Carnivora: The Primary Structures of Adult Lion
(Panthern leo) Hemoglobins*
Meeno Jahan, Aftab Ahmed, and Gerhard Braunitzer**
Abt. Proteinchemie Max-Planck-Institut für Biochemie,
D-8033, Martinsried bei München, FRG
Zafar H. Zaidi
H. E. J. Research Institut of Chemistry, University of Karachi. Karachi-32, Pakistan
Reinhard Göltenboth
Zoologischer Garten. D-1000 Berlin
Z. Naturforsch. 42b, 1465—1470 (1987); received July 23, 1987
Lion, Hemoglobin, HPLC, Primary Structure
Complete amino acid sequences of lion (Panthera leo) hemoglobins are reported. Polyacryl­
amide gel disc electrophoresis at pH 8.3 and also in the presence of 8 M urea and Triton-X100,
RP-HPLC, liquid and gas phase amino acid sequenator. and FAB Mass spectrometer are used.
Sequences are aligned with human hemoglobin (Hb A) and compared with other hemoglobins of
felidae.
Introduction
Electrophoresis
Lion is classified into the order of Carnivora, fam­
ily of Felidae, genus of Panthera. Electrophoresis of
hemoglobin on starch gel and cellulose acetate is re­
ported [2, 3]. Bunn et al. [4] have determined that
the erythrocytes from the members of order carni­
vora have very low red cell 2,3 DPG. Chemical
studies from genus Panthera have been limited to
amino acid composition of tryptic peptides from lion
hemoglobin [3, 5] and amino acid sequence of major
hemoglobin component of Amur leopard [6]. In this
communication we present complete amino acid se­
quence of lion hemoglobins.
Disc electrophoresis of crude hemoglobin was
done on 10% polyacrylamide gel in Tris/glycine buf­
fer pH 8.3 [7]. Electrophoretic separation of globin
chains achieved on Triton gel in the presence of 8 M
urea and Triton-X100 [8].
Experimental
Preparation of hemolysate
Blood sample from lion of African origin, born in
zoological garden Berlin was collected in heparinized
tube. Erythrocytes were washed with physiological
saline and lyzed with distilled water, for 1 h in cold
[1]. The solution was centrifuged and dialyzed
against distilled water for 5 h.
* Communication on hemoglobin.
** Reprint requests to Prof. G. Braunitzer.
Abbreviations:
Q uadrol
=
N,N.N,N-tetrakis(2-hydroxypropyl)ethylendiamine. Reagent IV = trisodium
7-(isothiocyanato)-naphthalene-1.3,5-trisulphonate.
Verlag der Zeitschrift für Naturforschung, D-7400 Tübingen
0932-0776/87/1100-1465/$ 01.00/0
Separation of globin chains
Hemoglobin was dehemed with acidic acetone [9].
The separation of globin chains was carried out with
the reversed phase HPLC method using a Beckman
liquid chromatograph-334, controller 421, pump
110 A, UV-detector Biotronik-BT-3030, injector
from Altex and integrator from Shimadzu CRI-A. A
column of LiChrosorb-RP 2, (E. Merck) was equili­
brated with 12% formic acid in 50 mM ammonium
acetate. Globin chains were eluted with a gradient of
acetonitrile from 35—60% in 60 min.
Enzymatic cleavage
Isolated globin chains were oxidized [10], en­
zymatically cleaved [11] with TPCK trypsin (E.C
3.4.21.2) from cooper Biomedical in 5:100 mg en­
zyme substrate ratio at pH 10.5 for 2 h and at pH 9.5
for 1 h. The pH of enzymatic hydrolysate was low­
ered by titrating with dilute acetic acid to pH 4.
Chromatography of tryptic peptides
Prefractionation of tryptic peptides was done on a
column of sephadex G-25 (fine). Prefractioned peaks
were further purified by RP-HPLC [12], using a
LiChrosorb RP-2 column, equilibrated with 50 mM
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M. Jahan et al. • Carnivora: The Primary Structures of Adult Lion (Panthera leo) Hemoglobins
ammonium acetate. Elution of peptides was
achieved by applying a gradient of acetonitrile from
0—60% in 60 min.
Sequence determination
Amino acid sequence of tryptic peptides were de­
termined by Edman degradation [13] in liquid phase
sequencer Beckman 890 B and 890 C. A Quadrol
programe [14] was used for the sequence of native
chains and lysine peptides. Lysine peptides were
modified by reagent IV [15] priot to sequence. A
3-(dimethylamine) propyne programe [16] was em­
ployed for arginine peptides. Some peptides were
also sequenced by gas phase sequenator [17]. PTH
derivatives of amino acid were identified by HPLC
[18]. Sequence of blocked peptide was determined
by FAB-mass spectroscopy.
peptides is presented in supplementary material
(Table I- III).
By FAB mass spectroscopy N-acetyl serine as a
N-terminal group of ß l chain was detected. The com­
plete amino acid sequence of globin chains are given
(Fig. 3). Sequences aligned with human (Hb A ) re­
vealed 23 (16.3%) substitution in a-chain, 29
(19.8%) in ß l and 28 (19.1%) in II chains. Two
differences observed between ß l and ß l l chains at
031/ ß l l ) : ß(NAl)Ac-Ser/Gly, /?(HC l)Arg/Lys. The
exchanges are distributed over the entire length of
molecule. Four a I ß I contact points: a34(B15)
Leu-Cys, a lll( G 1 8 ) Ala-Cys, 0123(H1) Thr-Asn,
Results and Discussions
Electrophoretic separation of hemoglobins on disc
at pH 8.3 and under dissociating condition in pres­
ence of 8 M urea and Triton X-100 showed two
hemoglobin components and three globin chains a,
ß l , and ß l l (Fig. 1). Presence of two hemoglobin
components was also observed on cellulose acetate
electrophoresis by Brimhall et al. [3]. Separation of
three globin chains achieved by reversed phase
HPLC (Fig. 2), supports the result of Triton gel elec­
trophoresis.
Prefractionation of tryptic peptides gave some of
the peptides in pure form. By rechromatography of
peptides mixture on RP-HPLC peptides obtained in
poure form. Amino acid composition of the tryptic
TIME [ m i n ]
A
B
Fig. 1. Electrophoretic pattern of crude lion hemoglobin
on polyacrylamide gel. A. Disc at pH 8.3 B. Under dis­
sociating condition. 8 M urea and Triton X 100.
Fig. 2. Elution pattern of lion globin
chains on RP-HPLC LiChrosorb RP-2
column; buffer: 12% formic acid/
50 mM ammoniumacetate; gradient:
35 —60% acetonitrile in 60 min.
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M. Jahan et al. ■Carnivora: The Primary Structures of Adult Lion (Panthera leo) Hemoglobins
NA
A________________________________________________________________ AB
Pro
Hu
X
PI
P1
Hu
X
Val-Leu-Ser-Ser-Ala-Asp-Lys-Asn-Asn-Val-Lys-Ala-Cys-Trp-Gly-Lys-I Ie-Gly-Ser-His-Ala-Gly-Glu-Tyr-Gly-AlaBIAcSer-Phe-Leu-Ser-Ala-Glu-Glu-Lys-Gly-Leu-Val-Asn-Gly-Leu-Trp-Ser-Lys-Val-Asn-Val-Asp-Glu-Val-Gly-GlyBA Val-His
Thr-Pro
Ser-Ala
Thr-Ala
20
NA
Thr
10
Ala
Val
B____________________________
Ala 20
A
26
B
CD
Met
Leu
40
Glu-Ala-Leu-Glu-Arg-Thr-Phe-Cys-Ser-Phe-Pro-Thr-Thr-Lys-Thr-Tyr-Phe-Pro-His-Phe-Asp-Leu-Ser-HisGlu-Ala-Leu-Gly-Arg-Leu-Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe-Phe-Gln-Ser-Phe-Gly-Asp-Leu-Ser-Ser-Ala-Asp40
Glu
Thr-Pro
C
CD
D
EF
Lys
Lys
Asn
70
Val-Asp-Gly-Ser-Ala-Gln-Val-Gln-Ala-His-Gly-Gln-Lys-Val-Ala-Asp-Ala-Leu-Thr-Lys-Ala-Val-Val-His-Ile-AsnAla-Ile-Met-Ser-Asn-Ala-Lys-Val-Lys-Ala-His-Gly-Lys-Lys-Val-Leu-Asn-Ser-Phe-Ser-Asp-Gly-Leu-Lys-Asn-Ile-AspVal
Gly
Pro
60
Gly-Ala 71
Ala-His-Leu
E_____________________________________________________________________________________EF
F
FG
G
Met
80
His 90
Leu
Asp-Leu-Pro-Asn-Ala-Leu-Ser-Asp-Leu-Ser-Asp-Leu-His-Ala-Tyr-Lys-Leu-Arg-Val-Asp-Pro-Val-Asn-Phe-Lys-Phe-LeuAsp-Leu-Lys-Gly-Ala-Phe-Ala-Lys-Leu-Ser-Glu-Leu-His-Cys-Asp-Lys-Leu-His-Val-Asp-Pro-Glu-Asn-Phe-Arg-Leu-LeuAsn
Thr
Thr
90
100
F
FG
GH
G
H
1101 ni
Ala
a
lcu
Leu
ni
Ala
a
i1201
Ser-His-Cys-Leu-Leu-Val-Thr-Leu-Ala-Cys-His-His-Pro-Glu-Glu-Phe-Thr-Pro-Ala-Val-His-Ala-Ser-Leu-Asp-Lys-Phe
Gly-Asn-Val-Leu-Val-Cys-Val-Leu-Ala-His-His-Phe-Gly-His-Glu-Phe-Asn-Pro-Gln-Val-Gln-Ala-Ala-Phe-Gln-Lys-Val
110
Lys
Thr
Pro
Tyr
GH
H
HC
Leu-Ala-Ser
140
Phe-Ser-Ala-Val-Ser-Thr-Val-Leu-Thr-Ser-Lys-Tyr-Arg
Val-Ala-Gly-Val-Ala-Ser-Ala-Leu-Ala-His-Arg-Tyr-His
Asn 140
Lys
HC
Fig. 3. Amino acid sequence of the a and ß\ chains of Lion (P 1) hemoglobin in alignment with the corresponding chain of
human (Hu). Only exchanges are given for human hemoglobin Hb-A. Hb-II differs with H b l at positions ß M ß W . /3NA 1
Ac-Ser/Gly, ß W C 1 Arg/Lys.
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M. Jahan et al. ■Carnivora: The Primary Structures of Adult Lion (Panthera leo) Hemoglobins
Table I. Amino acid composition of peptides from a chain of lion.
Peptides:
T pl
T p2
T p3
T p4
T p5
T p 6/7/8
T p9a
T p9b
T p 10
Tp 11
T p 12
Tp 13
T p 14
Position:
1-7
8-11
12-16
17-31
3 2- 40
41-61
62-68
69-90
91-92
9 3-99
100-127
128-139
140-14
_
Asx
1.05
Thr
-
1.91
_
_
_
1.08
1.00
4.85
_
1.95
1.16
-
-
-
2.83
0.91
0.93
-
-
-
1.89
1.90
_
-
-
0.95
1.10
1.85
-
1.95
-
-
1.98
2.81
_
3.20
-
-
-
-
2.17
-
_
-
1.90
-
_
Ser
1.89
Glx
-
-
-
3.30
Pro
-
-
-
-
1.03
0.93
-
1.09
G lv
-
-
1.00
2.92
-
2.10
-
-
-
-
-
-
_
-
0.83
2.95
-
2.04
1.99
3.16
-
-
3.02
1.05
_
-
-
-
A la
1.01
-
Cys*
-
-
0.79
-
0.95
Val
0.95
0.98
-
-
-
1.03
1.00
1.16
-
-
-
_
_
1.51
-
1.91
1.94
2.06
_
-
_
lie
-
-
-
0.95
-
-
0.97
-
-
-
Leu
0.84
-
-
1.03
-
1.01
1.01
4.20
0.98
-
5.10
Tyr
-
-
-
0.65
-
0.69
-
0.77
-
-
-
Phe
-
-
-
-
1.90
1.83
-
-
-
0.98
1.94
-
1.17
-
0.92
-
1.99
_
His
-
-
-
1.31
-
3.25
_
2.43
-
_
3.97
_
_
Trp
-
-
0.78
-
-
-
-
-
-
-
-
_
_
Lys
1.06
0.90
0.95
-
0.99
1.06
1.06
1.07
-
0.98
1.07
Arg
-
-
-
0.94
-
-
-
-
1.00
-
-
Sum
7
4
5
9
21
7
22
2
7
28
15
1.01
-
1.07
-
2
12
* D e t e r m in e d a fte r p e r f o r m ic a c id o x id a t io n .
Table II. Amino acid composition of peptides from ß l chain of lion.
Tpl
Pos.
1-8
TP 2
9-1 7
Tp3
T p4
Tp5
T p6
T p9a
T p9b
T p 10a
T p lO b Tp 11
3 1- 40
41- 59
60-61
T p7
62-65
T p8
18-30
66
67- 76
77-82
83-87
88-95
96-104
Tp 12 -13 T p 14
105- 132 133-144
T p 15
1 4 5 - 14(
Asx
_
1.12
1.81
_
2.83
_
_
_
2.03
2.83
_
1.02
1.96
2.02
-
-
Thr
-
-
-
1.21
-
-
-
-
-
-
-
-
-
-
-
-
Ser
1.68
0.94
-
-
3.22(4)
-
-
-
1.70
-
-
0.82
-
-
1.02
-
Glx
2.24
-
2.11
1.29
1.42
-
-
-
-
-
-
1.07
1.09
4.08
-
-
Pro
-
-
-
1.35
-
-
-
-
-
-
-
-
0.91
0.89
-
-
-
1.82
2.22(3)
-
1.34
-
1.00
-
1.12
-
0.96
-
-
2.09
1.16
-
0.99
-
1.07
-
2.84
-
1.35
-
-
-
1.93
-
-
2.82
3.86
-
-
-
Gly
Ala
Cys*
Val
-
-
-
-
-
-
-
-
-
1.16
-
1.44
-
-
-
1.13
2.69
1.76
-
0.98
-
-
0.99
-
-
-
1.01
3.87
2.53
-
Met*
-
-
-
-
1.18
-
-
-
-
-
-
-
-
-
-
-
lie
-
-
-
-
0.90
-
-
-
-
1.00
-
-
-
-
-
-
Leu
1.05
2.21
2.19
1.43
-
-
-
2.11
1.31
-
1.96
1.02
4.01
1.29
-
Tyr
-
-
-
1.01
-
-
-
-
-
-
-
-
-
-
-
0.87
Phe
0.99
-
-
-
2.66
-
-
-
0.98
-
1.04
-
0.95
2.82
-
-
1.12
His
-
-
-
-
-
-
0.93
-
-
-
-
0.96
0.99
2.98
1.09
Trp
-
0.84
-
0.85
-
-
-
-
-
-
-
-
-
-
-
-
Lys
Arg
1.02
0.98
-
-
1.13
0.87
1.00
0.90
1.07
1.06
1.08
0.98
-
0.94
-
-
-
-
0.95
1.20
-
-
-
-
-
-
-
-
0.94
-
Sum
8
9
19
2
4
1
10
6
5
8
9
13
10
28
1.03
12
1.12
-
2
* Determined after performic acid oxidation.
Numbers in parentheses denote amino acid residue found during sequencing.
/?125(H3) Pro-Gin, and one a \ß2 contact point
/343(CD2) Glu-Gln are alterated. One substitution is
found at heme contact point /370(E14) Ala-Ser.
Among the major 2,3 DPG binding sites following
exchanges are detected at /31(NA1) Val-Ac-Ser,
/32(NA2) His-Phe. Replacement of ß N A 2 His, a
hydrophilic residue, with Phe, a hydrophobic one,
would result in the alteration of secondary structure
hydrophilic residue, with Phe, a hydrophobic one,
would result in the altration of secondary structure
[19]. These exchanges are also found in leopard, ja­
guar, and cat hemoglobins [6, 20, 21], The minimum
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M. Jahan et al. • Carnivora: The Primary Structures of Adult Lion (Panthera leo) Hemoglobins
1469
Table III. Amino acid composition of peptides from /3II chain of lion.
T pl
S.
1-8
T p2
9-17
X
r
r
X
0
V
a
s*
.1
:t*
1.69
0.98
1.59
-
-
u
1.02
r
0.82
Tp3
T p4
Tp5
T p6
T p7
T p8
T p9a
T p9b
T p 10a T p 10b T p 11
T p 12 - 13 T p 14
T p 15
18-30
31- 40
41-59
60-61
62-65
66
67- 76
77-82
83-87
105- 132
133-144
145-146
1.02
1.86
-
-
1.07
3.01
-
-
-
-
-
2.80(4)
2.22
1.18
1.42
-
1.20
-
-
-
-
-
0.88
-
1.99
2.96
-
1.36
-
0.98
2.63
-
1.36
2.21(3)
-
1.07
0.95
1.97
2.63
-
-
1.68
1.22
-
1.84
1.88
-
-
-
-
-
-
0.98
1.10
1.14
-
3.50
1.24
-
2.09
1.33
1.98
-
-
2.80
3.80
-
0.91
-
-
0.73
-
-
-
-
-
1.02
-
3.53
-
2.80
-
1.01
-
-
1.58
-
0.89
0.95
-
-
-
-
0.82
-
-
-
-
0.95
-
-
-
-
-
-
1.98
1.15
2.15
1.44
-
-
-
1.09
-
-
1.15
-
3.65
-
1.40
-
-
-
2.07
-
1.04
-
1.03
2.79
-
-
-
1.01
0.93
2.98
1.20
1.12
-
-
-
-
-
-
-
0.71
-
-
-
-
-
2.96
-
-
-
-
-
1.04
-
0.96
-
-
-
0.71
0.89
-
-
-
-
-
-
-
0.86
s
1.10
-
1.06
0.99
-
1.00
_
-
96-104
-
1.78
-
e
1.22
1.11
-
g
-
-
0.91
m
8
9
P
s
-
-
88-95
10
13
-
0.76
1.00
0.99
0.91
1.02
1.07
1.00
1.00
0.89
-
0.86
1.13
-
-
-
-
-
-
-
-
-
0.97
-
-
-
19
2
4
1
10
6
5
8
9
28
12
2
* Determined after performic acid oxidation.
Numbers in parentheses denote amino acid residue found during sequencing.
Table IV. The minimum amino acid differences found in
different felidae hemoglobin.
Jaguar
a Globin chain
Lion
Jaguar
Leopard
ß\ Globin chain
Lion
Jaguar
Leopard
1
Leopard
1
0
0
3
3
are shown (Table IV). This is a reference work concerning panda hemoglobin.
Cat
9
8
8
6
6
5
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spectra of blocked peptide. Sincerely thanks Ms.
B. Schrank, Mr. R . Mentele, Ms. R . Gautsch, Ms.
A. Muhr, and Ms. E. Wottawa for their help in
amino acid analysis and sequence work. M. Jahan is
thankful to Max-Planck-Gesellschaft zur Förderung
der Wissenschaften e.V. for the award of a doctral
fellowship, A. Ahmed for a fellowship to D A A D .
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M. Jahan et al. • Carnivora: The Primary Structures of Adult Lion (Panthera leo) Hemoglobins
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Unauthenticated
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