be ruled out. This said, the observed constancy of hemoglobins in the present study may be explained by strong selection pressures maintaining hemoglobins within narrow limits of specificity or/and by the geologically recent subdivision of the Aegean area into islands i.e. after the end of Miocene (Sontaar & Boekschoten, 1967).
im
m
Fig. 1. Polyacrylamide gel electro ~horesis of Agama stellio hemoglobins. Electrophoresis was completed when the bromophenol blue marker reached the lower end of the tubes. From left to right: Aged hemolysate from Naxos; the same aged sample after reduction and alkylation ; fresh hemolysate from Chios; the same fresh sample after reduction and alkylation. made, each from the lizards collected from each of the following islands: Naxos, Paros, Chios, Kalimnos, Kos, Rodos and Kastellorizo. Rabbit antisera prepared against hemolysate pools from the islands of Paros, Chios and Kos gave continuous immunoprecipitation lines with hemolysates from the other islands (Fig. 2). Samples stored at 4°C or at - 2 5 ° C even for a few days showed very diffuse bands of hemoglobin. However the normal electrophoretic pattern was resumed if the samples were reduced and alkylated (Fig. 1). These results indicate disulphide formation and disulphide-exchange between hemoglobin molecules, which are then in an association equilibrium. The sieving effect of the gel tends to spread the components of this equilibrium while fl-mercaptoethanol and iodoacetamide by reduction and alkylation respectively prevent disulphide formation and lead to discrete bands. Mercaptoethanol and iodoacetamide had no effect on the electrophoretic profile of fresh hemolysates (Fig. I). Similar phenomena have been reported for hemoglobins of other reptilian species (Riggs et al., 1964; Sullivan & Riggs, 1964, 1967; Reischl & Diefenbach, 1976; Horton et al., 1972), and
0
0
0
O
odDo o Q o 0
0
0
0
Fig. 2. Ouchterlony plates showing the reaction between rabbit anti- A. stellio hemoglobins sera and hemoglobins of Agama stellio from several islands. Antisera were contained in central wells and hemolysates in the peripheral wells. Left plate: Central well; anti-Chios serum. Peripheral wells from "3 o'clock" and clockwise; Kastellorizo, Kos, Rodos, Kalimnos, Naxos, hemolysate from the turtle Testudo hermanni. Middle plate: Central well; anti-Paros serum. Peripheral wells from "12 o'clock"; Kastellorizo, Paros, Chios, Kos. Right plate: Central well; anti-Kos serum. Peripheral wells as in middle plate.
REFERENCES CAMPBELL D. H., GARVEY J. S., CREMER N. E. & SUSSDORF D. H. In Methods in Immunology. A Laboratory Text for Instruction and Research pp. 25(~260. Benjamin, New
York. DAAN S. (1967) Variation and taxonomy of the Hardun, Agama stellio (Linnaeus, 1758) (Reptilia, Agamidae). Beaufortia 14, 109-134. DESSAUER H. G. & Fox W. (1964): In Taxonomic Biochemistry and Serology (Edited by LEONE C. A.) pp. 625 647. Ronald Press, New York. GORMAN G. C. & SHOCHATD. (1972) A taxonomic interpretation of chromosomal and electrophoretic data on the Agamid lizards of Israel with notes on some East African species. Herpetologica 28, 106-112. GUTTMANS. I. (1970a). Hemoglobin electrophoresis and relationships within the lizard genus Sceloporus (Sauria: Iguanidae). Comp. Biochem. Physiol. 34, 563 568. GUTTMAN S. |. (1970b) An electrophoretic study of the hemoglobins of the sand lizards, Callisaurus, Holbrookia and Uma. Comp. Biochem. Physiol. 34, 569-574. GUTTMAN S. I. (1971) Analysis of hemoglobins of old and new world lizards. J. Herpet. 5, 11 16. HORTON B., FRASER R., DUPOURQUE D., BAILEY D. t~:
CHERNOFFA. (1972) An analysis of the hemoglobins from some common turtles. J. exp. Zool. 180, 373-384. MAURER R. H. In Disc Electrophoresis and Related Techniques of Polyacrylamide Gel Electrophoresis 2nd edn. pp. 46-47. Walter de Gruyter, Berlin. ONDRIASJ. C. (1968) Liste des amphibiens et de reptiles de la Gr6ce. Biol. Gallo-Hellenica l, 111-135. REISCHL E. & DIEFENBACHC. O. DA C. (1976) Heterogeneity and polymerization of hemoglobins of Caiman latirostris (Crocodylia: Reptilia). Comp. Biochem Physiol. 54B, 543 545. RlGGS A. B., SULLIVAN B. • AGEE J. R. (1964) Polymerization of frog and turtle hemoglobins. Proc. natn. Acad. Sci. U.S.A. 51, 1127-1134. SONTAAR P. Y. t~ BOEKSCHOTEN G. J. (1967) Quaternary
mammals in the south Aegean island arc; with notes on other fossil mammals from coastal regions of the Mediterranean (l). Konikle, N ederl. Akad. Van Wetenschappen, Amsterdam Set. B, 70, 556 564. SULLIVAN B. (1974) Reptilian hemoglobins. In Chemical Zoology (Edited by FLORKIN M. t~ SCHEER n.). Vol 9, Ch. 14, Academic Press, New York. SULLIVANB. & RIGGS A. (1964) Hemoglobin: Reversal of oxidation and polymerization in turtle red cells. Nature, Lond. 204, 1098 1099. SULLIVAN B. & RIGGS A. (1967) Structure, function and evolution of turtle hemoglobins 1. Distribution of heavy hemoglobins. Comp. Biochem. Physiol. 23, 437-447. SUNDERMANW. F. JR (1964) Procedure for electrophoretic fractionation of hemoglobins in starch gel, with quantitation of hemoglobin A2. In Hemoglobin (Edited by SUNDERMANW. F. & SUNDERMANW. F. JR) p. 104. Lippincott, Philadelphia. WEBSTERT. P., SELANDERR. K. & YANG S. Y. (1972). Genetic variability and similarity in the Anolis lizards of Bimini. Evolution 26, 523 535.
0305-0491/81/0201-0359102.00/0
© Pergamon Press Ltd 1981. Printed in Great Britain
ELECTROPHORETIC A N D I M M U N O L O G I C STUDY OF THE HEMOGLOBINS OF THE GREEK ISLAND POPULATIONS OF THE LIZARD AGAMA STELLIO ANGELA XYDA and A. A. HARITOS Zoological Laboratory and Museum, University of Athens, Panepistimiopolis, Athens 621, Greece
(Received 13 March 1980) Abstract--l. Polyacrylamide gel electrophoresis of 98 hemolysates of the lizard Agama stellio from 10
Greek islands, revealed a constant electrophoretic pattern consisting of two slow hemoglobin bands. 2. Aging of hemolysates produced a diffuse electrophoretic profile that was restored to normal by reduction and alkylation. 3. Rabbit antisera against Agama stellio hemolysates from three islands gave a continuous precipitin line in Ouchterlony plates with hemolysates from several islands.
INTRODUCTION
trophoretic run was that described by Maurer (1971). Samples were reduced and alkylated by incubation for 10min with fl-mercaptoethanol and subsequently for 10 min with iodoacetamide at final concentrations of 3~o (v/v) and 4~ (w/v) respectively. Gels were stained for hemoglobin with the benzidine stain (Sunderman, 1964). Rabbit antisera were prepared against hemolysates of Agama stellio by the following procedure. Equal volumes of hemolysate and Freund's conplete adjuvant (Difco) were emulsified and 1 ml of this emulsion was distributed subcutaneously into the neck region. The same injection was given 15 days later in the foot-pads. Four anamnestic injections with 1 ml of hemolysate were given on the 34, 38, 42 and 46th day after the primary immunization. The rabbits were bled 1 week after the last injection. Antisera were kept -25°C. Immunodiffusion was carried out in 0.85?/0 Ionoagar No 2 (Difco) prepared in borate-saline (Campbell et al., 1970). Immunoprecipitation arcs were stained with 0.2~o acid fuchsin solution in methyl alcohol, water, acetic acid (5/4/1, v/v/v).
Electrophoresis of hemolysates from various lizard species has shown the existence of one to three hemoglobin bands (Guttman 1970a,b 1971 ; Sullivan, 1974). Most species show a constant electrophoretic profile for hemoglobin, although the number of individuals tested for each species was usually limited. Hemoglobin polymorphism as revealed by electrophoresis has been reported for several lizards (Dessauer & Fox, 1964; Guttman, 1970a, 1971; Webster et al., 1972), as well as for Agama stellio from Israel (Gorman & Shochat, 1972). Aging of hemolysates from reptiles has been reported to lead possibly by polymerization to diffuse electrophoretic patterns that could be reversed to normal by reduction and alkylation (Sullivan & Riggs, 1967; Reischl & Diefenbach, 1976). However in vitro polymerization occurs also in other vertebrate groups. Agama stellio in Greece is present mainly in the islands of the Aegean Sea, with only one subspecies (Agama s. stellio) (Daan, 1967; Ondrias, 1968). The present investigation was intended to initiate the study of hemoglobins of the Greek lizards as well as to identify any effect of the geographic isolation on the electrophoretic and antigenic properties of Agama stellio hemoglobins.
RESULTS AND DISCUSSION
MATERIALSAND METHODS Blood was obtained by cardiac puncture and heparin was added as anticoagulant. The erythrocytes were collected by centrifugation at 3000 rpm in a bench centrifuge and washed three times with 3 vol of 0.85~o saline. Packed red cells were lysed with three volumes of cold distilled water. The hemolysate was centrifuged at 27,000g for 20 min at 4°C in a RC2-B Sorvall preparative centrifuge and the supernatant was immediately electrophoresed or stored at - 2 5 ° C Polyacrylamide gel electrophoresis was performed using a 4~ spacer and 7.5~o separation gel prepared with Cyanogum 41 (B.D.H.), in Tris-HCl buffer, pH 8.9. Gels were formed in 7.5 x 0.5 cm glass tubes. Electrode chambers were filled with Tris-glycine buffer, pH 8.3. Each gel was loaded with 52 of hemolysate mixed with 252 of sucrose solution 40~o. The procedure for gel preparation and elec359
The electrophoretic patterns of 98 hemolysates of Agama stellio (50d', 48~) from the islands of Mikonos (20), Dilos (6), Naxos (10), Paros (3), Chios (24), Leros (2), Kalimnos (8), Kos (16), Rodos (5) and Kastellorizo (4) were examined. With fresh hemolysates two bands were stained for hemoglobin, the faster being broader (Fig. 1). No difference in the relative mobilities of the two bands or in their relative staining intensity was observed between individuals of the same island or of different islands. G o r m a n & Shochat (1972) reported variation of the electrophoretic pattern of hemoglobin of Agama stellio collected from Israel, consisting of one, two or three major bands. This variability was not found to be geographically limited. However information about the number of individuals tested, locations of collection and subspecies found was not given. Daan (1967) reported the presence of Agama s. stellio and Agama s. brachydactyla in Iarael while only the former subspecies was found in the Greek islands (Ondrias, 1968). Immunodiffusion experiments offered a further evidence for the identity of hemoglobins of Agama stellio island populations. Seven pools of hemolysates were
360
ANGELA XYDA and A. A. HARITOS m
m
may simply imply the deterioration of the reducing environment of the hemolysate upon aging. Obviously variability of hemoglobins of Agama stellio Greek island populations that can be revealed by other techniques or by a considerably greater sample of animals, can not be ruled out. This said, the observed constancy of hemoglobins in the present study may be explained by strong selection pressures maintaining hemoglobins within narrow limits of specificity or/and by the geologically recent subdivision of the Aegean area into islands i.e. after the end of Miocene (Sontaar & Boekschoten, 1967).
im
m
Fig. 1. Polyacrylamide gel electro ~horesis of Agama stellio hemoglobins. Electrophoresis was completed when the bromophenol blue marker reached the lower end of the tubes. From left to right: Aged hemolysate from Naxos; the same aged sample after reduction and alkylation ; fresh hemolysate from Chios; the same fresh sample after reduction and alkylation. made, each from the lizards collected from each of the following islands: Naxos, Paros, Chios, Kalimnos, Kos, Rodos and Kastellorizo. Rabbit antisera prepared against hemolysate pools from the islands of Paros, Chios and Kos gave continuous immunoprecipitation lines with hemolysates from the other islands (Fig. 2). Samples stored at 4°C or at - 2 5 ° C even for a few days showed very diffuse bands of hemoglobin. However the normal electrophoretic pattern was resumed if the samples were reduced and alkylated (Fig. 1). These results indicate disulphide formation and disulphide-exchange between hemoglobin molecules, which are then in an association equilibrium. The sieving effect of the gel tends to spread the components of this equilibrium while fl-mercaptoethanol and iodoacetamide by reduction and alkylation respectively prevent disulphide formation and lead to discrete bands. Mercaptoethanol and iodoacetamide had no effect on the electrophoretic profile of fresh hemolysates (Fig. I). Similar phenomena have been reported for hemoglobins of other reptilian species (Riggs et al., 1964; Sullivan & Riggs, 1964, 1967; Reischl & Diefenbach, 1976; Horton et al., 1972), and
0
0
0
O
odDo o Q o 0
0
0
0
Fig. 2. Ouchterlony plates showing the reaction between rabbit anti- A. stellio hemoglobins sera and hemoglobins of Agama stellio from several islands. Antisera were contained in central wells and hemolysates in the peripheral wells. Left plate: Central well; anti-Chios serum. Peripheral wells from "3 o'clock" and clockwise; Kastellorizo, Kos, Rodos, Kalimnos, Naxos, hemolysate from the turtle Testudo hermanni. Middle plate: Central well; anti-Paros serum. Peripheral wells from "12 o'clock"; Kastellorizo, Paros, Chios, Kos. Right plate: Central well; anti-Kos serum. Peripheral wells as in middle plate.
REFERENCES CAMPBELL D. H., GARVEY J. S., CREMER N. E. & SUSSDORF D. H. In Methods in Immunology. A Laboratory Text for Instruction and Research pp. 25(~260. Benjamin, New
York. DAAN S. (1967) Variation and taxonomy of the Hardun, Agama stellio (Linnaeus, 1758) (Reptilia, Agamidae). Beaufortia 14, 109-134. DESSAUER H. G. & Fox W. (1964): In Taxonomic Biochemistry and Serology (Edited by LEONE C. A.) pp. 625 647. Ronald Press, New York. GORMAN G. C. & SHOCHATD. (1972) A taxonomic interpretation of chromosomal and electrophoretic data on the Agamid lizards of Israel with notes on some East African species. Herpetologica 28, 106-112. GUTTMANS. I. (1970a). Hemoglobin electrophoresis and relationships within the lizard genus Sceloporus (Sauria: Iguanidae). Comp. Biochem. Physiol. 34, 563 568. GUTTMAN S. |. (1970b) An electrophoretic study of the hemoglobins of the sand lizards, Callisaurus, Holbrookia and Uma. Comp. Biochem. Physiol. 34, 569-574. GUTTMAN S. I. (1971) Analysis of hemoglobins of old and new world lizards. J. Herpet. 5, 11 16. HORTON B., FRASER R., DUPOURQUE D., BAILEY D. t~:
CHERNOFFA. (1972) An analysis of the hemoglobins from some common turtles. J. exp. Zool. 180, 373-384. MAURER R. H. In Disc Electrophoresis and Related Techniques of Polyacrylamide Gel Electrophoresis 2nd edn. pp. 46-47. Walter de Gruyter, Berlin. ONDRIASJ. C. (1968) Liste des amphibiens et de reptiles de la Gr6ce. Biol. Gallo-Hellenica l, 111-135. REISCHL E. & DIEFENBACHC. O. DA C. (1976) Heterogeneity and polymerization of hemoglobins of Caiman latirostris (Crocodylia: Reptilia). Comp. Biochem Physiol. 54B, 543 545. RlGGS A. B., SULLIVAN B. • AGEE J. R. (1964) Polymerization of frog and turtle hemoglobins. Proc. natn. Acad. Sci. U.S.A. 51, 1127-1134. SONTAAR P. Y. t~ BOEKSCHOTEN G. J. (1967) Quaternary
mammals in the south Aegean island arc; with notes on other fossil mammals from coastal regions of the Mediterranean (l). Konikle, N ederl. Akad. Van Wetenschappen, Amsterdam Set. B, 70, 556 564. SULLIVAN B. (1974) Reptilian hemoglobins. In Chemical Zoology (Edited by FLORKIN M. t~ SCHEER n.). Vol 9, Ch. 14, Academic Press, New York. SULLIVANB. & RIGGS A. (1964) Hemoglobin: Reversal of oxidation and polymerization in turtle red cells. Nature, Lond. 204, 1098 1099. SULLIVAN B. & RIGGS A. (1967) Structure, function and evolution of turtle hemoglobins 1. Distribution of heavy hemoglobins. Comp. Biochem. Physiol. 23, 437-447. SUNDERMANW. F. JR (1964) Procedure for electrophoretic fractionation of hemoglobins in starch gel, with quantitation of hemoglobin A2. In Hemoglobin (Edited by SUNDERMANW. F. & SUNDERMANW. F. JR) p. 104. Lippincott, Philadelphia. WEBSTERT. P., SELANDERR. K. & YANG S. Y. (1972). Genetic variability and similarity in the Anolis lizards of Bimini. Evolution 26, 523 535.