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Electrophoretic analysis of the hemoglobins of four cetacean species
Comp. Biochem. PhysioL, 1975, VoL 51B, pp. 209 to 211. Pergamon Press. Printed in Great Britain
ELECTROPHORETIC ANALYSIS OF THE HEMOGLOBINS OF FOUR CETACEAN SPECIES SUSAN N. BORDER* Department of Biology, Occidental College, Los Angeles, California 90041, U.S.A. (Received 2 May, 1974) Abstract--l. Cellulose acetate electrophoresis was performed on the hemoglobins of four cetacean species: Globicephala scammoni, Lagenorhynchus obliquedens, Orcinus orca and Tursiops truncatus. 2. One species, O. orca, exhibited three hemoglobin bands; the other species, exhibited two bands. 3. Typical electropherograms for these species are presented and comparisons are made with previous studies.
INTRODUCTION THE HEMOGLOnINSof four cetacean species, Globicephala scammoni, Lagenorhynchus obliquedens, Orcinus orca and Tursiops truncatus, were studied by cellulose acetate electrophoresis. Similar studies on some or all of these species have been conducted previously; however, the results were contradictory and the present investigation is an attempt to clarify the interspecific relationships. Data on the comparative percentages of the fractions of each species will also be given. The blood for this investigation was obtained from animals maintained in captivity at Marineland of the Pacific over a 2-month period from 3 January through 21 February 1973. MATERIALS AND METHODS Blood was obtained by venipuncture, drawn into vacuum tubes treated with EDTA and stored in a refrigerator. The hemolysate was prepared by washing the red blood cells three times in physiological saline, lysing them by the addition of 1.5 ml water for each 1.0 ml packed cells and freezing. Removal of the stroma was accomplished by the addition of 0.4 ml chloroform per 1.0 ml cells and centrifuging the mixture. The hemolysate was drawn off and stored under refrigeration until use. The method used for electrophoresis was essentially that of Briere et al. (1965). The procedure differed in that electrophoresis proceeded at 350 V for 40 rain, and the strips, after destaining, were cleared by soaking for two l-rain periods in absolute methanol followed by a 50-sec treatment in 13~oacetic acid in methanol (v/v). They were then air dried on plastic plates. The dried strips were removed, and the optical densities of the hemoglobin bands were recorded by a Densicord Recording Electrophoresis Densitometer. Relative mobilities of the hemoglobins were determined by applying samples from two animals on the same cellulose acetate strip. Compar/sons * Present address: Department of Ecology, University of Minnesota, St. Paul, Minnesota 55108, U.S.A. 0
209
were made of all samples from the same animal, all animals of the same species, all cetacean species and comparing cetacean hemoglobins to human hemoglobin A. RESULTS AND DISCUSSION Typical electropherograms for each species are given in Fig. 1. Intraspecific comparisons gave consistent results in regard to number and mobility of the fractions. There was, however, some variation in the relative densities o f the different fractions within a given species. This is most a p p a r e n t in the case of L. obliquedens. The relative percentages of the fractions are given in Table 1, with the column arrangement corresponding to the relative mobility of the fractions. Figure 2 shows the mobilities of each species' hemoglobin relative to the others and to human hemoglobin A. F r o m Table 1 it is apparent that generally the differences in fraction percentages between animals of a given species is no greater than the variation among different samples from the same animal. The exceptions axe the case previously mentioned and one G. scammoni, MLP72-23, in which the percentage of the smaller fraction doubled in the 43 days between samples. This animal had been captured less than 1 month prior to the first sampling, and the change may reflect an adjustment to life in captivity, The differences in the other cases involving multiple samples from the same animal are small enough to be accounted for by the error in the densitometric tracing and its quantification. This error was determined to vary between 1 and 5 per cent. There have been three papers previously published containing data on electrophoretic mobility of the hemoglobins o f cetaceans to which this paper is pertinent. D e Monti & Pilleri (1968) have published an electropherogram of the hemoglobin of Delphinus delphis showing three fractions. Horvath et al. (1968)
210
Susm~ N. BOmaER Table 1. Relative percentages of hemoglobin bands Species
Animal
Sex
G. scammoni
MLP72-21
F
MLP72-22
M
MLP72-23
F
Date (1973)
Fractions (increasing mobility) -> +
3 January 15 February 3 January 15 February 21 February 3 January 15 February
31 22 20 23 22 13 26 22_+5
69 78 80 77 78 87 74 78+_5
5 February 15 February 21 February
12 8 6 9+2
88 92 94 91-+2
Mean +_S.D. T. truncatus
Ragged fin 4
M
Notch fin 3 Mean_+ S,D.
M
L. obliquedens Smooth fin Sam
O.
orca
? F
Ragged fin
M
Clean fin Mean +_S.D.
F
3 January 3 January 16 February 3 January 16 February 16 February
Corky
F M
3 January 3 January
Orky Mean_+S.D.
27 20 15 24 17 6 21_+4 3 4 4+1
73 80 85 76 83 94 (excluded from calculation) 79+_4 77 81 79-+2
20 15 18+2
Table 2. Summary of hemoglobin fractionation studies Species
Investigators
No. of samples
-
D. delphis
De Monti & Pilled Horvath eta/. Baluda et al.
1 3 1
T. truncatus
Horvath et al. Baluda et al. This study
2 3 3
Horvath et al. Baluda et al. This study
3 3 5
21 +4
(2) 100 79+4
Baluda et al. This study
6 2
4-+ 1
77 + 8 79 -+2
23+8 18_+2
G. scammoni
Baluda et al. This study
5 7
28 + 6 22 + 5
72_+6 78_+5
Human hemoglobin A (I and 2)
All studies
L. obliquedens
O. Orca
14-1 (3)
Fractions and relative percentages (increasing mobility) + 67.7 (1) 77.9
(1)
18.2 (2) 22.1 (2) ?
(1)
9+ 2
100 91+2
100
Numbers in parentheses refer to relative amounts when actual percentage amounts are not available. (I) refers to the major fraction, (2) to the secondary, etc.
Hemoglobin
Species 4Globicephalo
L agenorhynchus
scommoni
obliquedens
Orc/'nu5 orca
Tursiops trunool-us
Human Hemoglobin ,6.
Fig. 2. Electrophoretic patterns of four cetacean species and human hemoglobin A.
ELECTROPHORETIC ANALYSIS OF THE HEMOGLOBINS OF FOUR CETACEAN SPECIES SUSAN N. BORDER* Department of Biology, Occidental College, Los Angeles, California 90041, U.S.A. (Received 2 May, 1974) Abstract--l. Cellulose acetate electrophoresis was performed on the hemoglobins of four cetacean species: Globicephala scammoni, Lagenorhynchus obliquedens, Orcinus orca and Tursiops truncatus. 2. One species, O. orca, exhibited three hemoglobin bands; the other species, exhibited two bands. 3. Typical electropherograms for these species are presented and comparisons are made with previous studies.
INTRODUCTION THE HEMOGLOnINSof four cetacean species, Globicephala scammoni, Lagenorhynchus obliquedens, Orcinus orca and Tursiops truncatus, were studied by cellulose acetate electrophoresis. Similar studies on some or all of these species have been conducted previously; however, the results were contradictory and the present investigation is an attempt to clarify the interspecific relationships. Data on the comparative percentages of the fractions of each species will also be given. The blood for this investigation was obtained from animals maintained in captivity at Marineland of the Pacific over a 2-month period from 3 January through 21 February 1973. MATERIALS AND METHODS Blood was obtained by venipuncture, drawn into vacuum tubes treated with EDTA and stored in a refrigerator. The hemolysate was prepared by washing the red blood cells three times in physiological saline, lysing them by the addition of 1.5 ml water for each 1.0 ml packed cells and freezing. Removal of the stroma was accomplished by the addition of 0.4 ml chloroform per 1.0 ml cells and centrifuging the mixture. The hemolysate was drawn off and stored under refrigeration until use. The method used for electrophoresis was essentially that of Briere et al. (1965). The procedure differed in that electrophoresis proceeded at 350 V for 40 rain, and the strips, after destaining, were cleared by soaking for two l-rain periods in absolute methanol followed by a 50-sec treatment in 13~oacetic acid in methanol (v/v). They were then air dried on plastic plates. The dried strips were removed, and the optical densities of the hemoglobin bands were recorded by a Densicord Recording Electrophoresis Densitometer. Relative mobilities of the hemoglobins were determined by applying samples from two animals on the same cellulose acetate strip. Compar/sons * Present address: Department of Ecology, University of Minnesota, St. Paul, Minnesota 55108, U.S.A. 0
209
were made of all samples from the same animal, all animals of the same species, all cetacean species and comparing cetacean hemoglobins to human hemoglobin A. RESULTS AND DISCUSSION Typical electropherograms for each species are given in Fig. 1. Intraspecific comparisons gave consistent results in regard to number and mobility of the fractions. There was, however, some variation in the relative densities o f the different fractions within a given species. This is most a p p a r e n t in the case of L. obliquedens. The relative percentages of the fractions are given in Table 1, with the column arrangement corresponding to the relative mobility of the fractions. Figure 2 shows the mobilities of each species' hemoglobin relative to the others and to human hemoglobin A. F r o m Table 1 it is apparent that generally the differences in fraction percentages between animals of a given species is no greater than the variation among different samples from the same animal. The exceptions axe the case previously mentioned and one G. scammoni, MLP72-23, in which the percentage of the smaller fraction doubled in the 43 days between samples. This animal had been captured less than 1 month prior to the first sampling, and the change may reflect an adjustment to life in captivity, The differences in the other cases involving multiple samples from the same animal are small enough to be accounted for by the error in the densitometric tracing and its quantification. This error was determined to vary between 1 and 5 per cent. There have been three papers previously published containing data on electrophoretic mobility of the hemoglobins o f cetaceans to which this paper is pertinent. D e Monti & Pilleri (1968) have published an electropherogram of the hemoglobin of Delphinus delphis showing three fractions. Horvath et al. (1968)
210
Susm~ N. BOmaER Table 1. Relative percentages of hemoglobin bands Species
Animal
Sex
G. scammoni
MLP72-21
F
MLP72-22
M
MLP72-23
F
Date (1973)
Fractions (increasing mobility) -> +
3 January 15 February 3 January 15 February 21 February 3 January 15 February
31 22 20 23 22 13 26 22_+5
69 78 80 77 78 87 74 78+_5
5 February 15 February 21 February
12 8 6 9+2
88 92 94 91-+2
Mean +_S.D. T. truncatus
Ragged fin 4
M
Notch fin 3 Mean_+ S,D.
M
L. obliquedens Smooth fin Sam
O.
orca
? F
Ragged fin
M
Clean fin Mean +_S.D.
F
3 January 3 January 16 February 3 January 16 February 16 February
Corky
F M
3 January 3 January
Orky Mean_+S.D.
27 20 15 24 17 6 21_+4 3 4 4+1
73 80 85 76 83 94 (excluded from calculation) 79+_4 77 81 79-+2
20 15 18+2
Table 2. Summary of hemoglobin fractionation studies Species
Investigators
No. of samples
-
D. delphis
De Monti & Pilled Horvath eta/. Baluda et al.
1 3 1
T. truncatus
Horvath et al. Baluda et al. This study
2 3 3
Horvath et al. Baluda et al. This study
3 3 5
21 +4
(2) 100 79+4
Baluda et al. This study
6 2
4-+ 1
77 + 8 79 -+2
23+8 18_+2
G. scammoni
Baluda et al. This study
5 7
28 + 6 22 + 5
72_+6 78_+5
Human hemoglobin A (I and 2)
All studies
L. obliquedens
O. Orca
14-1 (3)
Fractions and relative percentages (increasing mobility) + 67.7 (1) 77.9
(1)
18.2 (2) 22.1 (2) ?
(1)
9+ 2
100 91+2
100
Numbers in parentheses refer to relative amounts when actual percentage amounts are not available. (I) refers to the major fraction, (2) to the secondary, etc.
Hemoglobin
Species 4Globicephalo
L agenorhynchus
scommoni
obliquedens
Orc/'nu5 orca
Tursiops trunool-us
Human Hemoglobin ,6.
Fig. 2. Electrophoretic patterns of four cetacean species and human hemoglobin A.