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Comparative studies on avian hemoglobins
Comp. Biochem. Physiol., 1965, Vol. 15, pp. 217 to 235. Pergamon Press Ltd. Printed in Great Britain
COMPARATIVE STUDIES ON AVIAN HEMOGLOBINS* A N I L S A H A t and J H A R N A G H O S H Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California, U.S.A. and Depa, i~ent of Applied Chemistry, University College of Science and Technology, Calcutta, India (Received 4 December 1964.; in revised form 8 ffanuary 1965) A b s t r a c t - - 1 . Studies o n
paper electrophoretic mobility of hemoglobins obtained from avian species representing thirteen natural orders show that avian erythrocytes possess one, two or three hemoglobins. 2. The avian hemoglobins may be categorized into five groups depending on the degree of mobility towards anode. Although the ratio of hemoglobin-1 to other hemoglobins varies considerably, hemoglobin-1 was found to be the major component. 3. The avian hemoglobins are relatively resistant to alkaline denaturation and the resistance characteristics depend on individual species. 4. Duck, chick, pigeon and dove show a comparable amino acid composition in hemoglobin-1 whereas a widely divergent amino acid composition has been found in hemoglobin-2 of duck and of chick. 5, Wide differences in amino acid composition of a~- and fl~-chains of duck, chick and pigeon have been observed. INTRODUCTION THE presence of multiple hemoglobins in birds like chick (Saha, 1956a; Saha et al., 1957), duck and guinea fowl was revealed by paper electrophoretic investigation of avian erythrocyte lysates. A comparative study showed that one of the avian hemoglobin components, hemoglobin-2, possessed paper electrophoretic mobility identical with one of the abnormal human hemoglobins, H b - E (Saha et al., 1957). Further investigation of the behavior of avian hemoglobins by employing paper electrophoresis revealed the presence of one, two or three hemoglobins in birds representing different avian species (Dutta et al., 1958). Attempts were made during the present study to seek certain relationships between the physico-chemical characteristics of hemoglobins obtained from different avian families and natural orders and the position of the avian natural orders in the evolutionary scale. Studies were thus undertaken to determine the number of hemoglobin components present in avian species and to evaluate the percentage distribution of such hemoglobin components. The resistance to alkaline denaturation was also determined to characterize the hemoglobins. Amino acid compositions of some avian hemoglobins and their sub-units were also reported to establish the inherent differences of the hemoglobins. * Contribution No. 3155. t Present address: McGill University, Royal Victoria Hospital, Montreal, Canada. 217
218
ANIL SAHA AND JHARNA GHOSH
EXPERIMENTAL Materials and methods Birds were captured alive in the region around Calcutta, India and brought to the laboratory without any delay. Erythrocytes were collected in heparinized isotonic saline at 5°C by cutting the jugular vein of the bird. Avian erythrocytes were washed by suspending four times with ten times their volume of isotonic saline at 5°C and spinning down each time in a refrigerated centrifuge at 600 g for 15 min. Packed cell volume was determined by centrifuging the erythrocytes at 1000 g for 15 rain at 5°C. The packed avian erythrocytes were then lysed with an equal volume of water and 0.4 volume of toluene, and kept overnight at 2°C. The suspension was centrifuged at 10,000 g for 15 rain at 5°C; clear hemoglobin solution was withdrawn with a syringe and centrifuged again at 10,000g for 15 rain. The hemoglobin solutions were stored in small vials at - 15°C and thawed prior to the electrophoretic runs. The hemoglobin solutions were diluted with the appropriate buffer to a solution (5 g Hb/100 ml) prior to the paper electrophoretic runs.
Paper electrophoresis Paper electrophoresis of avian hemoglobins was conducted in barbiturate buffer of pH 8"6 and ionic strength 0.05 for 16 hr at 220 V. Paper electrophoresis was carried out in a horizontal type apparatus with limited evaporation. The paper electrophoretic apparatus was kept in a large chamber maintained at 4°C and the entire electrophoretic procedure was carried out inside the cold chamber. The paper electropherograms, ~rhatman 3 mm paper, 18 x 46 cm, were removed immediately after the experiment, suspended by clamps in a horizontal position and dried quickly under infrared lamps. The relative mobility of a test avian hemoglobin was evaluated by applying chick hemoglobins and other hemoglobins possessing different mobilities as categorized under different groups (Table 1) on the same paper electropherogram and the electrophoretic run was conducted under identical experimental conditions. The relative paper electrophoretic mobilities were calculated on the basis of the mobility of a particular hemoglobin component towards the anode under the following conditions: barbiturate buffer of pH 8.6 and ionic strength 0.05, 220 V for 16 hr at 4°C. Hemoglobin-1 showed the slowest electrophoretic mobility toward anode while hemoglobin-3 showed the fastest mobility. The hemoglobin component categorized under Group V moved farthest toward the anode, while the one under Group I remained nearest to the cathode (Table 1). The paper electropherograms were scanned photometrically at 540 m/z with a Photovolt densitometer model 525. Alkaline denaturation Alkaline denaturation of hemoglobins was studied with the cell-free lysates of avian erythrocytes. Denaturation reaction was carried out with unfractionated hemoglobin solution according to the method of Singer et aL (1951). The alkaline denaturation was carried out at 20°C. After the completion of denaturation, the
Phalacrocoracidae (Eo) Accipitridae (Eo-Oligo) Ardeidae (Eo) Anatidae (Eo) Anatidae (Eo) Phasiandidae (Eo) Phasiandidae (Eo) Phasiandidae (Eo) Phasiandidae (Eo) Cuculidae (Eo-Oligo) Cuculidae (Eo-Oligo) Cuculidae (Eo-Oligo)
Pelecaniformes
Cuculiformes
Cuculiformes
Cuculiformes
Galliformes
Galliformes
Galliformes
Galliformes
Anseriformes
Anseriformes
Ciconiiformes
Falconiformes
Family
Natural order
.4ythya retina (Linn) (Plio) Numida meleagris (Linn) (Pleisto) Gallus gaihis (Plio) Coturnix coturnis (Linn) (Pleisto) Francolinus pondicerianus (Gmelin) (Pleisto) Cuculus varius vahl (Pleisto) Cuculus micropterus (Gould) (Pieisto) Eudynamys scolopacea (Linn)
Phalacrocorax niger (Viellot) (Oligo) Milvus migrans govinda (Sykes) (Mio) Bubulcus ibis coromandus (Bodd) Anas platyrhynchos
Zoological name
Cuckoo
Indian cuckoo
Hawk cuckoo
Grey partridge
Grey quail
Domestic fowl
Guinea fowl
Common pochard
Mallard duck
Cattle egret
Kite
Little cormorant
Common name
ridABLE 1 - - E L E C T R O P H O R E T I C M O B I L I T Y OF AVIAN H E M O G L O B I N S
I
lip
II
III
IV
V
Hemoglobin in increasing order of mobility
b~ ',O
0 0 r~ 0
<
z
O
O
Coraeiformes
Coraciformes
Strigiformes
Psittaciformes
Psittaciformes
Columbiformes
Columbiformes
Columbiformes
Columbiformes
Gruiformes
Gruiformes
Cuculiformes
Natural order
Cuculidae (Eo-Oligo) Rallidae (Eo) Railidae (Eo) Columbidae (Oligo) Columbidae (Oligo) Columbidae (Oligo) Columbidae (Oligo) Psittacidae (Mio) Psittacidae (Mio) Strigidae (Eo) Coraeiidae (Eo-Oligo) Alcedinidae (Oligo)
Family
Psittacula krameri borealis (Neum) Psittacula cyanocephala (Linn) Otus bakkamoena (Pennant) (Pleisto) Coracias benghalensis (Linn) Halcyon smyrnensis (Linn)
Streptopelia orientalis
Centr@us sinensis (Stephens) Amaurornis phoenicurus (Pennant) Fulica atra (Linn) (Pleisto) S tr ep topelia chinensis suratensis (Gmelin) Columba livia (Gmelin) (Pleisto) Terron phoenicoptera
Zoological name
Common name
HEMOGLOBINS--continued
Kingfisher
Bluejay
Collard scops owl
Blossom headed parakeet
Rose ringed parakeet
Rufous turtle dove
Bengal green pigeon
Pigeon
Spotted dove
Coot
Water hen
Kuko
r~'ABLE 1 - - E I , E C T R O P H O R E ' r l t g 1MOBILITY OF AVIAN
I
~t
II
II1
IV
V
Hemoglobin in increasing order of mobility
m
©
t~ 00 a,
>
to Ix9
Capitonidae
Capitonidae
Picidae (Mio) Pycnonotidae
Pycnonotidae
Campephagidae Muscicapidae
Muscicapidae
Muscicapidae
Muscicapidae
Ploceidae
Ploceidae
Ploceidae
Piciformes
Piciformes
Passeriformes
Passeriformes
Passeriformes Passeriformes
Passeriformes
Passeriformes
Passeriformes
Passeriformes
Passeriformes
Passeriformes
Family
Piciformes
Natural order
Megalaima lineata (Viellot) Megalaima asiatica (Latham) Dinopium benghalensis (Linn) Pycnonotus cafer (Linn) Pycnonotus jocosus (Linn) Coracina novaehollandiae Copsychus saularis (Linn) Chrysomma sinensis (Gmelin) A'axicola insignis (Hodgson) Turdoides somervillei (Sykes) Estrilda amandava flavidiventris (Linn) Lonchura malabarica (Linn) Lonchura punctulata (Linn)
Zoological name
Common name
IIEMOGLOBINS--Continued
Spotted munia
White throated munia
Indian red munia
Jungle babbler
Hodgson's bush-chat
Yellow eyed babbler
Kabasi Magpie robin
Red whiskered bulbul
Red vented bulbul
Goldenbacked woodpecker
Blue throated barbet
Lineated barbet
T A B L E 1 - - E L E C T R O P H O R E T I C MOBILITY OF AVIAN
I
*
II
,
,
*
IV
III
O
V
Hemoglobin in increasing order of mobility
to
0
N
x
0
0
Ploceidae
Sturnidae
Sturnidae
Sturnidae
Dicruridae
Oriolidae
Corvidae (Mio) Corvidae (Mio)
Passeriformes
Passeriformes
Passeriformes
Passeriformes
Passeriformes
Passeriformes
Passeriformes
Lonchura malacca (Linn.) Passer domesticus (Linn.) Sturnus pagodarum (Gmelin) Aeridotheres tristis (Gmelin) Gracula religiosa (Gmelin) Dicrurus macrocercus (Viellot) Oriohls xanthornus (Viellot) Crypsirina vagabunda (Latham) Corvus splendens splendeus (Veillot) (Plio)
Zoological name
I-touse crow
"Free pie
Black headed oriole
Black drongo
Bank myna
Common myna
Brahminy myna
Itouse sparrow
Black headed munia
Common name
HEMOGLOBINS--continued
I
•
II
,
III
,
IV
V
IIemoglobin in increasing order of mobility
T h e different natural orders are placed in Table I according to the evolutionary scale. Fossil record periods are shown in parentheses : Eo, Eocene; Oligo, Oligocene; Mio, Miocene; Plio, Pliocene; Pleisto, Pleistocene. T h e fossil records mentioned in the section of zoological names represent genus. T h e paper electrophoretic mobility was determined in barbiturate buffer, ionic strength 0"05 and p H 8'6, at 220 V for 16 hr, at 4°C on Whatman 3 m m paper with I,KB Paper Electrophoresis apparatus model 3276.
Passeriformes
Ploceidae
Family
Passeriformes
Natural order
T A B L E I - - E L E C T R O P I t O R E T I C M O B I L I T Y OF AVIAN
a
;3 :~
y.
tO tO tO
COMPARATIVE STLrDIF.,S ON AVIAN HEMOGLOBINS
223
hemoglobin solution was neutralized, filtered and stored at 2°C until the optical density of the filtered solution was determined. Calculations were made on the basis of per cent hemoglobin remaining undenatured at the end of a specified interval of time. Hemoglobin-I, from different avian species as isolated by paper electrophoresis, was subjected to alkaline denaturation in the same way that it was carried out in the case of unfractionated hemoglobin solution. Amino acid composition Amino acid composition of avian hemoglobins was determined with hemoglobin components isolated chromatographically. Ion-exchange chromatography on IRC-50 XE 64 was carried out as described previously (Saha, 1964). Peak zones were concentrated by centrifugation at 100,000g for 16 hr. The concentrated protein solutions were dialyzed free of salt; an aliquot was dried at ll0°C and subsequently used for acid hydrolysis. The sample was hydrolyzed at 110°C in 6N HC1 for 22, 48 and 70 hr in a sealed, evacuated glass tube. Amino acid analyses were carried out at 52"7°C with a Beckman model 120B amino acid analyzer. Amino acid composition reported in Table 6 was calculated by extrapolating the values obtained on 22, 48 and 70 hr hydrolysates. o~- and /3~-chains of duck, chick and pigeon hemoglobin-1 were obtained by IRC-50 chromatography of globins with urea gradient (Saha, 1964; Wilson & Smith, 1959). Heme moiety was removed from globin by treating hemoglobin with acetone and hydrochloric acid at - 2 0 ° C (Anson & Mirsky, 1930). The precipitated globin was washed free of hemin hydrochloride by acetone at -10°C, dissolved in a minimum quantity of water and dialyzed free of residual acetone and hydrochloric acid against several changes of water at 3°C. Globin (100--200 mg dry wt) was dialyzed twice against 10°/o formic acid (3 1. each time) and applied on an IRC-50 XE 64 column, 1-5 x 58 cm, which was previously equilibrated at room temperature with 10% formic acid. Five hundred ml of 6M urea, pH 1.88, was fed to a mixing vessel containing 125 ml of 2M urea, pH 1.9, followed by 600 ml of 8M urea, pH 1"8. Urea used herein was purified according to Benesch et al. (1955). Peak zones were dialyzed free of urea and then lyophilized. ~ - and /3~-chains represent the first and the second peaks as each emerged from the chromatographic column in the succeeding order. Amino acid composition of these sub-units was determined in the way as mentioned earlier and the amino acid residues were reported on the basis of molecular weight of the polypeptide moiety as 31,000. RESULTS
.an attempt has been made to categorize the avian hemoglobins on the basis of their electrophoretic migration on paper and the results have been presented in Table 1. Besides the degree of electrophoretic mobility of avian hemoglobins with respect t'o one another, Table I shows the number of hemoglobin components present in each bird mentioned therein. For the sake of comparison, the experimental conditions were maintained identical. The difference in the rate of electrophoretic migration of proteins results from the difference in the surface charge
224
A N I L SAHA AND JHARNA G H O S H
density of the molecules at a particular pH. T h a t a better resolution of avian hemoglobins could be achieved in alkaline buffers means that the hemoglobin moving faster toward the anode possesses a lesser a m o u n t of net positive charge in comparison with the hemoglobin showing a slower etectrophoretic mobility. T h e relative paper electrophoretic mobilities of the avian hemoglobins categorized u n d e r G r o u p I to G r o u p V in Table 1 were 1-00, 1.28, 1.42, 2.47 and 2.78 respectively. U n d e r identical conditions of paper electrophoresis, the avian hemoglobins TABLE 2raTHE
RELATIVE PROPORTIONS OF HEMOGLOBIN COMPONENTS IN DIFFERENT AVIAN SPECIES ARRANGED ACCORDING TO THE EVOLUTIONARY SCALE
Hemoglobin Natural order
Falconiformes Falconiformes Anseriformes Anseriformes Galliformes Galliformes Galliformes Coraciiforrnes Piciformes Piciformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes
Name of species
Kite Falcon Duck Common pochard Guinea fowl Chick Grey quail Kingfisher Lineated barbet Blue throated barbet Red vented bulbul Red whiskered bulbul Magpie robin Yellow eyed babbler Hodgson's bush-chat White throated munia Spotted munia Black headed munia House sparrow Common myna Bank myna Hill myna Black drongo
1
2
Ratio 1:2
69'7 66-6 62'0 78'4 63-0 70"0 76"3 75 "2 83.3 87.5 76"4 70.1 58"2 67"7 69"5 70"5 71"3 69-2 71 '8 61 "6 68.7 65-4 63"0
30"2 33 "3 38"0 20"8 37"0 30"0 23"6 24"8 16.6 13-4 23"5 29'8 41 "7 33-3 30"5 29"5 28.7 29'0 28-1 38"3 31"2 34"6 36'4
2-30 2-00 1"63 3"76 1'70 2'33 3"23 3"03 5-01 6"52 3'25 2.35 1.39 2"03 2-31 2-38 2-48 2-38 2'55 1 "60 2.20 1"89 1"75
The relative proportion of hemoglobin-1 and hemoglobin-2 in avian erythrocyte lysates was determined by employing paper electrophoresis, scanning the dry paper electropherograms at 540 m/z with a Photovolt Densitometer and calculating the relative areas under each peak with a planimeter. in G r o u p I I I and in G r o u p V showed electrophoretic mobilities identical with those of h u m a n hemoglobins E and A. This identical behavior during paper electrophoresis was substantiated further by subjecting to paper electrophoresis an equal mixture of avian hemoglobins and hemoglobins obtained from normal h u m a n adults ( H b - A ) and patients with hemogobin-E-thalassemia disease (Chatterjea
225
COMPARATIVE STUDIES ON AVIAN HEMOGLOBINS
et al., 1956, 1957). The charge differences as observed between the three little cormorant hemoglobins are very striking since the differences in relative mobility between the three little cormorant hemoglobins were found to be the maximum in comparison with the other systems containing multiple hemoglobins (Table I). Although most of the birds in the natural order Passeriformes possess two hemoglobins and some of them possess three hemoglobins, no member of this group has been found in possession of a single Fib in its blood system. The difference in the TABLE 3 - - T H E RELATIVE PROPORTIONS OF HEMOGLOBIN COMPONENTS IN DIFFERENT AVIAN SPECIES ARRANGED ACCORDING TO THE EVOLUTIONARY SCALE Hemoglobin Natural order
Pelecmaiformes Gruiformes Gruiformes Passeriformes Passeriformes Passeriformes
N a m e of species
Little cormorant Water hen Coot Black h e a d e d oriole T r e e pie Crow
1
2
3
40.6 67-3 74-2 50"0 45"1 42-8
25-0 8-4 12-1 25-1 32"6 35-7
34.2 24.0 13-5 25"6 22"3 21"5
Ratio 1:2:3 1.62 8.01 6"13 2"00 2"02 1-99
: : : : : :
1-00 1"00 1'00 1"00 1"46 1"66
: : : : : :
1"36 2"86 1"11 1"01 1-00 1'00
T h e relative p r o p o r t i o n s of i n d i v i d u a l h e m o g l o b i n s i n a t h r e e - h e m o g l o b i n c o m p o n e n t s y s t e m w e r e d e t e r m i n e d i n t h e m a n n e r d e s c r i b e d in T a b l e 2.
relative mobility between the hemoglobins in the two-hemoglobin system of Passeriformes (Group I and Group III) is larger than that observed in the other two-hemoglobin-containing systems, for example, the two hemoglobins of Anseriformes and Galliformes (Group II and Group Ill). With the exception of the three hemoglobins of the little cormorant, all the members in the threehemoglobin system are in Groups II, III, and IV, and electrophoretically these three hemoglobins are more closely related to each other than the three little cormorant hemoglobins. It may be noted that in Passeriformes hemoglobin 1 of the three-hemoglobin system (crow, tree pie and blackheaded oriole) has a higher electrophoretic mobility than hemoglobin-1 of the two-hemoglobin system. A better resolution of the hemoglobins of a three-component system as found in birds like crow, blackheaded oriole and woodpecker, could be achieved by conducting the paper electrophoresis in barbiturate buffers of higher alkalinity, for example, pH 8.8 and pH 9"0 and ionic strength 0-05. The differences in the relative mobilities of avian hemoglobins in the three-component system possibly originate from the distribution of carboxyl groups on the polypeptide chain, since the paper eleetropherograms revealed a single elongated zone with sodium acetate buffer of ionic strength 0-05 and pH's 5-0-5.4, where the carboxyl groups of a protein remain uncharged. However, with sodium phosphate buffer of ionic strength 0.05 and pH's 7-2, 6.8, 6.4 and 6-2, only two hemoglobins were revealed. t5
226
/~IL SAHA AND JHXRNA GHOSH
The relative distribution of major hemoglobin components present in avian species has been shown in Table 2. The per cent composition of the different hemoglobins was obtained by calculating the area under the zones which were evaluated from the optical density curves by means of a planimeter. The relative proportion of hemoglobin-2 in the system containing two hemoglobins was always found to be less than that of hemoglobin-1. The ratio of hemoglobin-1 to hemoglobin-2 ranged from 1.39 to 2.55 in thirteen out of twenty-three birds reported herein. All the birds in the order Passeriformes revealed the presence of multiple hemoglobins and the ratio of hemoglobin-1 to hemoglobin-2 ranged from 1.39 to 3.25. The avian hemoglobins obtained from the order Piciformes showed the TABLE 4---ALKALINE DENATURATION OF AVIAN HEMOGLOBINS
Natural order
Name
% Undenatured Hb
Group I (0-20% alkali-resistant hemoglobin) Falconiformes Kite 15 Falconiformes Falcon 14 Coraciiformes Kingfisher 8 Group II (20-50°'0 alkali-resistant hemoglobin) Galliformes Guinea fowl 39 Cuculiformes Indian cuckoo 36 Cuculiformes Hawk cuckoo 44 Columbiformes Turtle dove 43 Piciformes Woodpecker 44 Passeriformes Red whiskered bulbul 30 Passeriformes House sparrow 34 Passeriformes Hill myna 33 Passeriformes Common myna 37 Passeriformes Blackheaded oriole 33 Passeriformes Tree pie 41 Passeriformes. House crow 35 Group III (50% or higher alkali-resistant hemoglobin) Ciconiiformes Cattle egret 58 Anseriformes Domestic duck 84 Galliformes Chick 72 Columbiformes Pigeon 50 Alkaline denaturation of avian hemoglobins was determined on the avian erythrocyte lysates as described by Singer et al. (1951) and Saha (1956b). presence of a higher ratio of hemoglobin-1. Broadly, a close range in the value of the ratio of hemoglobins within a particular natural order might be noticed. An exception may, however, be observed in Anseriformes where the ratio of hemoglobins-1 and -2 in duck was 1.63 and that in common pochard was 3.76. In the system containing three hemoglobin components, hemoglobin-1 was found to be the major component as was also the case in the system containing two hemoglobins (Table 3). When compared individually to the relative proportion of hemoglobin-1 present in avian erythrocyte lysate, hemoglobins-2 and -3
COMPARATIVE STUDIES ON AVIAN HRMOGLOBIN$
227
appeared to be the minor components. Between hemoglobins-2 and -3 it is very difficult to choose the one to fit in the next position. In the Passeriformes, hemoglobin-2 showed a preponderance over hemoglobin-3 whereas in Gruiformes the reverse was true. There exists a certain congruity in the presence of multiple hemoglobins in a particular natural order. For example, all the birds in Passeriformes possess multiple hemoglobins and so also do the members of Piciformes and Anseriformes. A similar situation may be noted in Columbiformes and Cuculiformes where only one hemoglobin component was observed. The notable exception was in the order Coraciiformes; where bluejay possesses one hemoglobin component, kingfisher erythrocyte lysate revealed the presence of three hemoglobins. It may be noticed that habitat, locomotor adaptation and feeding habit do not indicate any correlationship with the occurrence of the single hemoglobin or multiple hemoglobins in a particular avian species. Cattle egret, dove and bluejay; kite, duck, fowl and myna; little cormorant, water hen, kingfisher, woodpecker and crow represent the diversity in the avian way of life. It seems difficult to find out any parallelism between the requirement of oxygen as demanded by the activity of a particular bird and the presence of multiple hemoglobins although the idea cannot be ignored completely. The fossil record as mentioned in Table 1 presents an over-simplified picture of evolutionary trend. For example, the fossil record shows the existence of the family Phalacrocoracidae during the middle Eocene period (38-57million years ago) and that of the genus Phalacrocorax during the low or middle Oligocene period (27-35 million years ago). Both Anatidae and Phasianidae are ascribed to Eocene while the genera comprising mallard duck and domestic fowl are ascribed to Miocene and low Pliocene (11-27 and 1.0--11 million years ago) respectively. Although the time span between the family and genus fossil records indicates a measure in the rate of evolution, the available information is too meagre to produce a complete picture. However, the primitiveness does not seem to lead to the reason as to the presence of multiple hemoglobins in avian species. Alkaline denaturation
The resistance of avian hemoglobins to alkaline denaturation varies considerably. The avian hemoglobins were divided into three groups based on the amount of hemoglobin left after alkaline denaturation for 1 min. The resistance to alkaline denaturation apparently does not follow the evolutionary trend (Table 1). The number of hemoglobins in a particular avian species does not influence the degree of resistance to alkaline denaturation as observed in the cases of kingfisher and cattle egret hemoglobins. Certain close relationships may be noticed in Passeriformes birds, most of them being in the group which contains 20-50o,"0 alkaliresistant hemoglobin. Similarly, two members of Columbiformes, dove and pigeon, contain 43 and 500/0 alkali-resistant hemogobin, respectively. Variation in the alkali-resistance property within the same natural order may be noted in cases of chick and guinea fowl. Chick hemoglobins are 1.85 times more resistant to alkaline denaturation than the guinea fowl hemoglobins.
228
ANIL SAHA AND JHARNA G n o s ~ I
Since some of the avian species possess multiple hemoglobins, it was of interest to find out the relative contribution of individual hemoglobin toward the cumulative resistance to alkaline denaturation. Isolation of hemoglobin-1 was undertaken as this particular hemoglobin could easily be obtained in a larger quantity. Avian hemoglobins were subjected to paper electrophoresis in Na-phosphate buffer, p H 6.8 and ionic strength 0-05 at 220 V for 20-22 hr at 4°C. After the completion of the electrophoretic run, only the peak zones were cut and eluted with the same TABLE 5~ALKALINE DI~qATURATION OF HEMOGLOBIN-1 OF DIFFERENT AVIAN SPECIES
(%)Undenatured Hb Natural order
Galliformes GaUiformes Piciformes Passeriformes Passeriforrnes
Species
Chick Guinea fowl Woodpecker House sparrow Red whiskered bulbul
1 rain
5 rain
10 rain
15 rain
62.5 19"3 15.0 20"1 12"1
28"3 14"8 3"2 12-0 3"6
23.9 4.0 3"0 8"2 3"0
7.6 -2"6 ---
Hemoglobin-1 was isolated by paper electrophoresis. The zone representing heraoglobin1 was cut and eluted chromatographically. The alkaline denaturation was then conducted as mentioned previously (Singer et al., 1951; Saha, 1956b). buffer used for paper electrophoresis by descending chromatography in a watersaturated, all-glass chromatographic chamber at 2-3°C. The hemoglobin solution was concentrated by centrifuging overnight at 100,000 g at 4°C in a Spinco model L ultracentrifuge. The concentration of hemoglobin was determined spectrophotometrically as carbonmonoxy-hemoglobin at 540 mtz and brought back to the required concentration with distilled water for alkaline denaturation studies. Results are presented in Table 5. Hemoglobin-1 may be considered to be alkali-resistant when compared with the alkali-resistance characteristics of human adult hemoglobin. In all cases hemoglobin-1 showed a value lower than that observed with the mixture of hemoglobins. The higher values obtained with the erythrocyte lysate may originate from the protein-protein association that possibly resisted the alkaline denaturation of proteins. Although electrophoresis of hemoglobins is not expected to alter the alkali-resistance property, the various experimental steps employed herein may, however, affect the proteins. Amino acid composition
Amino acid composition of hemoglobin-1 from duck, chick, pigeon and dove is presented in Table 6. The fossil record of these birds indicates that Anatidae is the most primitive of the four different avian species mentioned herein, followed by Phasiandidae and Columbidae. Similarity between duck and chick hemoglobins-1
229
COMPARATIVE STUDIES ON AVIAN HEMOGLOBINS
was closer than between hemoglobins-2 from these two species. Major differences between pigeon hemoglobin-1 and duck and chick hemoglobins-1 were observed in the number of glycine and methionine residues. Hemoglobins-2 in duck and chick differ mainly in histidine, threonine, serine, glutamic acid, alanine, valine, isoleucine, and leucine residues• Duck hemoglobin-2 has a rather large number of glutamic acid and alanine residues. It may be mentioned that duck hemoglobins possess a higher degree of resistance to alkaline denaturation than chick hemoglobins. Table 7 shows the amino acid residues in a~- and flt~-chains of duck, chick and pigeon hemoglobins-1 which belong to Group II (Table 1) in order of TABLE 6---AMINO ACID COMPOSITION OF AVIAN HEMOGLOBINS
Hemoglobin-1
Lys His Arg Asp Thr Ser Glu Pro Gly Ala Cys/2 Val Met Ileu Leu Tyr Phe
Hemoglobin-2
Duck
Chick
Pigeon
Dove
Duck
Chick
47 36 18 56 30 26 49 23 39 88 8 59 6 27 68 12 33
47 34 19 53 30 24 49 25 38 80 8 58 7 29 74 17 33
49 33 20 58 26 31 48 20 48 80 8 62 1 25 72 10 34
49 36 18 60 30 29 43 21 46 83 9 61 2 21 70 12 36
47 34 20 56 22 35 86 26 35 93 9 48 13 6 74 21 33
44 26 19 54 28 27 61 22 34 70 8 54 12 18 68 17 32
The amino acid residues were calculated on the basis of molecular weight of hemoglobin being 64,500. The hydrolytic losses were corrected by extrapolating the value to zero hour. The hemoglobins were obtained by IRC-50 XE 64- chromatography (Saha, 1964). electrophoretic mobility. The comparative study was further pursued by separating the a2- and fl~-chains of hemoglobin component-1 obtained from duck, chick and pigeon and then determining the distribution of amino acid residues in each chain (Table 7). Differences between duck and chick a~-chain may be found in the number of alanine, serine and leucine residues and those between chick and pigeon a~-chains in the number of aspartic acid, serine, proline, glycine and tyrosine residues. Between fl~-chains of duck and chick a noticeable difference was observed only in the number of aspartic acid residues, whereas between chick and pigeon ~X-chains differences were observed in the number of serine, proline, glycine,
--
4
14 37 9 16 2'7 : 1"9 : 1"0 1"2 : 1'0 0.7 : 1-0 2-5:1"8:1"0 2'6 : 1'0
3
13 31 7 17 3"0 : 2'4 : 1"0 1"08 : 1"0 0'6 : 1"0 2-8:1"8:1"0 2"4 : 1"0
15 36 6 16 2'3 : 1'6 : 1-0 1'3 : 1'0 0'4 : 1"0 2-6:1"8:1'0 2'4 : 1-0
25 18 11 30 14 16 24 10 24 41 3 29
Pigeon
24 17 9 27 15 11 23 13 19 40 4 29
24 19 8 26 14 14 24 12 20 47 3 31
Chick 23 17 10 30 15 12 25 12 18 40 5 28 3 13 36 6 17 2"3 : 1'7 : 1'0 1"2 : 1"0 0'7 : 1"0 2'4:1"7:1"0 2-8 : 1'0
Duck 23 16 I0 26 14 12 25 13 19 40 4 28 3 15 38 7 17 2-3 : 1"6 : 1"0 1"04 : 1"0 0-7 : 1"0 2"4:1"7:1-0 2"5 : 1"0
Chick
fl~t-chain
35 5 16 2-4 : 1'6 : 1"0 1"I : 1"0 0"5 : 1"0 2"4:2"0:I'0 3"2 : 1'0
11
24 16 10 27 13 16 24 10 22 38 5 33 --
Pigeon
T h e n u m b e r of amino acid residues in ~ - and fl~- chains were calculated on the basis of molecular weight of eq- and fl2-chains being 31,000. Globin was obtained from h e m o g l o b i n component-1 by a c e t o n e - h y d r o c h l o r i c acid t r e a t m e n t (Anson & Mirsky, 1930). T h e polypeptide chains were isolated by c h r o m a t o g r a p h y of globins on I R C - 5 0 X E 64 with urea gradient at p H 1-9 (Saha, 1964; Wilson & Smith, 1959). T h e first and the second peaks as e m e r g e d from the c h r o m a t o g r a p h i c columns were designated as ~xt- and fit-chains.
Lys His Arg Asp Thr Ser Glu Pro Gly Ala Cys/2 Val Met Ileu Leu Tyr Phe Lys : His : Arg Asp : GIu Pro : G l y Ala:Val:Phe Leu : Ileu
Duck
ACID RESIDUES IN o i l - A N D f l l - C I I A I N S
o~-chain
TABLE 7--AMINO
o
t" 00
bo Go
COMPARATIVE STUDIES ON AVIAN HEMOGLOBINS
231
valine, isoleucine and leucine residues. The extent of differences in the number of amino acid residues among ~-chains of chick and pigeon is greater than that among f~-chains. The relative distribution of the amino acid residues indicates that chick ~ - and f~-chains fit in the intermediate position between the ~ - and f~-chains from duck and pigeon. Although only one methionine residue was observed in pigeon hemoglobin-l, the present experimental conditions did not provide the conclusive proof as to which of the two chains contains the single residue. Despite the differences in the distribution pattern of the amino acid residues in o~- and fit-chains of duck, chick and pigeon hemoglobin-l, a similarity may be noticed among ~-chains and f~-chains when t,~e chains are being considered as a group However, the dissimilarity between ~]- and ft-chains in each species is quite obvious. Hill & Buettner-Janusch (1964) have discussed a similar situation which was with a~- and f~- chains of primate hemoglobins, where ~2-ehain peptides from the primate hemoglobins show few differences and f2-chain sequences show a greater degree of variability. DISCUSSION Classification of birds into twenty-seven different natural orders was based on the comparative morphological features. Comparative anatomy has established a relationship between birds of widely divergent characteristics. Although no direct evidence in fossil form is available to identify the developmental stages through which the evolutionary change from the reptilian skeleton to the avian form occurred, osteological similarities indicate a common ancestor for the primitive reptilian and avian species; it is considered probable that this common ancestor belonged to the order Mecondontia. Prior to the successful adaptation for arboreal habitat, the physiological variations in the avian species must have preceded the morphological adaptations. The adaptive radiation in the avian species must have started after the appearance of birds in the Mesozoic era (180-230 million years ago) and eventually diverged in many directions of speciation with respect to morphology and physiology. In all probability, the major speciations in the avian kingdom occurred in the earlier part of the Tertiary period. By the later part of the Eocene (38-57 million years ago) most orders of birds had appeared and by the end of the Miocene (11-27 million years ago) all modern avian families, at least non-passerine birds, were in existence. The classification of Aves as shown in Table 1 places the primitive orders at the top and the more recent ones at the bottom. In reality, the evolution of Ayes occurred in a three-dimensional pattern and not in two-dimensional form as described in Table 1. The two-dimensional presentation of the natural orders beginning with the primitive orders and leading towards the recent ones does not elaborate the degree of evolutionary changes in different families and orders. Furthermore, adaptive radiation resulting from a number of orienting factors like locomotor adaptation, feeding habits, etc., produced a wider divergence in the morphological characteristics. The influence of zoogeographic distribution was also of primary importance. It is quite plausible that the evolutionary changes did
232
ANIL SARAANDJnam~A Gnosu
not parallel the changes in the morphological features and the mutation in the amino acid conformation of proteins. Biochemical evolution of a protein is relatively resistant to environmental pressure. Studies of the characteristics of avian hemoglobins reveal the similarities and dissimilarities and thereby the interrelationship between the different orders and families with respect to one of the respiratory. pigments, hemoglobin. An excellent review on the electrophoretic patterns of avian egg-white proteins as taxonomic characters has been published by Sibley (1960). A definite relation between the presence of a number of hemoglobin components detected in the avian erythrocyte lysate and the position of a particular avian species in the evolutionary scale could not be obtained, although some similarities in the behavior could be traced. Among the Passeriformes birds, there exist two distinct groups: one containing two hemoglobins and the other with three hemoglobins, whereas in birds of the order Galliformes or Anseriformes, only one group containing two hemoglobins has been found. The normal habitat of an avian species does not reflect on the occurrence of multiple hemoglobins. Hemoglobins-1 and -2 representing Groups I and III respectively (Table 1) show a greater chargedifference among themselves except in cases of three-hemoglobin components representing Groups II, III and IV. It indicates that the differences in the amino acid composition of the hemoglobins in the two-component system are greater than those in the three-component systems. Chick hemoglobins-1 and -2 contain 100 and 102 residues of basic amino acids (lysine, histidine, and arginine) and 89 and 115 residues of acidic amino acids (aspartic and glutomic acids) (Saha, 1964). Notable differences have been found in histidine, glutamic acid, alanine, methionine, and isoleucine content. The amino acid composition of ~2- and fl2-chains of chick hemoglobins reflects the similar differences. ~2- and ~2-chains of chick hemoglobin-1 resemble each other more closely than either %- and fl~-chains of chick hemoglobin-2. It has already been reported that there exists a differential rate of bio-synthesis of chick hemoglobins-1 and -2 (Saha & Ghosh, 1960; Saha, 1960). In vivo incorporation of radioactive amino acids is higher in chick hemoglobin-2 while the reverse is the case with in vitro experiments. That the nucleated avian erythrocytes contain the required genetic information for the biosynthesis of all the sub-units may be inferred from the in vitro biosynthesis of chick hemoglobins. All the components that were found in adult chicks were found in the young ones. It implies that the hemoglobins and their sub-units are being synthesized in the avian system irrespective of age and difference in amino acid composition and sequence. The presence of embryonic hemoglobins in chick other than the two hemoglobins as found in the literature (Dunlap et al., 1956; Saha, 1956a; Rodnan & Ebaugh, 1957; Saha et al., 1957; Helm & Huisman, 1958) has been reported. D'Amelio & Salvo (1961) detected the presence of two distinct embryonic hemoglobin components at the first 68 hr of development by means of starch and agar electrophoresis, and immunoelectrophoresis. Wilt (1962) observed two kinds of hemoglobins in the adult chicks which are also present in 2- and 3-day-old embryos
COMPARATIVE STUDIES ON AVIAN HEMOGLOBINS
233
and in addition, a third immunologicaUy distinct globin-like component persisted only during the first 48 hr of development. Based on the vertical starch-gel electrophoresis, ManweU et al. (1963) have reported the presence of a hemoglobin in early chick embryo. A similar observation was reported by Fraser (1964) employing the electrophoresis of cyanomethemoglobin on cellulose acetate paper. The amino, acid composition of hemoglobins from little cormorant, woodpecker, crow and tree pie, revealed that no definite resemblance could be found between the different hemoglobins of a bird or between the hemoglobins from different species (unpublished observation, Saha & Ghosh). No correlation has, however, been found between the hemoglobins in the different groups (Table 1). The oxygen equilibria studies reveal that all these hemoglobins possess the biological characteristics, i.e. oxygenation and deoxygenation under differential air pressure. But this still leaves us with the question: Why do hemoglobins with different structures and in different proportions appear in mature erythrocytes of different avian species ? It seems obvious that there exists a rate-determining factor or factors to control the production of multiple hemoglobins in a particular avian species. There are four sub-units per hemoglobin molecule and twelve sub-units in a system containing three hemoglobins. Any hindrance in the formation of polypeptide chain with the participation of enzymes or in the release of polypeptide moiety from the ribosomal template would result in decreased production. A competitive system of protein biosynthesis may prevail in the avian system, considering the biological necessity of the bird. One may speculate whether or not all the different hemoglobins are produced in the same cell or in different cells.. The existence of a particular type of cell producing a particular type of hemoglobin can neither be proved nor be discredited despite the fact that the ratio of radioactive amino acid incorporation in chick hemoglobins-1 and -2 could be influenced by the amino acid composition of the incubating media (Saha, 1960; Saha & Ghosh, 1960). Manwell et al. (1963) have observed the discontinuation of the biosynthesis of an embryonic type hemoglobin in chick after the first 5 days of incubation in White Leghorn, New Hampshire and Columbian chicks and after approximately 6 days in Bantam chicks. In the turkey, adult hemoglobin begins to appear only after 8 days of incubation whereas in red-winged black bird, adult and embryonic hemoglobins are synthesized simultaneously from the beginning. That a controller gene is operating seems quite obvious and is in conformation with the experimental observations (Saha, 1960; Saha & Ghosh, 1960; Manwell et al., 1963). Ion-exchange chromatography of tryptie peptides and ehymotryptic peptides from the trypsin-resistant core of chick hemoglobins-1 and -2 reveal the similar and dissimilar peptide zones of the two cognate proteins (Saha, 1964). Growing attention is being paid to locate the genie mutation by finding out the substituted amino acids in the homologous proteins in the different species. Hemoglobins from different species have been investigated to locate the amino acid substitution (Hill , t al., 1963; Pauling & Zuckerkandl, 1963). The genic mutation along the protein chain reflects the cumulative pressure on the template for mutation factors, such as unfavorable metabolic activity requiring change in performance of oxygen
234
ANIL SAHA AND JHARNA GHOSH
equilibration, or change in internal milieu due to feeding habits, which would contribute considerably toward this process. If non-lethal mutation be the general procedure, then the steric effect and side-chain interaction between the sub--units, the polarization of charged groups along the chain, and the stabilization of integral protein structure will have to be considered. One should also consider the divergence and convergence to the point mutation which would bring the original amino acids back to position. A greater understanding could be achieved by determining the amino acid sequence of hemoglobin sub-units obtained from different avian species. Interrelationship between the order, family and species of the avian kingdom could possibly be established. SUMMARY Studies on paper electrophoretic mobility of hemoglobins obtained from avian species representing thirteen natural orders show that avian erythrocytes possess one, two or three hemoglobins. The avian hemoglobins may be categorized into five groups depending on the degree of mobility towards anode. Although the ratio of hemoglobin-1 to other hemoglobins varies considerably, hemoglobin-1 was found to be the major component. The avian hemoglobins are relatively resistant to alkaline denaturation and the resistance characteristics depend on individual species. Duck, chick, pigeon and dove show a comparable amino acid composition in hemoglobin-1 whereas a widely divergent amino acid composition has been found in hemoglobin-2 of duck and of chick. Noticeable difference was observed in the number of following amino acid residues: alanine, serine and leucine (between ~-chains of duck and chick); aspartic acid, serine, proline, glycine and tyrosine (between ~-chains of chick and pigeon) ; aspartic acid, serine, proline, glycine and tyrosine (between ~l-chains of chick and pigeon); aspartic acid (ill-chains of duck and chick); and serine, proline, glycine, valine, isoleucine and leucine (between fl~-chains of chick and pigeon). Acknowledgements--The authors are thankful to the Indian Council for Medical Research for a research grant. The authors are indebted to Dr. B. Biswas of the Indian Museum, Calcutta and to Dr. H. Friedmann and Dr. Hildegaard Howard of the Los Angeles County Museum for the fossil record and the arrangement of different natural orders in the evolutionary scale. The authors are greatly indebted to the late Professor B. C. Guha for his kind advice and support during this investigation. One of the authors (A. S.) is grateful to Dr. Dan H. Campbell and Dr. Walter A. Schroeder for their kind patronage.
REFERENCES ANSO~," M. L. & Mn~sk~ A. E. (1930) Protein coagulation and its reversal; preparation of insoluble globin, soluble globin and heine..~. Gen. Physiol. 13, 469--476. BENF.SCHR. E., LARDYH. A. & BENESCIqR. (1955) The sulfhydryl groups of crystalline proteins I. Some alb.mnins, enzymes and hemoglobins. ~. Biol. Chem. 216, 663. CHATanmJEAJ. B., SAHAA. K., RoY R. N. & GHOS~ S. K. (1956) Electrophoretic analysis of hemoglobin in Cooley's anemia (Thalassemia). Evidences of interaction of Thalassemia gene with that of an abnormal hemoglobin. Bull. Calcutta Sch. Trop. ivied. 4, 103-105.
COMPARATIVE STUDIES O N AVIAN H E M O G L O B I N S
235
CHATTERIEA J. B., SAHA A. K., RoY R. N. & GHOSH S. K. (1957) Hemoglobin EThalasaemia disease. IndianJ. Med. Sci. 11, 554-564. D'AMELIO, V. & SALVOA. M. (1961) Further studies on the embryonic chick hemoglobin. An electrophoretic and immunoelectrophoretic analysis. Acta Embryol. Morph. Exp. 4, 250-259. DUNLAP J. S., JOHNSON V. L. & FARN~ D. S. (1956) Multiple hemoglobins in birds. Experientia 12, 352-353. DUTTA R., GHOSH J. & GUHA B. C. (1958) Electrophoretic behavior of avian hemoglobins. Nature 181, 1204-1052. FRASER R. C. (1964) Electrophoretic characteristics and cell content of the hemoglobins of developing chick embryos. ~t. Exp. Zool. 156, 185-196. HELM H. J. VANV~t and HUISMANT. H. J. (1958) The two hemoglobin components of the chicken. Science 127, 762-763. HILL R. L. & BU~TTN~a-JANUSCHJ. (1964) Evolution of hemoglobin. Fed. Proc. 23, 12361242. HILL R. L., BUETTNER-JANUSCHJ. & BUETTNER-JANUSCHV. (1963) Evolution of hemoglobin in primates. Proc. Nat. Acud. Sci. Wash. 50, 885-893. MANWELLC., BAKERC. M. A., ROSLANSKYJ. D. & FOUGHTM. (1963) Molecular genetics of avian proteins. II. Control genes and structural genes for embryonic and adult hemoglobins. Proc. ,Vat. Acad. Sci. Wash. 49, 496-503. PAULINe L. & ZUCrmaKANDLE. (1963) Chemical Paleogenetics: Molecular Restoration. Studies of extinct forms of life. Acta Chem. Scan& 17, $9-S19. RODNm'~ G. P. & EBAUGHF. G. (1957) Paper electrophoresis of small animal hemoglobin. Proc. Exp. Biol. ,~/ied. 95, 397--401. SAHAA. (1956a) Studies on chick hemoglobin by means of paper electrophoresis. Science Culture, Calcutta 21, 756-758. SAHAA. (1956b) Studies on the alkaline denaturation of chick hemoglobin. Indian ]. Physiol. 10, 87-93. SAHAA. (1960) In vitro biosynthesis of chick hemoglobins. Biochim. Biophys. Res. Comm. 2, 450-454. SAHA A. (1964) Comparative studies on chick hemoglobins. Biochim. Biophys. Acta 93, 573-584. SAHA A, DUTTA R. & GHOSH, J. (1957) Paper electrophoresis of avian and mammalian hemoglobins. Science 125, 447-448. SAHA A. & GHOSHJ. (1960) Biosynthesis of chick hemoglobins. Science 132, 468-470. SIBLEY C. G. (1960) The electrophoretic patterns of avian egg-white proteins as taxonomic characters. Science 102, 215-284. SINGER K., CHERNOFF A. I. &StNGER L. (1951) Studies on abnormal hemoglobins. I. Their demonstration in sickle cell anemia and other hemolytic disorders by means of alkali denaturation. II. Their identification by means of the method of fractional denaturation. Blood 6, 413--428, 429-435. WILSON S. & SMITH D. B. (1959) Separation of the valyl-leucyl and valyl-glutamyl polypeptide chains of horse globin by fractional precipitation and column chromatography. Canad. J. Biochem. Physiol. 13, 405-416. WmT F. H. (1962) The ontology of chick embryo. Proc. Nat. Acad. Sci. Wash. 48, 1582-1590.
COMPARATIVE STUDIES ON AVIAN HEMOGLOBINS* A N I L S A H A t and J H A R N A G H O S H Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California, U.S.A. and Depa, i~ent of Applied Chemistry, University College of Science and Technology, Calcutta, India (Received 4 December 1964.; in revised form 8 ffanuary 1965) A b s t r a c t - - 1 . Studies o n
paper electrophoretic mobility of hemoglobins obtained from avian species representing thirteen natural orders show that avian erythrocytes possess one, two or three hemoglobins. 2. The avian hemoglobins may be categorized into five groups depending on the degree of mobility towards anode. Although the ratio of hemoglobin-1 to other hemoglobins varies considerably, hemoglobin-1 was found to be the major component. 3. The avian hemoglobins are relatively resistant to alkaline denaturation and the resistance characteristics depend on individual species. 4. Duck, chick, pigeon and dove show a comparable amino acid composition in hemoglobin-1 whereas a widely divergent amino acid composition has been found in hemoglobin-2 of duck and of chick. 5, Wide differences in amino acid composition of a~- and fl~-chains of duck, chick and pigeon have been observed. INTRODUCTION THE presence of multiple hemoglobins in birds like chick (Saha, 1956a; Saha et al., 1957), duck and guinea fowl was revealed by paper electrophoretic investigation of avian erythrocyte lysates. A comparative study showed that one of the avian hemoglobin components, hemoglobin-2, possessed paper electrophoretic mobility identical with one of the abnormal human hemoglobins, H b - E (Saha et al., 1957). Further investigation of the behavior of avian hemoglobins by employing paper electrophoresis revealed the presence of one, two or three hemoglobins in birds representing different avian species (Dutta et al., 1958). Attempts were made during the present study to seek certain relationships between the physico-chemical characteristics of hemoglobins obtained from different avian families and natural orders and the position of the avian natural orders in the evolutionary scale. Studies were thus undertaken to determine the number of hemoglobin components present in avian species and to evaluate the percentage distribution of such hemoglobin components. The resistance to alkaline denaturation was also determined to characterize the hemoglobins. Amino acid compositions of some avian hemoglobins and their sub-units were also reported to establish the inherent differences of the hemoglobins. * Contribution No. 3155. t Present address: McGill University, Royal Victoria Hospital, Montreal, Canada. 217
218
ANIL SAHA AND JHARNA GHOSH
EXPERIMENTAL Materials and methods Birds were captured alive in the region around Calcutta, India and brought to the laboratory without any delay. Erythrocytes were collected in heparinized isotonic saline at 5°C by cutting the jugular vein of the bird. Avian erythrocytes were washed by suspending four times with ten times their volume of isotonic saline at 5°C and spinning down each time in a refrigerated centrifuge at 600 g for 15 min. Packed cell volume was determined by centrifuging the erythrocytes at 1000 g for 15 rain at 5°C. The packed avian erythrocytes were then lysed with an equal volume of water and 0.4 volume of toluene, and kept overnight at 2°C. The suspension was centrifuged at 10,000 g for 15 rain at 5°C; clear hemoglobin solution was withdrawn with a syringe and centrifuged again at 10,000g for 15 rain. The hemoglobin solutions were stored in small vials at - 15°C and thawed prior to the electrophoretic runs. The hemoglobin solutions were diluted with the appropriate buffer to a solution (5 g Hb/100 ml) prior to the paper electrophoretic runs.
Paper electrophoresis Paper electrophoresis of avian hemoglobins was conducted in barbiturate buffer of pH 8"6 and ionic strength 0.05 for 16 hr at 220 V. Paper electrophoresis was carried out in a horizontal type apparatus with limited evaporation. The paper electrophoretic apparatus was kept in a large chamber maintained at 4°C and the entire electrophoretic procedure was carried out inside the cold chamber. The paper electropherograms, ~rhatman 3 mm paper, 18 x 46 cm, were removed immediately after the experiment, suspended by clamps in a horizontal position and dried quickly under infrared lamps. The relative mobility of a test avian hemoglobin was evaluated by applying chick hemoglobins and other hemoglobins possessing different mobilities as categorized under different groups (Table 1) on the same paper electropherogram and the electrophoretic run was conducted under identical experimental conditions. The relative paper electrophoretic mobilities were calculated on the basis of the mobility of a particular hemoglobin component towards the anode under the following conditions: barbiturate buffer of pH 8.6 and ionic strength 0.05, 220 V for 16 hr at 4°C. Hemoglobin-1 showed the slowest electrophoretic mobility toward anode while hemoglobin-3 showed the fastest mobility. The hemoglobin component categorized under Group V moved farthest toward the anode, while the one under Group I remained nearest to the cathode (Table 1). The paper electropherograms were scanned photometrically at 540 m/z with a Photovolt densitometer model 525. Alkaline denaturation Alkaline denaturation of hemoglobins was studied with the cell-free lysates of avian erythrocytes. Denaturation reaction was carried out with unfractionated hemoglobin solution according to the method of Singer et aL (1951). The alkaline denaturation was carried out at 20°C. After the completion of denaturation, the
Phalacrocoracidae (Eo) Accipitridae (Eo-Oligo) Ardeidae (Eo) Anatidae (Eo) Anatidae (Eo) Phasiandidae (Eo) Phasiandidae (Eo) Phasiandidae (Eo) Phasiandidae (Eo) Cuculidae (Eo-Oligo) Cuculidae (Eo-Oligo) Cuculidae (Eo-Oligo)
Pelecaniformes
Cuculiformes
Cuculiformes
Cuculiformes
Galliformes
Galliformes
Galliformes
Galliformes
Anseriformes
Anseriformes
Ciconiiformes
Falconiformes
Family
Natural order
.4ythya retina (Linn) (Plio) Numida meleagris (Linn) (Pleisto) Gallus gaihis (Plio) Coturnix coturnis (Linn) (Pleisto) Francolinus pondicerianus (Gmelin) (Pleisto) Cuculus varius vahl (Pleisto) Cuculus micropterus (Gould) (Pieisto) Eudynamys scolopacea (Linn)
Phalacrocorax niger (Viellot) (Oligo) Milvus migrans govinda (Sykes) (Mio) Bubulcus ibis coromandus (Bodd) Anas platyrhynchos
Zoological name
Cuckoo
Indian cuckoo
Hawk cuckoo
Grey partridge
Grey quail
Domestic fowl
Guinea fowl
Common pochard
Mallard duck
Cattle egret
Kite
Little cormorant
Common name
ridABLE 1 - - E L E C T R O P H O R E T I C M O B I L I T Y OF AVIAN H E M O G L O B I N S
I
lip
II
III
IV
V
Hemoglobin in increasing order of mobility
b~ ',O
0 0 r~ 0
<
z
O
O
Coraeiformes
Coraciformes
Strigiformes
Psittaciformes
Psittaciformes
Columbiformes
Columbiformes
Columbiformes
Columbiformes
Gruiformes
Gruiformes
Cuculiformes
Natural order
Cuculidae (Eo-Oligo) Rallidae (Eo) Railidae (Eo) Columbidae (Oligo) Columbidae (Oligo) Columbidae (Oligo) Columbidae (Oligo) Psittacidae (Mio) Psittacidae (Mio) Strigidae (Eo) Coraeiidae (Eo-Oligo) Alcedinidae (Oligo)
Family
Psittacula krameri borealis (Neum) Psittacula cyanocephala (Linn) Otus bakkamoena (Pennant) (Pleisto) Coracias benghalensis (Linn) Halcyon smyrnensis (Linn)
Streptopelia orientalis
Centr@us sinensis (Stephens) Amaurornis phoenicurus (Pennant) Fulica atra (Linn) (Pleisto) S tr ep topelia chinensis suratensis (Gmelin) Columba livia (Gmelin) (Pleisto) Terron phoenicoptera
Zoological name
Common name
HEMOGLOBINS--continued
Kingfisher
Bluejay
Collard scops owl
Blossom headed parakeet
Rose ringed parakeet
Rufous turtle dove
Bengal green pigeon
Pigeon
Spotted dove
Coot
Water hen
Kuko
r~'ABLE 1 - - E I , E C T R O P H O R E ' r l t g 1MOBILITY OF AVIAN
I
~t
II
II1
IV
V
Hemoglobin in increasing order of mobility
m
©
t~ 00 a,
>
to Ix9
Capitonidae
Capitonidae
Picidae (Mio) Pycnonotidae
Pycnonotidae
Campephagidae Muscicapidae
Muscicapidae
Muscicapidae
Muscicapidae
Ploceidae
Ploceidae
Ploceidae
Piciformes
Piciformes
Passeriformes
Passeriformes
Passeriformes Passeriformes
Passeriformes
Passeriformes
Passeriformes
Passeriformes
Passeriformes
Passeriformes
Family
Piciformes
Natural order
Megalaima lineata (Viellot) Megalaima asiatica (Latham) Dinopium benghalensis (Linn) Pycnonotus cafer (Linn) Pycnonotus jocosus (Linn) Coracina novaehollandiae Copsychus saularis (Linn) Chrysomma sinensis (Gmelin) A'axicola insignis (Hodgson) Turdoides somervillei (Sykes) Estrilda amandava flavidiventris (Linn) Lonchura malabarica (Linn) Lonchura punctulata (Linn)
Zoological name
Common name
IIEMOGLOBINS--Continued
Spotted munia
White throated munia
Indian red munia
Jungle babbler
Hodgson's bush-chat
Yellow eyed babbler
Kabasi Magpie robin
Red whiskered bulbul
Red vented bulbul
Goldenbacked woodpecker
Blue throated barbet
Lineated barbet
T A B L E 1 - - E L E C T R O P H O R E T I C MOBILITY OF AVIAN
I
*
II
,
,
*
IV
III
O
V
Hemoglobin in increasing order of mobility
to
0
N
x
0
0
Ploceidae
Sturnidae
Sturnidae
Sturnidae
Dicruridae
Oriolidae
Corvidae (Mio) Corvidae (Mio)
Passeriformes
Passeriformes
Passeriformes
Passeriformes
Passeriformes
Passeriformes
Passeriformes
Lonchura malacca (Linn.) Passer domesticus (Linn.) Sturnus pagodarum (Gmelin) Aeridotheres tristis (Gmelin) Gracula religiosa (Gmelin) Dicrurus macrocercus (Viellot) Oriohls xanthornus (Viellot) Crypsirina vagabunda (Latham) Corvus splendens splendeus (Veillot) (Plio)
Zoological name
I-touse crow
"Free pie
Black headed oriole
Black drongo
Bank myna
Common myna
Brahminy myna
Itouse sparrow
Black headed munia
Common name
HEMOGLOBINS--continued
I
•
II
,
III
,
IV
V
IIemoglobin in increasing order of mobility
T h e different natural orders are placed in Table I according to the evolutionary scale. Fossil record periods are shown in parentheses : Eo, Eocene; Oligo, Oligocene; Mio, Miocene; Plio, Pliocene; Pleisto, Pleistocene. T h e fossil records mentioned in the section of zoological names represent genus. T h e paper electrophoretic mobility was determined in barbiturate buffer, ionic strength 0"05 and p H 8'6, at 220 V for 16 hr, at 4°C on Whatman 3 m m paper with I,KB Paper Electrophoresis apparatus model 3276.
Passeriformes
Ploceidae
Family
Passeriformes
Natural order
T A B L E I - - E L E C T R O P I t O R E T I C M O B I L I T Y OF AVIAN
a
;3 :~
y.
tO tO tO
COMPARATIVE STLrDIF.,S ON AVIAN HEMOGLOBINS
223
hemoglobin solution was neutralized, filtered and stored at 2°C until the optical density of the filtered solution was determined. Calculations were made on the basis of per cent hemoglobin remaining undenatured at the end of a specified interval of time. Hemoglobin-I, from different avian species as isolated by paper electrophoresis, was subjected to alkaline denaturation in the same way that it was carried out in the case of unfractionated hemoglobin solution. Amino acid composition Amino acid composition of avian hemoglobins was determined with hemoglobin components isolated chromatographically. Ion-exchange chromatography on IRC-50 XE 64 was carried out as described previously (Saha, 1964). Peak zones were concentrated by centrifugation at 100,000g for 16 hr. The concentrated protein solutions were dialyzed free of salt; an aliquot was dried at ll0°C and subsequently used for acid hydrolysis. The sample was hydrolyzed at 110°C in 6N HC1 for 22, 48 and 70 hr in a sealed, evacuated glass tube. Amino acid analyses were carried out at 52"7°C with a Beckman model 120B amino acid analyzer. Amino acid composition reported in Table 6 was calculated by extrapolating the values obtained on 22, 48 and 70 hr hydrolysates. o~- and /3~-chains of duck, chick and pigeon hemoglobin-1 were obtained by IRC-50 chromatography of globins with urea gradient (Saha, 1964; Wilson & Smith, 1959). Heme moiety was removed from globin by treating hemoglobin with acetone and hydrochloric acid at - 2 0 ° C (Anson & Mirsky, 1930). The precipitated globin was washed free of hemin hydrochloride by acetone at -10°C, dissolved in a minimum quantity of water and dialyzed free of residual acetone and hydrochloric acid against several changes of water at 3°C. Globin (100--200 mg dry wt) was dialyzed twice against 10°/o formic acid (3 1. each time) and applied on an IRC-50 XE 64 column, 1-5 x 58 cm, which was previously equilibrated at room temperature with 10% formic acid. Five hundred ml of 6M urea, pH 1.88, was fed to a mixing vessel containing 125 ml of 2M urea, pH 1.9, followed by 600 ml of 8M urea, pH 1"8. Urea used herein was purified according to Benesch et al. (1955). Peak zones were dialyzed free of urea and then lyophilized. ~ - and /3~-chains represent the first and the second peaks as each emerged from the chromatographic column in the succeeding order. Amino acid composition of these sub-units was determined in the way as mentioned earlier and the amino acid residues were reported on the basis of molecular weight of the polypeptide moiety as 31,000. RESULTS
.an attempt has been made to categorize the avian hemoglobins on the basis of their electrophoretic migration on paper and the results have been presented in Table 1. Besides the degree of electrophoretic mobility of avian hemoglobins with respect t'o one another, Table I shows the number of hemoglobin components present in each bird mentioned therein. For the sake of comparison, the experimental conditions were maintained identical. The difference in the rate of electrophoretic migration of proteins results from the difference in the surface charge
224
A N I L SAHA AND JHARNA G H O S H
density of the molecules at a particular pH. T h a t a better resolution of avian hemoglobins could be achieved in alkaline buffers means that the hemoglobin moving faster toward the anode possesses a lesser a m o u n t of net positive charge in comparison with the hemoglobin showing a slower etectrophoretic mobility. T h e relative paper electrophoretic mobilities of the avian hemoglobins categorized u n d e r G r o u p I to G r o u p V in Table 1 were 1-00, 1.28, 1.42, 2.47 and 2.78 respectively. U n d e r identical conditions of paper electrophoresis, the avian hemoglobins TABLE 2raTHE
RELATIVE PROPORTIONS OF HEMOGLOBIN COMPONENTS IN DIFFERENT AVIAN SPECIES ARRANGED ACCORDING TO THE EVOLUTIONARY SCALE
Hemoglobin Natural order
Falconiformes Falconiformes Anseriformes Anseriformes Galliformes Galliformes Galliformes Coraciiforrnes Piciformes Piciformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes
Name of species
Kite Falcon Duck Common pochard Guinea fowl Chick Grey quail Kingfisher Lineated barbet Blue throated barbet Red vented bulbul Red whiskered bulbul Magpie robin Yellow eyed babbler Hodgson's bush-chat White throated munia Spotted munia Black headed munia House sparrow Common myna Bank myna Hill myna Black drongo
1
2
Ratio 1:2
69'7 66-6 62'0 78'4 63-0 70"0 76"3 75 "2 83.3 87.5 76"4 70.1 58"2 67"7 69"5 70"5 71"3 69-2 71 '8 61 "6 68.7 65-4 63"0
30"2 33 "3 38"0 20"8 37"0 30"0 23"6 24"8 16.6 13-4 23"5 29'8 41 "7 33-3 30"5 29"5 28.7 29'0 28-1 38"3 31"2 34"6 36'4
2-30 2-00 1"63 3"76 1'70 2'33 3"23 3"03 5-01 6"52 3'25 2.35 1.39 2"03 2-31 2-38 2-48 2-38 2'55 1 "60 2.20 1"89 1"75
The relative proportion of hemoglobin-1 and hemoglobin-2 in avian erythrocyte lysates was determined by employing paper electrophoresis, scanning the dry paper electropherograms at 540 m/z with a Photovolt Densitometer and calculating the relative areas under each peak with a planimeter. in G r o u p I I I and in G r o u p V showed electrophoretic mobilities identical with those of h u m a n hemoglobins E and A. This identical behavior during paper electrophoresis was substantiated further by subjecting to paper electrophoresis an equal mixture of avian hemoglobins and hemoglobins obtained from normal h u m a n adults ( H b - A ) and patients with hemogobin-E-thalassemia disease (Chatterjea
225
COMPARATIVE STUDIES ON AVIAN HEMOGLOBINS
et al., 1956, 1957). The charge differences as observed between the three little cormorant hemoglobins are very striking since the differences in relative mobility between the three little cormorant hemoglobins were found to be the maximum in comparison with the other systems containing multiple hemoglobins (Table I). Although most of the birds in the natural order Passeriformes possess two hemoglobins and some of them possess three hemoglobins, no member of this group has been found in possession of a single Fib in its blood system. The difference in the TABLE 3 - - T H E RELATIVE PROPORTIONS OF HEMOGLOBIN COMPONENTS IN DIFFERENT AVIAN SPECIES ARRANGED ACCORDING TO THE EVOLUTIONARY SCALE Hemoglobin Natural order
Pelecmaiformes Gruiformes Gruiformes Passeriformes Passeriformes Passeriformes
N a m e of species
Little cormorant Water hen Coot Black h e a d e d oriole T r e e pie Crow
1
2
3
40.6 67-3 74-2 50"0 45"1 42-8
25-0 8-4 12-1 25-1 32"6 35-7
34.2 24.0 13-5 25"6 22"3 21"5
Ratio 1:2:3 1.62 8.01 6"13 2"00 2"02 1-99
: : : : : :
1-00 1"00 1'00 1"00 1"46 1"66
: : : : : :
1"36 2"86 1"11 1"01 1-00 1'00
T h e relative p r o p o r t i o n s of i n d i v i d u a l h e m o g l o b i n s i n a t h r e e - h e m o g l o b i n c o m p o n e n t s y s t e m w e r e d e t e r m i n e d i n t h e m a n n e r d e s c r i b e d in T a b l e 2.
relative mobility between the hemoglobins in the two-hemoglobin system of Passeriformes (Group I and Group III) is larger than that observed in the other two-hemoglobin-containing systems, for example, the two hemoglobins of Anseriformes and Galliformes (Group II and Group Ill). With the exception of the three hemoglobins of the little cormorant, all the members in the threehemoglobin system are in Groups II, III, and IV, and electrophoretically these three hemoglobins are more closely related to each other than the three little cormorant hemoglobins. It may be noted that in Passeriformes hemoglobin 1 of the three-hemoglobin system (crow, tree pie and blackheaded oriole) has a higher electrophoretic mobility than hemoglobin-1 of the two-hemoglobin system. A better resolution of the hemoglobins of a three-component system as found in birds like crow, blackheaded oriole and woodpecker, could be achieved by conducting the paper electrophoresis in barbiturate buffers of higher alkalinity, for example, pH 8.8 and pH 9"0 and ionic strength 0-05. The differences in the relative mobilities of avian hemoglobins in the three-component system possibly originate from the distribution of carboxyl groups on the polypeptide chain, since the paper eleetropherograms revealed a single elongated zone with sodium acetate buffer of ionic strength 0-05 and pH's 5-0-5.4, where the carboxyl groups of a protein remain uncharged. However, with sodium phosphate buffer of ionic strength 0.05 and pH's 7-2, 6.8, 6.4 and 6-2, only two hemoglobins were revealed. t5
226
/~IL SAHA AND JHXRNA GHOSH
The relative distribution of major hemoglobin components present in avian species has been shown in Table 2. The per cent composition of the different hemoglobins was obtained by calculating the area under the zones which were evaluated from the optical density curves by means of a planimeter. The relative proportion of hemoglobin-2 in the system containing two hemoglobins was always found to be less than that of hemoglobin-1. The ratio of hemoglobin-1 to hemoglobin-2 ranged from 1.39 to 2.55 in thirteen out of twenty-three birds reported herein. All the birds in the order Passeriformes revealed the presence of multiple hemoglobins and the ratio of hemoglobin-1 to hemoglobin-2 ranged from 1.39 to 3.25. The avian hemoglobins obtained from the order Piciformes showed the TABLE 4---ALKALINE DENATURATION OF AVIAN HEMOGLOBINS
Natural order
Name
% Undenatured Hb
Group I (0-20% alkali-resistant hemoglobin) Falconiformes Kite 15 Falconiformes Falcon 14 Coraciiformes Kingfisher 8 Group II (20-50°'0 alkali-resistant hemoglobin) Galliformes Guinea fowl 39 Cuculiformes Indian cuckoo 36 Cuculiformes Hawk cuckoo 44 Columbiformes Turtle dove 43 Piciformes Woodpecker 44 Passeriformes Red whiskered bulbul 30 Passeriformes House sparrow 34 Passeriformes Hill myna 33 Passeriformes Common myna 37 Passeriformes Blackheaded oriole 33 Passeriformes Tree pie 41 Passeriformes. House crow 35 Group III (50% or higher alkali-resistant hemoglobin) Ciconiiformes Cattle egret 58 Anseriformes Domestic duck 84 Galliformes Chick 72 Columbiformes Pigeon 50 Alkaline denaturation of avian hemoglobins was determined on the avian erythrocyte lysates as described by Singer et al. (1951) and Saha (1956b). presence of a higher ratio of hemoglobin-1. Broadly, a close range in the value of the ratio of hemoglobins within a particular natural order might be noticed. An exception may, however, be observed in Anseriformes where the ratio of hemoglobins-1 and -2 in duck was 1.63 and that in common pochard was 3.76. In the system containing three hemoglobin components, hemoglobin-1 was found to be the major component as was also the case in the system containing two hemoglobins (Table 3). When compared individually to the relative proportion of hemoglobin-1 present in avian erythrocyte lysate, hemoglobins-2 and -3
COMPARATIVE STUDIES ON AVIAN HRMOGLOBIN$
227
appeared to be the minor components. Between hemoglobins-2 and -3 it is very difficult to choose the one to fit in the next position. In the Passeriformes, hemoglobin-2 showed a preponderance over hemoglobin-3 whereas in Gruiformes the reverse was true. There exists a certain congruity in the presence of multiple hemoglobins in a particular natural order. For example, all the birds in Passeriformes possess multiple hemoglobins and so also do the members of Piciformes and Anseriformes. A similar situation may be noted in Columbiformes and Cuculiformes where only one hemoglobin component was observed. The notable exception was in the order Coraciiformes; where bluejay possesses one hemoglobin component, kingfisher erythrocyte lysate revealed the presence of three hemoglobins. It may be noticed that habitat, locomotor adaptation and feeding habit do not indicate any correlationship with the occurrence of the single hemoglobin or multiple hemoglobins in a particular avian species. Cattle egret, dove and bluejay; kite, duck, fowl and myna; little cormorant, water hen, kingfisher, woodpecker and crow represent the diversity in the avian way of life. It seems difficult to find out any parallelism between the requirement of oxygen as demanded by the activity of a particular bird and the presence of multiple hemoglobins although the idea cannot be ignored completely. The fossil record as mentioned in Table 1 presents an over-simplified picture of evolutionary trend. For example, the fossil record shows the existence of the family Phalacrocoracidae during the middle Eocene period (38-57million years ago) and that of the genus Phalacrocorax during the low or middle Oligocene period (27-35 million years ago). Both Anatidae and Phasianidae are ascribed to Eocene while the genera comprising mallard duck and domestic fowl are ascribed to Miocene and low Pliocene (11-27 and 1.0--11 million years ago) respectively. Although the time span between the family and genus fossil records indicates a measure in the rate of evolution, the available information is too meagre to produce a complete picture. However, the primitiveness does not seem to lead to the reason as to the presence of multiple hemoglobins in avian species. Alkaline denaturation
The resistance of avian hemoglobins to alkaline denaturation varies considerably. The avian hemoglobins were divided into three groups based on the amount of hemoglobin left after alkaline denaturation for 1 min. The resistance to alkaline denaturation apparently does not follow the evolutionary trend (Table 1). The number of hemoglobins in a particular avian species does not influence the degree of resistance to alkaline denaturation as observed in the cases of kingfisher and cattle egret hemoglobins. Certain close relationships may be noticed in Passeriformes birds, most of them being in the group which contains 20-50o,"0 alkaliresistant hemoglobin. Similarly, two members of Columbiformes, dove and pigeon, contain 43 and 500/0 alkali-resistant hemogobin, respectively. Variation in the alkali-resistance property within the same natural order may be noted in cases of chick and guinea fowl. Chick hemoglobins are 1.85 times more resistant to alkaline denaturation than the guinea fowl hemoglobins.
228
ANIL SAHA AND JHARNA G n o s ~ I
Since some of the avian species possess multiple hemoglobins, it was of interest to find out the relative contribution of individual hemoglobin toward the cumulative resistance to alkaline denaturation. Isolation of hemoglobin-1 was undertaken as this particular hemoglobin could easily be obtained in a larger quantity. Avian hemoglobins were subjected to paper electrophoresis in Na-phosphate buffer, p H 6.8 and ionic strength 0-05 at 220 V for 20-22 hr at 4°C. After the completion of the electrophoretic run, only the peak zones were cut and eluted with the same TABLE 5~ALKALINE DI~qATURATION OF HEMOGLOBIN-1 OF DIFFERENT AVIAN SPECIES
(%)Undenatured Hb Natural order
Galliformes GaUiformes Piciformes Passeriformes Passeriforrnes
Species
Chick Guinea fowl Woodpecker House sparrow Red whiskered bulbul
1 rain
5 rain
10 rain
15 rain
62.5 19"3 15.0 20"1 12"1
28"3 14"8 3"2 12-0 3"6
23.9 4.0 3"0 8"2 3"0
7.6 -2"6 ---
Hemoglobin-1 was isolated by paper electrophoresis. The zone representing heraoglobin1 was cut and eluted chromatographically. The alkaline denaturation was then conducted as mentioned previously (Singer et al., 1951; Saha, 1956b). buffer used for paper electrophoresis by descending chromatography in a watersaturated, all-glass chromatographic chamber at 2-3°C. The hemoglobin solution was concentrated by centrifuging overnight at 100,000 g at 4°C in a Spinco model L ultracentrifuge. The concentration of hemoglobin was determined spectrophotometrically as carbonmonoxy-hemoglobin at 540 mtz and brought back to the required concentration with distilled water for alkaline denaturation studies. Results are presented in Table 5. Hemoglobin-1 may be considered to be alkali-resistant when compared with the alkali-resistance characteristics of human adult hemoglobin. In all cases hemoglobin-1 showed a value lower than that observed with the mixture of hemoglobins. The higher values obtained with the erythrocyte lysate may originate from the protein-protein association that possibly resisted the alkaline denaturation of proteins. Although electrophoresis of hemoglobins is not expected to alter the alkali-resistance property, the various experimental steps employed herein may, however, affect the proteins. Amino acid composition
Amino acid composition of hemoglobin-1 from duck, chick, pigeon and dove is presented in Table 6. The fossil record of these birds indicates that Anatidae is the most primitive of the four different avian species mentioned herein, followed by Phasiandidae and Columbidae. Similarity between duck and chick hemoglobins-1
229
COMPARATIVE STUDIES ON AVIAN HEMOGLOBINS
was closer than between hemoglobins-2 from these two species. Major differences between pigeon hemoglobin-1 and duck and chick hemoglobins-1 were observed in the number of glycine and methionine residues. Hemoglobins-2 in duck and chick differ mainly in histidine, threonine, serine, glutamic acid, alanine, valine, isoleucine, and leucine residues• Duck hemoglobin-2 has a rather large number of glutamic acid and alanine residues. It may be mentioned that duck hemoglobins possess a higher degree of resistance to alkaline denaturation than chick hemoglobins. Table 7 shows the amino acid residues in a~- and flt~-chains of duck, chick and pigeon hemoglobins-1 which belong to Group II (Table 1) in order of TABLE 6---AMINO ACID COMPOSITION OF AVIAN HEMOGLOBINS
Hemoglobin-1
Lys His Arg Asp Thr Ser Glu Pro Gly Ala Cys/2 Val Met Ileu Leu Tyr Phe
Hemoglobin-2
Duck
Chick
Pigeon
Dove
Duck
Chick
47 36 18 56 30 26 49 23 39 88 8 59 6 27 68 12 33
47 34 19 53 30 24 49 25 38 80 8 58 7 29 74 17 33
49 33 20 58 26 31 48 20 48 80 8 62 1 25 72 10 34
49 36 18 60 30 29 43 21 46 83 9 61 2 21 70 12 36
47 34 20 56 22 35 86 26 35 93 9 48 13 6 74 21 33
44 26 19 54 28 27 61 22 34 70 8 54 12 18 68 17 32
The amino acid residues were calculated on the basis of molecular weight of hemoglobin being 64,500. The hydrolytic losses were corrected by extrapolating the value to zero hour. The hemoglobins were obtained by IRC-50 XE 64- chromatography (Saha, 1964). electrophoretic mobility. The comparative study was further pursued by separating the a2- and fl~-chains of hemoglobin component-1 obtained from duck, chick and pigeon and then determining the distribution of amino acid residues in each chain (Table 7). Differences between duck and chick a~-chain may be found in the number of alanine, serine and leucine residues and those between chick and pigeon a~-chains in the number of aspartic acid, serine, proline, glycine and tyrosine residues. Between fl~-chains of duck and chick a noticeable difference was observed only in the number of aspartic acid residues, whereas between chick and pigeon ~X-chains differences were observed in the number of serine, proline, glycine,
--
4
14 37 9 16 2'7 : 1"9 : 1"0 1"2 : 1'0 0.7 : 1-0 2-5:1"8:1"0 2'6 : 1'0
3
13 31 7 17 3"0 : 2'4 : 1"0 1"08 : 1"0 0'6 : 1"0 2-8:1"8:1"0 2"4 : 1"0
15 36 6 16 2'3 : 1'6 : 1-0 1'3 : 1'0 0'4 : 1"0 2-6:1"8:1'0 2'4 : 1-0
25 18 11 30 14 16 24 10 24 41 3 29
Pigeon
24 17 9 27 15 11 23 13 19 40 4 29
24 19 8 26 14 14 24 12 20 47 3 31
Chick 23 17 10 30 15 12 25 12 18 40 5 28 3 13 36 6 17 2"3 : 1'7 : 1'0 1"2 : 1"0 0'7 : 1"0 2'4:1"7:1"0 2-8 : 1'0
Duck 23 16 I0 26 14 12 25 13 19 40 4 28 3 15 38 7 17 2-3 : 1"6 : 1"0 1"04 : 1"0 0-7 : 1"0 2"4:1"7:1-0 2"5 : 1"0
Chick
fl~t-chain
35 5 16 2-4 : 1'6 : 1"0 1"I : 1"0 0"5 : 1"0 2"4:2"0:I'0 3"2 : 1'0
11
24 16 10 27 13 16 24 10 22 38 5 33 --
Pigeon
T h e n u m b e r of amino acid residues in ~ - and fl~- chains were calculated on the basis of molecular weight of eq- and fl2-chains being 31,000. Globin was obtained from h e m o g l o b i n component-1 by a c e t o n e - h y d r o c h l o r i c acid t r e a t m e n t (Anson & Mirsky, 1930). T h e polypeptide chains were isolated by c h r o m a t o g r a p h y of globins on I R C - 5 0 X E 64 with urea gradient at p H 1-9 (Saha, 1964; Wilson & Smith, 1959). T h e first and the second peaks as e m e r g e d from the c h r o m a t o g r a p h i c columns were designated as ~xt- and fit-chains.
Lys His Arg Asp Thr Ser Glu Pro Gly Ala Cys/2 Val Met Ileu Leu Tyr Phe Lys : His : Arg Asp : GIu Pro : G l y Ala:Val:Phe Leu : Ileu
Duck
ACID RESIDUES IN o i l - A N D f l l - C I I A I N S
o~-chain
TABLE 7--AMINO
o
t" 00
bo Go
COMPARATIVE STUDIES ON AVIAN HEMOGLOBINS
231
valine, isoleucine and leucine residues. The extent of differences in the number of amino acid residues among ~-chains of chick and pigeon is greater than that among f~-chains. The relative distribution of the amino acid residues indicates that chick ~ - and f~-chains fit in the intermediate position between the ~ - and f~-chains from duck and pigeon. Although only one methionine residue was observed in pigeon hemoglobin-l, the present experimental conditions did not provide the conclusive proof as to which of the two chains contains the single residue. Despite the differences in the distribution pattern of the amino acid residues in o~- and fit-chains of duck, chick and pigeon hemoglobin-l, a similarity may be noticed among ~-chains and f~-chains when t,~e chains are being considered as a group However, the dissimilarity between ~]- and ft-chains in each species is quite obvious. Hill & Buettner-Janusch (1964) have discussed a similar situation which was with a~- and f~- chains of primate hemoglobins, where ~2-ehain peptides from the primate hemoglobins show few differences and f2-chain sequences show a greater degree of variability. DISCUSSION Classification of birds into twenty-seven different natural orders was based on the comparative morphological features. Comparative anatomy has established a relationship between birds of widely divergent characteristics. Although no direct evidence in fossil form is available to identify the developmental stages through which the evolutionary change from the reptilian skeleton to the avian form occurred, osteological similarities indicate a common ancestor for the primitive reptilian and avian species; it is considered probable that this common ancestor belonged to the order Mecondontia. Prior to the successful adaptation for arboreal habitat, the physiological variations in the avian species must have preceded the morphological adaptations. The adaptive radiation in the avian species must have started after the appearance of birds in the Mesozoic era (180-230 million years ago) and eventually diverged in many directions of speciation with respect to morphology and physiology. In all probability, the major speciations in the avian kingdom occurred in the earlier part of the Tertiary period. By the later part of the Eocene (38-57 million years ago) most orders of birds had appeared and by the end of the Miocene (11-27 million years ago) all modern avian families, at least non-passerine birds, were in existence. The classification of Aves as shown in Table 1 places the primitive orders at the top and the more recent ones at the bottom. In reality, the evolution of Ayes occurred in a three-dimensional pattern and not in two-dimensional form as described in Table 1. The two-dimensional presentation of the natural orders beginning with the primitive orders and leading towards the recent ones does not elaborate the degree of evolutionary changes in different families and orders. Furthermore, adaptive radiation resulting from a number of orienting factors like locomotor adaptation, feeding habits, etc., produced a wider divergence in the morphological characteristics. The influence of zoogeographic distribution was also of primary importance. It is quite plausible that the evolutionary changes did
232
ANIL SARAANDJnam~A Gnosu
not parallel the changes in the morphological features and the mutation in the amino acid conformation of proteins. Biochemical evolution of a protein is relatively resistant to environmental pressure. Studies of the characteristics of avian hemoglobins reveal the similarities and dissimilarities and thereby the interrelationship between the different orders and families with respect to one of the respiratory. pigments, hemoglobin. An excellent review on the electrophoretic patterns of avian egg-white proteins as taxonomic characters has been published by Sibley (1960). A definite relation between the presence of a number of hemoglobin components detected in the avian erythrocyte lysate and the position of a particular avian species in the evolutionary scale could not be obtained, although some similarities in the behavior could be traced. Among the Passeriformes birds, there exist two distinct groups: one containing two hemoglobins and the other with three hemoglobins, whereas in birds of the order Galliformes or Anseriformes, only one group containing two hemoglobins has been found. The normal habitat of an avian species does not reflect on the occurrence of multiple hemoglobins. Hemoglobins-1 and -2 representing Groups I and III respectively (Table 1) show a greater chargedifference among themselves except in cases of three-hemoglobin components representing Groups II, III and IV. It indicates that the differences in the amino acid composition of the hemoglobins in the two-component system are greater than those in the three-component systems. Chick hemoglobins-1 and -2 contain 100 and 102 residues of basic amino acids (lysine, histidine, and arginine) and 89 and 115 residues of acidic amino acids (aspartic and glutomic acids) (Saha, 1964). Notable differences have been found in histidine, glutamic acid, alanine, methionine, and isoleucine content. The amino acid composition of ~2- and fl2-chains of chick hemoglobins reflects the similar differences. ~2- and ~2-chains of chick hemoglobin-1 resemble each other more closely than either %- and fl~-chains of chick hemoglobin-2. It has already been reported that there exists a differential rate of bio-synthesis of chick hemoglobins-1 and -2 (Saha & Ghosh, 1960; Saha, 1960). In vivo incorporation of radioactive amino acids is higher in chick hemoglobin-2 while the reverse is the case with in vitro experiments. That the nucleated avian erythrocytes contain the required genetic information for the biosynthesis of all the sub-units may be inferred from the in vitro biosynthesis of chick hemoglobins. All the components that were found in adult chicks were found in the young ones. It implies that the hemoglobins and their sub-units are being synthesized in the avian system irrespective of age and difference in amino acid composition and sequence. The presence of embryonic hemoglobins in chick other than the two hemoglobins as found in the literature (Dunlap et al., 1956; Saha, 1956a; Rodnan & Ebaugh, 1957; Saha et al., 1957; Helm & Huisman, 1958) has been reported. D'Amelio & Salvo (1961) detected the presence of two distinct embryonic hemoglobin components at the first 68 hr of development by means of starch and agar electrophoresis, and immunoelectrophoresis. Wilt (1962) observed two kinds of hemoglobins in the adult chicks which are also present in 2- and 3-day-old embryos
COMPARATIVE STUDIES ON AVIAN HEMOGLOBINS
233
and in addition, a third immunologicaUy distinct globin-like component persisted only during the first 48 hr of development. Based on the vertical starch-gel electrophoresis, ManweU et al. (1963) have reported the presence of a hemoglobin in early chick embryo. A similar observation was reported by Fraser (1964) employing the electrophoresis of cyanomethemoglobin on cellulose acetate paper. The amino, acid composition of hemoglobins from little cormorant, woodpecker, crow and tree pie, revealed that no definite resemblance could be found between the different hemoglobins of a bird or between the hemoglobins from different species (unpublished observation, Saha & Ghosh). No correlation has, however, been found between the hemoglobins in the different groups (Table 1). The oxygen equilibria studies reveal that all these hemoglobins possess the biological characteristics, i.e. oxygenation and deoxygenation under differential air pressure. But this still leaves us with the question: Why do hemoglobins with different structures and in different proportions appear in mature erythrocytes of different avian species ? It seems obvious that there exists a rate-determining factor or factors to control the production of multiple hemoglobins in a particular avian species. There are four sub-units per hemoglobin molecule and twelve sub-units in a system containing three hemoglobins. Any hindrance in the formation of polypeptide chain with the participation of enzymes or in the release of polypeptide moiety from the ribosomal template would result in decreased production. A competitive system of protein biosynthesis may prevail in the avian system, considering the biological necessity of the bird. One may speculate whether or not all the different hemoglobins are produced in the same cell or in different cells.. The existence of a particular type of cell producing a particular type of hemoglobin can neither be proved nor be discredited despite the fact that the ratio of radioactive amino acid incorporation in chick hemoglobins-1 and -2 could be influenced by the amino acid composition of the incubating media (Saha, 1960; Saha & Ghosh, 1960). Manwell et al. (1963) have observed the discontinuation of the biosynthesis of an embryonic type hemoglobin in chick after the first 5 days of incubation in White Leghorn, New Hampshire and Columbian chicks and after approximately 6 days in Bantam chicks. In the turkey, adult hemoglobin begins to appear only after 8 days of incubation whereas in red-winged black bird, adult and embryonic hemoglobins are synthesized simultaneously from the beginning. That a controller gene is operating seems quite obvious and is in conformation with the experimental observations (Saha, 1960; Saha & Ghosh, 1960; Manwell et al., 1963). Ion-exchange chromatography of tryptie peptides and ehymotryptic peptides from the trypsin-resistant core of chick hemoglobins-1 and -2 reveal the similar and dissimilar peptide zones of the two cognate proteins (Saha, 1964). Growing attention is being paid to locate the genie mutation by finding out the substituted amino acids in the homologous proteins in the different species. Hemoglobins from different species have been investigated to locate the amino acid substitution (Hill , t al., 1963; Pauling & Zuckerkandl, 1963). The genic mutation along the protein chain reflects the cumulative pressure on the template for mutation factors, such as unfavorable metabolic activity requiring change in performance of oxygen
234
ANIL SAHA AND JHARNA GHOSH
equilibration, or change in internal milieu due to feeding habits, which would contribute considerably toward this process. If non-lethal mutation be the general procedure, then the steric effect and side-chain interaction between the sub--units, the polarization of charged groups along the chain, and the stabilization of integral protein structure will have to be considered. One should also consider the divergence and convergence to the point mutation which would bring the original amino acids back to position. A greater understanding could be achieved by determining the amino acid sequence of hemoglobin sub-units obtained from different avian species. Interrelationship between the order, family and species of the avian kingdom could possibly be established. SUMMARY Studies on paper electrophoretic mobility of hemoglobins obtained from avian species representing thirteen natural orders show that avian erythrocytes possess one, two or three hemoglobins. The avian hemoglobins may be categorized into five groups depending on the degree of mobility towards anode. Although the ratio of hemoglobin-1 to other hemoglobins varies considerably, hemoglobin-1 was found to be the major component. The avian hemoglobins are relatively resistant to alkaline denaturation and the resistance characteristics depend on individual species. Duck, chick, pigeon and dove show a comparable amino acid composition in hemoglobin-1 whereas a widely divergent amino acid composition has been found in hemoglobin-2 of duck and of chick. Noticeable difference was observed in the number of following amino acid residues: alanine, serine and leucine (between ~-chains of duck and chick); aspartic acid, serine, proline, glycine and tyrosine (between ~-chains of chick and pigeon) ; aspartic acid, serine, proline, glycine and tyrosine (between ~l-chains of chick and pigeon); aspartic acid (ill-chains of duck and chick); and serine, proline, glycine, valine, isoleucine and leucine (between fl~-chains of chick and pigeon). Acknowledgements--The authors are thankful to the Indian Council for Medical Research for a research grant. The authors are indebted to Dr. B. Biswas of the Indian Museum, Calcutta and to Dr. H. Friedmann and Dr. Hildegaard Howard of the Los Angeles County Museum for the fossil record and the arrangement of different natural orders in the evolutionary scale. The authors are greatly indebted to the late Professor B. C. Guha for his kind advice and support during this investigation. One of the authors (A. S.) is grateful to Dr. Dan H. Campbell and Dr. Walter A. Schroeder for their kind patronage.
REFERENCES ANSO~," M. L. & Mn~sk~ A. E. (1930) Protein coagulation and its reversal; preparation of insoluble globin, soluble globin and heine..~. Gen. Physiol. 13, 469--476. BENF.SCHR. E., LARDYH. A. & BENESCIqR. (1955) The sulfhydryl groups of crystalline proteins I. Some alb.mnins, enzymes and hemoglobins. ~. Biol. Chem. 216, 663. CHATanmJEAJ. B., SAHAA. K., RoY R. N. & GHOS~ S. K. (1956) Electrophoretic analysis of hemoglobin in Cooley's anemia (Thalassemia). Evidences of interaction of Thalassemia gene with that of an abnormal hemoglobin. Bull. Calcutta Sch. Trop. ivied. 4, 103-105.
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CHATTERIEA J. B., SAHA A. K., RoY R. N. & GHOSH S. K. (1957) Hemoglobin EThalasaemia disease. IndianJ. Med. Sci. 11, 554-564. D'AMELIO, V. & SALVOA. M. (1961) Further studies on the embryonic chick hemoglobin. An electrophoretic and immunoelectrophoretic analysis. Acta Embryol. Morph. Exp. 4, 250-259. DUNLAP J. S., JOHNSON V. L. & FARN~ D. S. (1956) Multiple hemoglobins in birds. Experientia 12, 352-353. DUTTA R., GHOSH J. & GUHA B. C. (1958) Electrophoretic behavior of avian hemoglobins. Nature 181, 1204-1052. FRASER R. C. (1964) Electrophoretic characteristics and cell content of the hemoglobins of developing chick embryos. ~t. Exp. Zool. 156, 185-196. HELM H. J. VANV~t and HUISMANT. H. J. (1958) The two hemoglobin components of the chicken. Science 127, 762-763. HILL R. L. & BU~TTN~a-JANUSCHJ. (1964) Evolution of hemoglobin. Fed. Proc. 23, 12361242. HILL R. L., BUETTNER-JANUSCHJ. & BUETTNER-JANUSCHV. (1963) Evolution of hemoglobin in primates. Proc. Nat. Acud. Sci. Wash. 50, 885-893. MANWELLC., BAKERC. M. A., ROSLANSKYJ. D. & FOUGHTM. (1963) Molecular genetics of avian proteins. II. Control genes and structural genes for embryonic and adult hemoglobins. Proc. ,Vat. Acad. Sci. Wash. 49, 496-503. PAULINe L. & ZUCrmaKANDLE. (1963) Chemical Paleogenetics: Molecular Restoration. Studies of extinct forms of life. Acta Chem. Scan& 17, $9-S19. RODNm'~ G. P. & EBAUGHF. G. (1957) Paper electrophoresis of small animal hemoglobin. Proc. Exp. Biol. ,~/ied. 95, 397--401. SAHAA. (1956a) Studies on chick hemoglobin by means of paper electrophoresis. Science Culture, Calcutta 21, 756-758. SAHAA. (1956b) Studies on the alkaline denaturation of chick hemoglobin. Indian ]. Physiol. 10, 87-93. SAHAA. (1960) In vitro biosynthesis of chick hemoglobins. Biochim. Biophys. Res. Comm. 2, 450-454. SAHA A. (1964) Comparative studies on chick hemoglobins. Biochim. Biophys. Acta 93, 573-584. SAHA A, DUTTA R. & GHOSH, J. (1957) Paper electrophoresis of avian and mammalian hemoglobins. Science 125, 447-448. SAHA A. & GHOSHJ. (1960) Biosynthesis of chick hemoglobins. Science 132, 468-470. SIBLEY C. G. (1960) The electrophoretic patterns of avian egg-white proteins as taxonomic characters. Science 102, 215-284. SINGER K., CHERNOFF A. I. &StNGER L. (1951) Studies on abnormal hemoglobins. I. Their demonstration in sickle cell anemia and other hemolytic disorders by means of alkali denaturation. II. Their identification by means of the method of fractional denaturation. Blood 6, 413--428, 429-435. WILSON S. & SMITH D. B. (1959) Separation of the valyl-leucyl and valyl-glutamyl polypeptide chains of horse globin by fractional precipitation and column chromatography. Canad. J. Biochem. Physiol. 13, 405-416. WmT F. H. (1962) The ontology of chick embryo. Proc. Nat. Acad. Sci. Wash. 48, 1582-1590.