Comparative studies on chick hemoglobins

BIOCKIMICA ET BIOPHYSICA ACTA

573

BBA 2 5 1 5 1

C O M P A R A T I V E S T U D I E S ON C H I C K H E M O G L O B I N S ANIL SAHA

Gates and Crellin Laboratories of Chemistry*, California Institute of Technology, Pasadena, Calif. (U.S.A.) (Received March i6th, 1964)

SUMMARY

Studies on chick Hemoglobins I and 2 were carried out by means of ion-exchange chromatography and amino acid analysis of the proteins and their subunits, and, chromatography of peptides obtained b y tryptic hydrolysis of the proteins and by chymotryptic hydrolysis of the trypsin-resistant core. Amino acid analysis of hemoglobins reveals that chick Hemoglobin I contains a greater number of amino acid residues in comparison with chick Hemoglobin 2. Amino acid composition of c%- and fl2-chains of Hemoglobin I and Hemoglobin 2 bears the same relationship. The number of changes in amino acid residues between ~2- and fl2-chains has been found to be 15 and 35 in Hemoglobin I and Hemoglobin 2, respectively. The resemblance between the two subunits of one hemoglobin is greater than that between the chains from the other hemoglobin. The amino acid composition of the peptides as isolated b y peptide chromatography distinguishes the similar and dissimilar features in the structure of Hemoglobins I and 2.

INTRODUCTION

Chick erythrocytes contain two hemoglobins which differ in their electrophoretic mobilities in paper electrophoresis and free-boundary electrophoresis, and, also in their elution characteristics on ion-exchange columns 1-5. They have also been shown to differ in their resistance to alkaline denaturation 6. The relative proportion of Hb I in chick erythrocytes decreases with embryonic development while that of chick Hb 2 increases 1. The rate of incorporation of radioactive amino acids into chick Hb I in vivo is slower in comparison with that of chick Hb 2 (ref. 7). In view of these experimental observations it was of interest to investigate the structure of these two cognate proteins. This communication describes the results of the preliminary chemical investigation on chick Hbs I and 2. MATERIALS AND METHODS

Citrated blood was collected by venepuncture from adult white leghorn chicks. Chick erythrocytes were washed thrice with four times their volume of 1 % NaC1 solution at 5 ° and were then lysed with an equal volume of distilled water and o. 4 volume of * Contribution No. 3098.

Biochim. Biophys. Acta, 93 (1964) 573-584

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A. s AI-IA

toluene e. Clear hemoglobin solution was collected the next day by centrifugation twice at IOOOO x g for 30 min at 5 °. Ion-exchange chromatography on IRC-5o XE-64 was employed to separate the hemoglobins. Hemoglobin solution (2 ml, 5O-lOO mg) was applied to an IRC-5o XE-64 column, 1.5 cm >.:: 35 cm, which was previously equilibrated with sodium citrate buffer (pH 6.5) containing o.15 N Na + (ref. 8). The column was developed with the initial developer (500 ml of sodium citrate buffer (pH 6.5) containing o.I5 N Na +) and changed to sodium citrate buffer (pH 6.5) of higher Na + concentrations. Buffer solutions used in chromatographic development contained 20 mg of KCN per 1.5-ml fractions were collected and absorbancy of each fraction was determined at 415 mt~ with a Beckman spectrophotometer. The peak zones were pooled and concentrated by centrifugation of the dilute solution at IOOOOO × g for 16 h. Concentrated hemoglobin solution thus obtained was freed of salt by dialyzing against several changes of distilled water (8 1) adjusted to pH 8.0 with a few drops of 4 N NH4OH. Aliquots containing approx. 5 mg hemoglobin (dry wt.) were used for the determination of amino acid composition. Hydrolysis of protein samples (dried at IiO °) was carried out in a sealed tube with 6.0 N HC1 at IIO ° for 22 and 7 ° h. After completion of hydrolysis, HC1 in the solution was driven off by blowing air directly into the hydrolysis tube which was placed in a water bath at 45 °. The dried sample was then dissolved in an appropriate volume (5 or IO ml) of sodium citrate buffer (pH 2.2) containing 0.2 N Na +. Amino acid analysis was carried out in a Beckman model 12o B amino acid analyzer using an accelerated procedure of amino acid analysis on 0.6 cm × io cm and 0. 9 cm × 60 cm columns with a buffer flow-rate of 40 ml/h and ninhydrin-reagent flow-rate of 20 ml/h at 52.7 °. Performic acid oxidation of hemoglobins was carried out according to MOORE9 and carbamidomethylation was carried out according to GOLDSTEINet al. 1°. Carbamidomethylated and oxidized hemoglobin samples were hydrolyzed in 6.0 N HC1 at IiO ° for 24 h and the amino acid analysis was carried out as mentioned earlier.

Enzymatic hydrolysis Tryptic hydrolysis of chick hemoglobins denatured for 4 rain at 95 ° was carried out at pH 8.0 for 8 h at 38° in a pH-stat (Radiometer Autotitrator, Copenhagen, Denmark). Trypsin (EC 3-4.4.4) (Worthington Biochemical Corporation) at an enzyme per substrate ratio of I:IOO (w/w), was added at o, 1.5, and 4 h. At the termination of enzymatic hydrolysis, the solution was adjusted to pH 6.5 with o.I N HC1. The precipitate (trypsin-resistant core) that resulted was spun down and the clear supernatant was used for chromatographic fractionation of peptides. The trypsin-resistant core was washed twice with distilled water, resuspended in water and adjusted to pH 7.8 with 0.2 N NaOH. Chymotryptic digestion of the trypsin-resistant core was carried out for 4 h at 38° in the pH-stat. Chymotrypsin (EC 3.4.4.5) (Worthington Biochemical Corporation) (enzyme/substrate weight ratio of 1:5o) was added at o h and at 1.5 h. After the completion of enzymatic hydrolysis, the solution was adjusted to pH 6.5 with o.I N HC1 and the precipitate that resulted was removed by centrifugation. The soluble peptides contained in the supernatant were fractionated by ionexchange chromatography. Chromatographic separation of the soluble peptides was performed on a column of dimension o. 9 cm × 22 cm, containing Amberlite IR-I2O of size 19/~ (supplied by Beckman Instruments, Inc.) at 52.7 ° in a Beckman amino acid analyzer equipped Biochirn. Biophvs. dcta, 93 (1964) 573 584

CHICK HEMOGLOBINS I AND 2

575

with a stream-divider p u m p assembly. Sample (2-3 ml in volume) was adjusted to p H 2.2 with HC1 and applied on the column which was initially equilibrated with o.15 N pyridine-acetic a c i d - w a t e r (pH 2.97) 11. The column was then developed with a linear gradient of buffers, o.15 N pyridine-acetic a c i d - w a t e r (pH 2.97), and 2.o N pyridine-acetic a c i d - w a t e r (pH 5.o). For comparative studies of the peptides obtained b y enzymatic hydrolysis of lO-12 mg hemoglobin, the flow of buffer and of ninhydrin reagent was maintained at a rate of 22.5 ml/h each. For preparative purposes the peptides from 6o-7o mg hemoglobin were applied on the column and the buffer flow-rate through the chromatographic column was maintained at 4o ml/h. A continuous withdrawal at the rate of 6 ml/h was made from the column effluent by the stream-divider p u m p assembly and mixed with a flow (34 ml/h) of sodium citrate buffer (pH 5.26) containing o.2 N Na ÷ and a flow of 2o ml/h of ninhydrin reagent. The recording chart speed was maintained at 6 in/h and fractions were collected at 6-rain intervals. The fractions containing the peptide zone were collected, dried under air current, and hydrolyzed in 6 N HC1 at IiO ° for 24 h. Amino acid composition of the peptides was determined by using the accelerated procedure of amino acid analyses.

Separation of polypeptide chains The polypeptide chains of chick hemoglobins were separated by ion-exchange chromatography according to WILSON"AND SMITH12. Hemoglobin solution as isolated by chromatography on IRC-5o XE-64 was treated with H C l - a c e t o n e 1~ at - - 2 0 °. The precipitated globin was dissolved in water and dialyzed against several changes of water at 3 °. Globin (7O-lOO mg) solution was dialyzed twice against IO % formic acid (3 1 each time) at 3 ° and applied on an IRC-5o XE-64 column, 0. 9 c m x 58 cm, which was previously equilibrated at room temperature with IO % formic acid. 500 ml of 6 M urea (pH 1.9) was fed to a mixing vessel containing 125 ml of 2 M urea (pH 1.9), followed by 600 ml of 8 M urea (pH 1.9). Chromatography with buffer containing urea was carried out at room temperature with a flow rate of 20 ml/h, and Io-ml fractions were collected throughout the chromatographic procedure. The zones containing the peaks were pooled, dialyzed against several changes of water until free of urea and then lyophilized. The lyophilized material was further dialyzed against water adjusted to p H 8.0 with 4 N NH4OH and an aliquot (approx. 5 mg globin, dry wt.) was used for amino acid analysis as mentioned earlier, c%- and fi2-chains represent the first and the second peaks as they emerged from the column in the succeeding order. RESULTS

Chromatography of chick hemoglobins Chromatography of hemoglobins on ion-exchange columns depends on various factors, e.g., ionic strength of buffer, pH, size of the column, and amount of protein present in the sample 14. Fig. IA represents the elution characteristics of chick Hbs I and 2 (50-60 rag) chronaatographed on IRC-5o XE-64 column with sodium citrate buffer. The initial developer has been sodium citrate buffer (pH 6.5), Na + concentration of o.16 N (18o ml), followed b y o.17 N (600 nal), 0.2 N (625 ml) and 0.25 N (Fig. IA). The zone containing chick Hb 2 ranged from 360 to 720 ml and that containing chick Hb i from 880 to 15oo ml. The plateau between the two peaks contained

Biochim. Bioph),s. Acta, 93 (1964) 573-584

576

A. SAint

approx. IOO ml. Reduction in the steps of development b y using the initial developer of higher ionic strength reduces the plateau. Fig. IB shows the elution characteristics of chick hemoglobins (5o-6o mg) with the initial developer sodium citrate buffer (pH 6.5) of o.18 N Na + (37 ° ml), followed b y sodium citrate buffer (pH 6.5) of Na +

m m Eo

[ oz ozoNN/ o ~oo

soo

900 EFFLUENT

Izoo

VOLUME(ml)

Fig. i a. I

I

I

I

I

6.0

~t

4o < m

E0

025N

0 I00

..

300

500

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700

VOLUME (rod

Fig, ] b. Fig. I. C h r o m a t o g r a p h y of chick hemoglobins oi1 IRC-5o XE-64.

ion concentration 0.2o N (55 ° ml) and 0.25 N (700 ml). The effluent volume containing each zone was smaller compared to the chromatogram as seen in Fig. IA. In the experiments designed to separate larger amounts of chick Hbs I and 2 from chick erythrocyte lysate a column, 3.o cm × 25 cm, was equilibrated with sodium citrate buffer (pH 6.5) containing o.18 N Na +. After the application of the sample on the column, the column was developed with 3oo ml of the initial developer and followed by sodium citrate buffer (pH 6.5, o.2 N Na +) until chick Hb 2 was displaced from the column. Chick Hb I was then eluted with sodium citrate buffer (pH 8.o, o.2 N Na+). Under these conditions the zone containing chick Hb I could be collected in a small volume. Although minor peaks containing heme-pigment, as distinguished by the absorbancy of the fractions at 28o and 415 m/x, emerged from the column preceding the main Biochi,,. Biophys. Acla, 93 (1964) 573-584

CHICK HEMOGLOBINS I A N D 2

577

peaks, attention could not be paid to those peaks. Starting agent development on IRC-5o (refs. 3, 4), pH-g radienta'5, and salt-gradienO 5 development on CM-cellulose have been reported. The relative proportion of Hb I and Hb 2 as separated in Calcutta, India, from chick erythrocyte lysate was 60 and 40 %, respectively; whereas the value obtained in this laboratory was approx. 80 and 20 %, and that reported by VAN DER HELM AND HUISMAN4 was 85 and 15 3/0. Studies have, however, not yet been made as to whether or not the relative proportion of these hemoglobins is related to zoogeographical speciation.

Amino acid composition of chick hemoglobins Table I presents the amino acid composition of chick Hbs I and 2. It is interesting to note that the total number of amino acid residues in chick Hb I (626) is greater in comparison with that in chick Hb 2 (594). Table I shows that there exists a considerable difference in the amino acid composition of these two hemoglobins. There are TABLE

I

AMINO ACID RESIDUES IN CHICK H b s I ANn 2 R e s u l t s w e r e e x p r e s s e d as a m i n o acid residues in h e m o g l o b i n m o l e c u l e of m o l e c u l a r w e i g h t 64 500. C y s t e i c acid w a s d e t e r m i n e d after p e r f o r m i c acid o x i d a t i o n 9 a n d c a r b a m i d o m e t h y l c y s t e i n e w a s d e t e r m i n e d a c c o r d i n g to GOLDSTEIN el al. 1°. Amino acid

Lys His Arg CySH Asp Thr Ser Glu Pro Gly Ala Val Met Ileu Leu Tyr Phe C a r b a m i d o m e t h y l CyS

Hb z

Hb 2

47 34 19 8 53 3o 24 49 25 38 80 58 7 29 74 17 33 7

44 26 19 8 54 28 27 61 22 34 7° 54 12 18 68 17 32 6

eight residues of cysteic acid obtained from each hemoglobin after performic acid oxidation. However, carbamidomethylation of chick hemoglobins produced seven and six residues of carbamidomethyl cysteine in case of chick Hb I and Hb 2, respectively. The numbers of basic (lysine, histidine, and arginine) and acidic (aspartic and glutamic acid) amino acids are IOO, lO2, and 89 and 115 for Hb I and Hb 2, respectively. It m a y be mentioned that the results reported by VAN DER HELM AND HUISMAN4 varied considerably from the results presented herein. Isoleucine is present in human fetal hemoglobin and not in adult human hemoglobin. In analogy, chick Hb 2 which increases in relative proportion during embryonic development 1 contains lesser amount of isoleucine. Amino acid composition of these two hemoglobins is in good agreement Biochim.

Biophys.

A c t a , 93 (1964) 573-584

578

A.

SAI-[A

with their electrophoretic behavior and elution characteristics. In comparison with hemoglobins from mammalian species like man1",17, elephanO ~, horse 19, guinea pig 18, mouse is, and some fish hemoglobins 2°, chick Hb I and H b 2 contain larger amounts of glutamic acid, S-containing amino acids, and isoleucine.

Comparison of soluble peptides Fig. z shows the chromatographic separation of the soluble peptides obtained by tryptic hydrolysis of chick hemoglobins (lO-12 mg hemoglobin). There were 31 tryptic peptides which could be discerned in the case of chick Hb I while under similar con9

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Fig. 2. C h r o m a t o g r a p h i c s e p a r a t i o n of soluble t r y p t i c peptides o b t a i n e d from chick H b s 1 a n d 2.

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peptides from trypsin-resistant core

ditions 2 9 peptides could be recognized with chick Hb 2. There were 15 peptides which were numbered I to 15 in the succeeding order of emergence from the column and possessed the c o m m o n elution characteristics besides an equal n u m b e r of peptides with dissimilar characteristics. The peptides n u m b e r e d A to I were specific for Hb I. In comparison with an early investigation on the fingerprints of soluble tryptic peptides obtained from chick hemoglobins 21, the separation presented herein is more distinctive and the differences are larger in number. A comparative s t u d y of the c h y m o t r y p t i c peptides obtained from the trypsinresistant core of H b I and H b 2 was also undertaken and the results were shown in Fig. 3. E a c h prime on a peak represents a peptide zone with c o m m o n elution characteristics. The n u m b e r of peptides with comparable elution characteristics was relatively large in this case. The presence of acidic amino acids (Table I) proportionately in a greater quantity, possibly produces a core resistant to the enzymatic action of Biochim. Biophys. Acla, 93 (1964) 573 -584

CHICK HEMOGLOBINS I AND 2

579

trypsin. Similar to the results as presented in Fig. 2, there were peptides specific to the individual hemoglobin, but they were relatively few in comparison with those obtained in the case of tryptic peptides. Fig. 3 indicates the possibility of a greater accordance in the structures of Hb I and Hb 2 around leucine and phenylalanine residues.

Amino acid compositio~, of peptides Tables II and III present the amino acid composition of some of the peptides as obtained by the tryptic digestion of chick hemoglobin and by the chymotryptic digestion of trypsin-resistant core, and isolated by the preparative ion-exchange chromatography. Not every peptide could be analyzed as the yield of such peptides was too small to determine the amino acid composition with the amino acid analyzer under the present experimental conditions. An even distribution of aspartic acid, TABLE II AMINO ACID COMPOSITION OF TRYPTIC PEPTIDES FROM CHICK I{b I

PeptideNo. I 2 I 3

4

Aminoacids Asp, Glu, Gly Asp,Thr2 Asp,Ala, Ileu Lys, Asp, Glu, Pro, Gly, Ala,Val, Ileu, Leu

Lys, Asp, Gly, Ala2,Val~,Met,Leu,Phe

H

Lys2,Thr 2, Alas,Met, Ileu,Leu

G F E 9

Lys, Asp,Ser, Glu,Ala2,Val,Leu Lys, Asp2,Glu,Ala, CyS,Ileu,Leu Lys, His, Asp, Glu2,Gly, Ala2,Val,Leu2 Lys,Asp,Ala, Leu

IO

Lys2, His,Thr, Gly, lleu, Phe

C II B

His,Arg, Asp,Glu,Pro,Gly, Ala,Val,Leu His,Arg,Ala2,Val,Leu Lys, His,Glu~,Ala,Val

A

Lys, His2,Asp,Ala,Leu 2

12 13 14 15

His~,Tyr Arg,Leu Lys, His,Gly, Ala Lys, His,Gly

alanine, and leucine has been found in the tryptic peptides and trypsin-resistant core peptides whereas the core peptides contain a larger quantity of glutamic acid and valine. The amino acid composition of chymotryptic peptides obtained from trypsin-resistant core indicates that the trypsin-resistant core is highly charged and hydrophobic in nature. Although only glycine was found in Peptides B and 36, it seems likely that Peptide B is a diglycyl or a triglycyl residue. The presence of free amino acids like glycine and alanine or dipeptides like Gly-Gly, Ala-Ala, and Val-Val (Peptides B, 26, 38, Table III) in the chymotryptic digest of trypsin-resistant core may be attributed to the configuration of the insoluble tryptic peptides and to the action of chymotrypsin depending on the position of leucine and phenylalanine.

Amino acid composition of chick-hemoglobin chains Table IV shows the amino acid composition of c%- and fl2-ehains of chick hemoglobins. It may be observed that ~ - and/~2-chains vary considerably in their amino Biochim. Biophys. Acia, 93 (1964) 573-584

580

A. SAHA TABLE III AMINO

ACID

COMPOSITION

OF

CHYMOTRYPTIC CORE

OF

PEPTIDES

CHICK

Hb

FROM

TRYPSIN-RESISTANT

I

Peptide No.

A mino acids

I-2 5

Asp.a,Glu,Pro, Gly, Ala2,Val, lleu, Leu Asp, Ala,Met, Leu

A

Asp,Glu,Gly, Ala,Val,lleu,Leu 2

7

Asp, Glu,Leu

8 9

Asp,Thr, Ser, Gly,Val,Ileu,Leu,Phe Asp, Ser, Glu,Ala,Phe Thr, Ser, Pro, Ala, Phe

IO Ii 13 5 t6 I7-18 21 22 B 23 26 27 29 C 31 36 38

Asp, Ala,Val,Leu Asp,Glu, Ala,Val, l l e u , L e u Ser, Glu, Gly Glu,Ala

Gly, Ala2,Val2,Leu Gtu,Ala, P h e Asp,Glu, Gly, Ala,Val, Leu Gly Glu, Ala, Val, P h e Ala AIa, Leu Glu Thr,Ser, Olu, Gly, Ala,Val, Leu Thr, Glu, Gly, Ala,Val2, Leu Gly Val

T A B L E IV AMINO ACID R E S I D U E S IN ~ 2 - A N D f l ~ - C H A I N S OF CHICK H b s I A N D 2 R e s u l t s were e x p r e s s e d as t h e n u m b e r of residues in a molecule of m o l e c u l a r w e i g h t 31ooo. Cysteie acid was d e t e r m i n e d b y p e r f o r m i c acid o x i d a t i o n 9. Hb •

Hb 2

A m i n o acid

Lys His Arg CySH Asp Thr Ser Glu Pro Gly Ala Val Met Ileu Leu Tyr Phe

24 17 9 4 27 15 1i 23 13 19 4° ;9 4 14 37 9 I0

23 16 io 4 26 14 12 25 13 19 4° 28 3 15 38 7 17

23 14 9 3 27 14 l4 33 1I 16 36 29 7 7 34 8 15

21 ii io 5 26 13 13 27 1i 17 32 25 5 io 34 9 17

Biochim. Biophys. Avla, 93 (1964) 573-584

CHICK HEMOGLOBINS I AND 2

581

acid content, el_, ]~21_, (X22 ' and/~2~-chains contain 311, 31o, 300, and 286 amino acid residues, respectively, suggesting thereby that ~ 1 and/~l-chains are slightly larger in dimension in comparison with c~22-and/3~-ehains. %1_ and ~22-chains contain a few residues more than/3a 1- and/322-chains, a21- and/322-chains resemble each other to a greater extent in comparison with either ~22- or/3~2-chains. Comparing a2- and /32-chains with respect to the number of individual amino acids replaced in each polypeptide chain, it is 15 between =21 and [331 while the number of amino acids replaced between ~22- and/322-chains is 35- The number of amino acid residue substitntions between ~21 and e 2 is 42 while that between ~1 and/322 is 45. Likewise there are 44 amino acid substitutions between /~21 and ~22, and 40 between /3~1 and /322. Glutamic acid content is m a x i m u m in ~2~ and minimum in c~21.The number of basic amino acids is m a x i m u m in c~21, while that of acidic amino acid is m a x i m u m in c~22. Although cysteic acid content of c~1- and/3~l-chains is the same, the ~2 and/~2-chains contain three and five residues of cysteic acid, respectively. The number of changes in amino acid residues between eA and/3 A, (xA and yF, /3A and 7 F have been reported to be 37, 37, and 30 (refs. 16, 17). Table IV shows that the number of isoleueine and glycine residues reaches a minimum in ~2-chain. The amino acid composition of a2 A-, /32*-, and 72r-chains of human hemoglobin provides a comparable situation where the number of glycine residue is minimum and isoleucine residue is nil with a2A-chain. The hybridization of chick hemoglobins produced three hemoglobins, as observed in free-boundary electrophoresis 7. The view that one of the half molecules m a y possibly be common to both Hb I and Hb 2 appears untenable on the basis of the amino acid composition of =~- and/3~-chains. Considering the charge difference between the hybrid molecule and H b I and Hb 2 it seems reasonable to postulate that the molecule formed during the hybridization resulted from the combination of a~l-chain of chick Hb I and/3z~-chain of chick H b 2. In this connection one should consider the complementarity between ~,- and /~2-chains of hemoglobins ~2 and the failure of these molecules to form the complementary structure, i.e., the misfit m a y be one of the main reasons for observing only three distinguishable species of molecules on hybridization. DISCUSSION Studies on chick hemoglobins present a situation where eight chains of polypeptide moiety, a and 13, incorporated into two hemoglobins are synthesized in the bird. It has been reported b y WILT23 that there are two globin-like components in the unincubated blastoderm, one of them appears to be minor adult globin component while the other is immunologically distinguishable from both adult components and disappears at 36-48 h of incubation. The presence of adult hemoglobins has been observed after approx. 48 h of incubation 23,24-2s. Despite the wide variation in their amino acid composition, the concurrent persistence of Hb I and Hb 2 was observed throughout the life-period of a chick. Earlier investigation has revealed the existence of a differential rate of biosynthesis of Hb I and Hb 2 b y nucleated chick erythrocytes under different conditions of milieu 7,~7; and under the experimental conditions studied so far, both hemoglobins were found to be synthesized. This observation suggests that the mechanism of the alignment of amino acids in a polypeptide chain in a definite Biochim. Biophys. Acta, 93 (I964) 573-584

582

A. S A H A

pattern can withstand a tremendous strain which might reflect in the differential rate of biosynthesis, depending upon the degree of severity. This adaptive mechanism may in all probability provide the means for the supply of oxygen during the multiphase changes in the avian life although it cannot be determined with certainty whether the presence of multiple hemoglobins is a phenomenon of evolutionary inheritance or a biological adaptation, followed by mutational changes. TABLE ELECTROPHORETIC

MOBILITY

V OF

AVIAN

HEMOGLOBINS

P a p e r e l e e t r o p h o r e s i s o f a v i a n h e m o g l o b i n s w a s c a r r i e d o u t i n b a r b i t u r a t e b u f f e r ( p H 8.6, I 0 . 0 5 ) a t 4 ° a t 2 2 o V f o r i 6 h (ref. 2). A v i a n h e m o g l o b i n s w e r e c a t e g o r i z e d o n t h e b a s i s o f t h e i n o b i l i t i e s towards anode. Order of mobility Natural order

English name z

Pelicaniformes Ciconiiformes Anseriformes Anseriformes Falconiformes Galliformes Galliformes Galliformes Galliformes Gruiformes Gruiformes Columbiformes Columbiformes Columbiformes Columbiformes Psittaciformes Psittaciformes Cuculiformes Cuculiformes Strigiformes Coraciiformes Coraciiformes Picitormes Piciformes Piciformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes

Little cormorant Cattle egret Domestic duck Common pochard Kite Guinea fowl Domestic fowl Grey quail Grey partridge Water hen Coot Spotted dove Pigeon Bengal crown pigeon Turtle dove Rose-ringed parakeet Black-headed parakeet Hawk cuckoo Indian cuckoo Owl Blue jay Kingfisher Lineated barbet Blue-throated barbet Golden-back woodpecker Red-vented bulbul Red-whiskered bulbul Kabasi Magpie robin Yellow-eye babbler Hogkin's bushchat Jungle babbler Indian red munia \Vhite-throated munia Spotted munia Black-headed munia House sparrow Common inyna Bank mvna Hill myna Black-headed oriole Tree pie House crow

2

* * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *

3

* * * * * * * * * *

4

5

*

* *

* * * * * * * * * * * *

* * *

*

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* * *

*

Biochim. Biophys. Acta,

93 ( t 9 6 4 ) 5 7 3 - 5 8 4

CHICK HEMOGLOBINS

I AND 2

583

Table V shows the electrophoretic mobility 2s of different avian hemoglobins as determined according to a method published previouslyl,E The avian hemoglobins mentioned in Table V represent a very small cross-section of the total number of birds present in the region around Calcutta, India. These birds represent the Himalayan mountain chain of the oriental region and m a y be narrowly classified as representing the Gangetic basin of Eastern India sub-region. Most of the birds possess two hemoglobins while the number of avian hemoglobins ranges from one to three in a particular bird. The electrophoretic behavior would reflect on the amino acid composition of these avian hemoglobins. The natural orders in Table V have been arranged from the primitive order to the recent ones. Table V shows that the number of hemoglobins in a particular avian species does not correspond to the evolutionary scale. It has not been possible to find out a bird like little Cormorant in possession of three hemoglobins with a wide difference in the electrophoretic mobility. I t m a y be noted that electrophoretically chick Hb I and Hb 2 belong to a rather large group of birds. The interrelationships of these hemoglobins m a y be evaluated b y the study of the amino acid conformation. A comparative study of the peptide chromatograms m a y provide information regarding the genic evolution resulted in the mutational change in a conservative protein like hemoglobin in the different avian species. Study along this line has already been suggested b y PAULING AND ZUCKERKANDL29. Preliminary studies on the peptide pattern of hemoglobins from a single mammalian order, the Primates, ranging from more primitive forms to recent ones, have been reported by HILL et al. a°. A similar comparative study in avian species has been carried out and will be reported elsewhere. The amino acid compositions of Hb I and Hb 2 are in conformity with the observations revealing the dissimilarities in the characteristics of the molecules, e.g., electrophoresis, chromatography, resistance to alkaline denaturation. The comparison of the chromatograms (Figs. 2 and 3) indicates that there are several zones of similarity between the peptides obtained from chick Hb I and Hb 2. Although the peptides with comparable elution characteristics m a y possess a relatively identical structure, the accordance could only be accepted with the complete amino acid sequence of each peptide. However, useful information could be obtained b y carrying on the investigation as outlined herein, and attempts could be made to understand the molecular evolution of hemoglobin in the avian kingdom.

ACKNOWLEDGEMENTS

The author expressed his thanks to Dr. R. T. JONES for peptide chromatography using amino acid analyzer and his gratitude to Dr. W. A. SCHROEDER for his suggestions and advice during this investigation. The author is grateful to Dr. D. H. CAMPBELL for his generous help. REFERENCES 1 A. 2 A. 3 A. 4 H. 5 T. 6 A.

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