Cetacean hemoglobins: Electrophoretic findings in nine species

Comp. Biochon. Physiol., 1972, Vol. 41B, pp. 647 to 653. Pergamon Press. Printed in Great Britain

CETACEAN HEMOGLOBINS: ELECTROPHORETIC FINDINGS IN NINE SPECIES* M A R Y E L L E N C. B A L U D A , D E B O R A H D U F F I E L D R O B E R T S. S P A R K E S t

K U L U and

Departments of Medicine, Pediatrics and Psychiatry, UCLA School of Medicine, Los Angeles, California 90024 (Received 7 M a y 1971) A b s t r a c t - - 1 . Hemoglobin samples from nine cetacean species (including

dolphins, porpoises and whales) were studied by electrophoresis on cellulose acetate and starch gel; cellulose acetate patterns are shown. 2. Human hemoglobin SA was used as a standard control to describe the relative migration of the cetacean hemoglobins. All hemoglobin samples were quantitated with a densitometer. 3. Each cetacean species demonstrated an essentially constant electrophoretic pattern. Three species have a single hemoglobin band and six have two hemoglobin bands. The possible relationship of these differences to diving habits, maturation, etc. needs further evaluation. INTRODUCTION CONSIDERABLErecent interest has focused on the unique diving abilities and oxygen requirements of cetaceans. However, relatively little work has been reported on the analysis of their hemoglobin, the oxygen-carrying component of the blood. This genetically determined protein is of particular interest because it may be related to their diving abilities and may be used to gain insight into their systematics and evolutionary history. We have, therefore, studied the electrophoretic characteristics of the hemoglobin of twenty-seven animals from nine cetacean species (suborder Odontoceti). MATERIALS AND METHODS Blood samples were collected in heparin from twenty-seven cetaceans, including Inia geoffremis (Amazon fresh-water dolphin), Lagenorhynchus obliquidens (Pacific white-striped porpoise), Tursiops truncatus (Atlantic bottle-nose porpoise), Tursiops gilli (Pacific bottlenose porpoise), Delphinus delphis (Common dolphin), Steno bredanensis (Rough-toothed dolphin), Stenella microps (Spinner porpoise), Orcinus orca (Pacific killer whale) and Globicephala scammoni (Pacific pilot whale). * Supported in part by Grant No. MR-0504A69 from the Division of Mental Retardation, S.R.S., H.E.W. and by the Institute of Child Health and Human Development Grant No. DO-4612, Mental Retardation Research Center, N.P.I., UCLA. t Reprint requests to: Robert S. Sparkes, M.D., Department of Medicine, UCLA School of Medicine, Los Angeles, California 90024. 647

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MARYELLENC. BALUDA,DEBORAHDUFFIELD KULU AND ROBERT S. SPARKES

Hemoglobin electrophoresis was performed using both cellulose acetate and starch gel. Preparation of the hemolysates and conditions for cellulose acetate electrophoresis are described in detail in the method of Sparkes et al. (1969). The red blood cells were washed once by the addition of 11 ml of 0.9% NaC1 to 1 ml of whole blood followed by centrifugation for 20 min at 600 g. The supernatant was discarded and the cells were lysed with a digitonin-buffer solution. The hemolysates were applied to Sepraphore I I I cellulose polyacetate strips (Gelman) and electrophoresed for I hr at 450 V at room temperature using cold EBT buffer (Gelman Hemo), pH 9"1. All hemolysates were run on two strips: one strip had the hemoglobin applied as a single application and this strip was used for quantitation; the second strip was used with a split applicator so that direct comparison of the animal hemoglobin with a known human control could be made on the same strip. Human SA (sickle cell and normal adult) hemoglobin was used as a control Following electrophoresis, the cellulose acetate strips were stained for 10 min with the protein stain, Ponceau S, and rinsed with 5% acetic acid until the background was white. The strips were dried with absolute methanol and cleared using 15 % acetic acid in methanol

(v/v). Quantitation of the hemoglobin bands was performed by the use of a Beckman Analytrol densitometer with a Scanatron (Gelman) attachment to record and integrate the electrophoretic patterns. The cellulose acetate strips with the single application were used for quantitation. Most of the samples were also electrophoresed on starch gel (Boyer, 1964). Electrostarch (Otto Hiller) was used with an EBT buffer, pH 8"6, in a vertical system. The conditions for electrophoresis were 180 V for 18 hr at 4°C. Following electrophoresis the gel was sliced, stained with benzidine (Smith, 1968) and photographed. RESULTS

H u m a n hemoglobin SA (HbSA) was used as a control for the cetacean h e m o globins. I t was necessary to have a consistent control because the animal samples were obtained over a period of several months and could not be compared to one another. Further, the S and A hemoglobins migrate differently and afford two bands of reference. T h e migration of h u m a n hemoglobin from the origin in cellulose acetate system was 4.8 cm for hemoglobin A (HbA) and 4.0 cm for hemoglobin S (HbS). H u m a n fetal hemoglobin ( H b F ) migrates slightly slower than H b A at a position of 4.5 cm from the origin. T h e relative migrations of the animal hemoglobins as compared to the h u m a n SA control can be seen in Figs. 1 and 2. T h e results are summarized in T a b l e 1. T h r e e of the species had hemoglobins that migrated as single bands, although none of the three were alike in position. Inia had the fastest migrating hemoglobin with a position about 3 m m ahead of H b A . Lagenorhynchus migrated as a single band in the position of H b S and T. truncatus was about 3 m m slower than H b A in the position of h u m a n H b F . T w o similar hemoglobin bands were observed for T. gilli, Delphinus, Steno, Stenella, Orcinus and Globicephala. T h e position of the faster migrating h e m o globin band of these animals was in the position of H b F while the slower moving hemoglobin band was in the position of H b S . T h e relative percentages of h e m o globin in the electrophoretic patterns of these animals are shown in T a b l e 1. T. gilli, Delphinus, Steno, Stenella and Orcinus all had similar patterns with the major portion of the hemoglobin in the position of H b S and the minor portion in

Origin

Inia I

Lagenorhynchus

I Delphinus

Human Hb SA

Orcinus

!

Globicephala

FIc. 1. Cellulose acetate strips showing migration of hemoglobin samples of Inia, Lagenorhynchus, Delphinus, Orcinus and Globicephala. The dark bands are the stained hemoglobins. H u m a n SA hemoglobin was used as the control; the HbS band is closer to the origin than is the HbA.

Origin

$

Stenella _

J

Steno

!

T. g i l l i

T. truncatus

Human Hb SA

FIG. 2. Cellulose acetate strips showing migration of hemoglobin samples of

SteneUa, Steno, T. gilli and T. truncatus. Human SA hemoglobin was used as the control.

Mahina Nani Nohea Pikake 155 154 Orky Corky Snorky Patches Small Large 3 H-2 H-1

Pacific bottle-nose dolphin

Common dolphin

Pacific white-striped porpoise

Rough-toothed dolphin Spinner porpoise

Pacific killer whale

Pacific pilot whale

T. gilli

D. delph~ L. obliquidens

S. bredanensis S. microps

O. orca

G. scammoni

Dolphin 70-19-2 70-21-2 70-23

183 Wave Buzz Kaipo Hanauma Noe Noe

Atlantic bottle-nose dolphin

T. truncatus

Stubby Pee Wee

Individual names

Amazon fresh water dolphin

Common name

I. geoffrensis

Formal name

69"6 89"8 72.0 79"7 68"0 83 "6 23"5 26-2 25 "4 26.6 37.5

64"9 65"7 60-4 79"1

77-9 100 100 100

0 0 0 69"0 72"0 69"5

0 0

H b in Position of HbS (%)

30"4 10"2 28"0 20"3 32"0 16 "4 76.5 73"8 74"6 73.4 62.5

35"1 34"3 39"6 20"9

22"1 0 0 0

100 100 100 31.0 28"0 30"5

0 0

Hb in Position of H b F (%)

TABLE 1 - - R E S U L T S OF HEMOGLOBIN ELECTROPHORESIS

0 0 0 0 0 0 0 0 0 0 0

0 0 0 0

0 0 0 0

0 0 0 0 0 0

100 100

H b sl. Faster than H b A (%)

M M M M F F M M M M F

F F M F

F F F F

F F M F M F

M M

Sex

Point Mugu Point Mugu Point Mugu Hawaii Hawaii

Point Mugu Point Mugu Marineland Marineland Marineland Marineland

Hawaii Hawaii Hawaii Hawaii

Point Mugu Marineland Marineland Marineland

Point Mugu Point Mugu Point Mugu Hawaii Hawaii Hawaii

Marineland Marineland

Source

~0

0

0 0

650

MARYELLENC. BALUDA,DEBORAHDUFFIELD KULU AND ROBERT S. SPARKES

the position of HbF. Globicephala differed in that its major hemoglobin band migrated in the position of HbF, while the minor portion was in the position of HbS. After staining with Ponceau S (a protein stain), some animals had one or two very faint bands which could be seen between the hemoglobin and the origin. Because these bands did not show up on the densitometric tracings, they constitute less than 1 per cent of the total hemoglobin. Further, it is uncertain whether they are hemoglobin or some other protein. The hemoglobin patterns in starch gel showed corresponding migration patterns to those on cellulose acetate. D. delphis is the only one of the species whose hemoglobin was not also electrophoresed on starch gel. DISCUSSION The cetaceans have been previously evaluated with regard to their physiological requirement for oxygen in relation to their diving abilities, fast swimming and thermoregulation in cold water (Ridgway & Johnston, 1966; Horvath et al., 1968). Since blood is the means of oxygen transport, routine hematological studies have also been done by many investigators (Medway & Geraci, 1964, 1965 ; Medway & Moldovan, 1966; Ridgway et al., 1970), without finding any outstanding differences among the different cetaceans. However, there have been few reports on the nature of their hemoglobin which is the oxygen-carrying protein of the blood. We have used standard electrophoretic systems as developed for human blood to compare the cetacean hemoglobins with those of human SA hemoglobin. Cellulose acetate electrophoresis requires a relatively short time to perform, is easy to set up and the pattern produced is the result of molecule separation by electrical charges. Cellulose acetate strips can also be easily examined densitometrically to quantitate the relative proportions of hemoglobins in a sample. Because starch gel electrophoresis separates molecules based on their size as well as charge, it may detect molecular differences not seen on the cellulose acetate electrophoresis. However, no differences between the two methods were noted in our hemoglobin studies. The hemoglobins from the animals could not be directly compared at the time of electrophoresis because their blood samples were obtained over a period of several months. Therefore, a known human hemoglobin SA was used as a standard control. Although the relative mobilities of the animal hemoglobins were similar to those of the human control, we do not mean to imply that these are the same hemoglobins. This is merely a temporary means to designate the hemoglobin bands until a standard hemoglobin nomenclature is developed for these animals. Further, similar electrophoretic migration patterns in the different species does not necessarily mean these are identical protein molecules in the different species. The hemoglobin pattern of the Inia showed a single band that migrated slightly faster than human HbA. This animal is the only fresh-water animal in the study. Two animals were studied and the fast moving band was seen on both cellulose acetate and on starch gel.

CETACEAN HEMOOLOBINS

651

T. tru~catus, the Atlantic bottle-nose porpoise, had one hemoglobin band in the position of human HbF. This species has been previously studied electrophoretically by Harkness & Grayson (1969) who found one band with nearly the same mobility as HbA at pH 8-6. Horvath et al. (1968) also studied the hemoglobin of T. trumatu~ but converted the hemoglobin to the carbon monoxide form and electrophoresed it at pH 9.1 on cellulose acetate. They observed one hemoglobin band with practically the same mobility as the human control. Thus, all studies on this species are essentially consistent, since the position of HbA and HbF are very close. The hemoglobin of L. obliquidens migrated as a single band in the position of human HbS in all three of the animals examined. Horvath et al. (1968) also noted that the hemoglobin of the Lagenorhynchus migrated more slowly than HbA using the carbon monoxide form of hemoglobin. One D. delphi*, three S. microps, one S. bredanemis and three T. gilli all showed similar electrophoretic patterns: two hemoglobin bands with the major band migrating in the position of HbS and the minor band migrating faster, approximately in the position of HbF. All of these animals are found in the Pacific Ocean. T. gilli, the Pacific bottle-nose porpoise, had a different pattern than the Atlantic bottle-nose porpoise (T. truncatus), which had a single hemoglobin band. Besides different hemoglobin patterns, these two species vary in size with T. gilli being the larger of the two. The hemoglobin of D. delphi, is the only one of the Pacific animals that had been previously studied. Horvath et al. (1968) converted its hemoglobin to the carbon monoxide form and compared the result to the human. It appeared to have one major hemoglobin band which migrated slower than HbA and two faint bands, one on each side of the major band. No attempt was made at quantitation. De Monte & Pilleri (1968) electrophoresed hemoglobin from D. delphi, on "Cellogel" for a long period of time at a low voltage. They compared the animal findings to human hemoglobin which had a relative mobility of 0.49. The Delphinus had a fast moving band which constituted 18.2 per cent of the hemoglobin with a relative mobility of 0.45, a slower moving band which constituted 67-7 per cent of the hemoglobin with a relative mobility of 0-37 and a trailing band which accounted for 14.1 per cent. This is a pattern similar to that found in our study where 22 per cent of the hemoglobin moved in the position of HbF which corresponds to the 18-2 per cent fast moving band of De Monte & Pilleri. However, no trailing was seen in our cellulose acetate system. The reason for the apparent difference of these findings to those of Horvath et al. (1968) is not evident. Two types of Pacific dwelling whales were studied. O. orca showed a hemoglobin pattern similar to the other Pacific cetaceans which exhibited two bands. The major portion of the hemoglobin migrated in the position of HbS while the minor portion was in the position of HbF. This pattern was seen with all six O. orca. The hemoglobins from G. scammoni migrated in the same positions as O. orca, but the proportions of the fast and slow bands were reversed. The main portion of the hemoglobin of the five Globicephala migrated in the position of HbS.

652

MARYELLENC. BALUDA,DEBORAH DUFFIELD KULU AND ROBERT S. SPARKES

The Orcinu~ and the Globicephala are both found in the Pacific waters; the main difference between the two is that the Orcinus is a much larger animal. The ages of the animals in this study were unknown except for two Orcinus. Both "Patches" and "154" were young animals and had a higher proportion of hemoglobin in the slower migrating position than the other four Orcinus studied. It is interesting to consider whether this difference could be age related with the slower migrating hemoglobin decreasing with maturity perhaps similar to the decrease in HbF with maturity as seen in humans. Medway & Geraci (1964) were the first to publish hematological values on T. truncatus; Medway & Moldovan (1966) followed with the values for Globicephala melaena, the Atlantic pilot whale. Recently, Ridgway et al. (1970) published a large study on several cetaceans which included T. truncatus, L. obliquidens, Phocoenoides dalli, O. orca, G. scammoni and L geoffrensis. From their study it was observed that freshly captured L. obliquidens have increased red blood cell counts, hemoglobin concentrations and blood volumes with a decrease in red blood cell diameter as compared to animals who had been in captivity for a longer time. Furthermore, one captive animal that was trained in deep water had values more similar to the freshly captured animals. Thus, some hematological changes may be related to the stimulation of deep diving in the wild vs. the lack of this in captive animals. Unfortunately, the animals in our study were all kept in captivity for unknown periods of time, so there is no way to correlate changes in the relative proportions of the electrophoretic types to any changes in the physiological states of these animals. It is not certain from our limited studies whether all animals of a species have the same hemoglobin electrophoretic patterns. However, except for possible age related differences in O. orca, all members of a species demonstrated similar patterns. There is no indication that sex significantly affects the hemoglobin types in a species. The finding of two electrophoretic hemoglobin types in several species is particularly intriguing. Although their significance is not known, it is interesting to consider the possibility that these hemoglobins have physiological significance. The possibility that the two hemoglobins may permit the animals to function well both at the water surface as well as at great depths requires consideration. Perhaps evaluation of animals both at the time of capture and later after being in captivity for some time might give some clue, as might physiological evaluation of the oxygen, carbon dioxide and nitrogen binding properties of the red cells or the hemoglobin. The finding of different electrophoretic hemoglobin patterns in the different species may permit the use of the hemoglobin types in taxonomy and possibly in evaluation of their evolutionary relationships, although the latter may require more detailed analysis of the hemoglobins, such as peptide mapping and amino acid sequencing.

Acknowledgements--The animal samples were made available through the kind cooperation of the staff at: Marineland of the Pacific, Palos Verdes Peninsula, California; the Marine Research Facility, U.S. Naval Underseas Warfare Center, Point Mugu, California; and Sea Life Park, Oahu, Hawaii.

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653

REFERENCES BoYEa S. H. (1964) Instructions for Starch Gel Vertical Electrophoresis. Buchler Instruments, Inc., Fort Lee, New Jersey. DE MONTE T. & PILLEaI G. (1968) Haemoglobin of Delphinus delphis L. and plasmaprotein fractions in some species of the family Delphinidae, determined by microelectrophoresis on cellulose acetate gel. Blut, XVlI Seite 25-30... HARKNESSD. R. & GRAYSONV. (1969) Erythrocyte metabolism in the bottle-nosed dolphin, Tursiops truncatus. Comp. Biochem. Physiol. 28, 1289-1301. HORVATH S. M., CHIODI H., RIDGWAYS. H. k AZAR S. (1968) Respiratory and electrophoretic characteristics of hemoglobin of porpoises and sea lion. Comp. Biochem. Physiol. 24, 1027-1033. MEOWAY W. & GERACIJ. R. (1964) Hematology of the bottlenose dolphin (Tursiops truncatus). A m . 3 . Physiol. 207, 1367-1370. MEDWAY W. & GERACI J. R. (1965) Blood chemistry of the bottlenose dolphin (Tursiops truncatus). Am. jT. Physiol. 209, 169-172. MEDWAYW. and MOLDOVANF. (1966) Blood studies on the North Atlantic pilot (pothead) whale, Globicephala melaena. Physiol. Zo~l. 39, 110-116. RIDGWAY S. H. & JOHNSTON D. G. (1966) Blood oxygen and ecology of porpoises of three genera. Science 151, 456-457. RIDGWAY S. H., SIMPSON J. G., PATTON G. S. & GILMARTINW. G. (1970) Hematological findings in certain small Cetaceans..7.A.V.M.A. 157, 566-575. SMITH I. (1968) Chromatographic and Electrophoretic Techniques, Vol. II, p. 233. WileyInterscienee, New York. SPARKESR. S., BALUDAM. C. & TOWNSENDD. E. (1969) Cellulose acetate electrophoresis of human glucose-6-phosphate dehydrogenase..7. Lab. Clin. Med. 73, 531-534.

Key Word Index--Hemoglobin---cetacean; cetacea--Hb ; electrophoresis of Hb ; dolphins--Hb; porpoises--Hb ; whale--Hb; lnia geoffremis ; Lagenorhynchus obliquidens ; Tursiops gilli ; Delphinus delphis ; Steno bredanensis ; Orcinus orca ; Stenella microps ; Globicephala scammoni.