Carnivora: The Primary Structure of the Major and Minor

Carnivora:
The Primary Structure of the Major and Minor Hemoglobin Components
of Adult North Persian Leopard (Panthera pardus sexicolor)
A f t a b A h m e d , Meeno Jahan, Gerhard Braunitzer*
A b t . Proteinchemie Max-Planck-Institut für Biochemie,
D-8033 Martinsried bei München, West Germany
Reinhard Göltenboth
Zoologischer Garten Berlin, D-1000 Berlin
Z. Naturforsch. 4 3 b , 1341-1346 (1988); received May 24, 1988
Leopard, Hemoglobin, Primary Structure, Identical Chains
The complete primary structure of the two hemoglobin components of the adult North Persian
Leopard are presented. The m a j o r component Hb-I accounts for 80—90% and the minor component Hb-II accounts for 20—10% of the total hemoglobin. Reversed phase H P L C was used for the
separation of the polypeptide chains. The amino acid sequences were established by automated
E d m a n degradation of the globin chains and of the tryptic peptides in liquid- and gas-phase
sequenators. The sequences are aligned with those of human H b - A . O u r result shows that the
hemoglobins of North Persian Leopard and Jaguar are identical in amino acid sequence.
Introduction
Experimental
The family Felidae (Cat and allies) is divided in to
three subfamilies. The subfamily of greater cat has
two genera. The genus Uncia with one specie Snow
Leopard (Uncia uncia) and the genus Panthera with
four species Leopard (Panthera pardus), Jaguar
(Panthera onco), Tiger (Panthera tigris) and Lion
(Panthera leo). The complete primary structures of
the hemoglobins are reported from Lion [1], Jaguar
[2] and the major component from Amur Leopard
[3], The present study is intended to be the fourth
communication aimed to determine the complete
primary structures of the hemoglobins from North
Persian Leopard (Panthera pardus sexicolor), to fill
the gap in the existing sequences of the genus
Panthera.
Preparation of
* Reprint requests to Prof. G. Braunitzer.
Abbreviations:
Quadrol = N,N,N',N'-tetrakis-(2-hydroxypropyl)ethylendiamine.
Reagent IV = trisodium-7-(isothiocyanato)naphthalene1,3,5-trisulphonate.
Propyne = 3-(dimethylamino)propyne.
TosPheCH 2 Cl = (N-tosyl-L-Phenylalanyl)chloromethane.
R P — H P L C = Reversed-phase high-performance liquid
chromatography.
Verlag der Zeitschrift für Naturforschung, D-7400 Tübingen
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hemolysate
Blood obtained from an adult Leopard (Zoological Garden, Berlin) in heparin was centrifuged and
washed three times with physiological saline (0.9%
NaCl). The erythrocytes were lysed with distilled
water for 1 h in the cold.
Electrophoresis
The hemolysate was analysed by Polyacrylamide
disc electrophoresis [4] using 10% gels and Tris/glycine buffer at p H 8.3. The globin chains were identified under dissociating conditions in the presence of
8 M urea and Triton X-100 [5].
Separation of globin
chains
The globin chains were separated by RP—HPLC
using a 342 Gradient Liquid Chromatograph, Controller 421 (Beckman) and a column of Nucleosil-C4
(Macherey & Nagel). Elution was carried out with
0.1% aqueous trifluoroacetic acid (TFA) with a
linear gradient of 0—35% acetonitrile within 2 min,
followed by 35—60% in 60 min, with a flow rate of
1 ml/min.
Tryptic digestion and separation of
peptides
The globin chains, oxidized with performic acid
[6], were digested with trypsin (TosPheCH 2 Cltreated, Worthington) [7] for 3 h at p H 10.5 and
p H 9.5 with an enzyme/substrate concentration of
5 :100. After 3 h the reaction was stopped by titration
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1342
A. A h m e d et al. • The Primary Structure of the Major and Minor Hemoglobin Components
to pH 4. The tryptic peptides were separated by
RP—HPLC [8] on a column of LiChrosorb R P 2
equilibrated with 0.05 M ammonium acetate. Peptides were eluted by applying a linear gradient of
0—40% acetonitrile in 60 min, with a flow rate of
1 ml/min.
Amino
acid analyses
The peptides separated by RP—HPLC were hydrolysed in 5.7 M HCl at 110 °C for 20 h, and analysed in an amino acid analyser (LC 5000, Biotronik). Tryptophan was determined in the presence
of 6% thioglycolic acid, and methionine and cysteine
after performic acid oxidation.
Sequence
Hb-i-
mm
.-(31
Hb-i- mm
-pn
• -a
determinations
Amino acid sequences were determined by automatic Edman degradation [9] in liquid-phase sequencers (Models 890B and 890C, Beckman). A
modified Quadrol program [10] was used for the sequencing of the native chains and lysine peptides.
The lysine peptides were sequenced after modification with reagent IV [11]. A 3-(dimethylamino)propyne [12] program was employed for arginine
peptides. Some peptides were also sequenced by a
gas-phase sequencer [13]. The phenylthiohydantoin
derivatives of amino acid were identified by H P L C
[14]Results and Discussion
The hemoglobin of North Persian Leopard consists of two components, as verified by Polyacrylamide gel disc electrophoresis (Fig. l a ) . The major
component accounting for 80—90% of the total
hemoglobin and the minor component making up for
the rest. Triton gel electrophoresis showed three globin chains namely a,ßI, and ßll (Fig. l b ) .
RP—HPLC of the hemolysate on a Nucleosil-C4
column resulted in the separation of three globin
chains confirmed the results of Triton electrophoresis. The separation profile is presented in
Fig. 2.
Separation of the tryptic peptides achieved by
R P - H P L C on a LiChrosorb R P 2 column. Some of
the contaminated peptides were rechromatographed
on a Vydac C18 column led to pure peptides. Table I
shows the hemoglobins with alteration at these key
positions observed in different mammalian species.
The peptides analysed for their amino acid composition are presented as Supplementary Material
(Tab. I I - I V ) .
Fig. 1. Electrophoretic pattern of North Persian Leopard
hemolysate on Polyacrylamide gel. (a) Disc electrophoresis
at pH 8.3. (b) Under dissociating conditions, in 8 M urea
and Triton X-100.
Heme.
PI
JJ
PI
T i m e ( min )
Fig. 2. Separation profile of the whole hemolysate by
RP-HPLC
on a column of Nucleosil C 4 size
(4.6x250 mm). Buffer: 0.1% trifluoroaceticacid; gradient
0 - 3 5 % acetonitrile in 2 min. followed by 3 5 - 6 0 %
acetonitrile in 60 min; flow rate 1 ml/min.
The primary structures of the globin chains were
established to some extent by sequencing the N-terminal part of the native chains up to position 42, but
mainly by sequencing the tryptic peptides. The complete amino acid sequence of the globin chains presented in Fig. 3. The sequence aligned with that of
human Hb-A showed 22 amino acid exchanges occur
in the a, 29 in the ßl and 28 in the ßII chains. Within
the two ß chains only two differences have been located at (ßl/ßll): ß N A 1 A c - S e r / G l y and ß H C l
Arg/Lys. These exchanges affect on eight functionally important positions; four in the a 1/31, one in the
alß2, and one in the heme contact points.
1343 A. A h m e d et al. • The Primary Structure of the M a j o r and Minor Hemoglobin Components
Mammal
/?1(NA 1) /?2(NA2) /?82(EF6)
/3143(H21)
Human
Mountain Zebra
Low Land Tapir
Nine-Banded Armadillo
Ring Tailed Lemur
Brown Lemur
Cat (ß A)
Cat (ßB)
Persian Leopard (ßl)
Persian Leopard (/3II)
Virginia White Tailed D e e r
G r a n d Galogo (ßl)
West European Hedgehog
Val
Val
Val
Val
Thr
Thr
Gly
Ac-Ser
Ac—Ser
Gly
His
His
His
His
His
His
His
His
His
His
His
His
Ala
-
Val
Val
His
Gin
Glu
Asn
Phe
Leu
Phe
Phe
Phe
Phe
Met
His
His
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys
Cys
Lys
Table I. Mammalian hemoglobins with
alteration at contact points for 2,3diphosphoglycerate.
Table II. Amino acid composition of peptides from a chain of Leopard.
Pos.
Asx
Thr
Ser
Glx
Pro
Gly
Ala
Cys*
Val
lie
Leu
Tyr
Phe
His
Trp
Lys
Arg
Sum
Tpl
1-7
Tp2
8-11
Tp3
12-16
Tp4
17-31
Tp5
32-40
Tp 6/7/8 Tp9a
41-61 62-68
-
1.03
0.89
1.87
3.19
0.89
1.93
1.97
1.81
_
_
_
-
-
-
1.82
-
-
-
-
-
0.89
3.23
2.79
1.00
-
-
-
-
-
1.13
-
-
1.00
-
2.79
3.08
-
-
1.02
0.97
0.97
-
0.95
0.86
0.88
-
-
-
-
-
-
1.08
-
-
-
-
-
0.96
1.14
0.81
-
-
-
-
-
1.91
-
1.01
-
-
-
-
-
-
1.06
0.90
0.78
1.13
-
-
-
7
4
5
-
_
Tpll
93-99
Tpl4
Tp 13
Tpl2
1 0 0 - 1 2 7 1 2 8 - 139140-141
_
1.85
-
-
-
-
-
Tp9b
69-90
TplO
91-92
4.68
-
-
-
-
-
-
1.03
-
1.21
1.21
1.90
2.12
2.24
2.19
-
-
-
-
-
-
-
1.85
3.85
-
-
1.25
-
-
-
-
-
-
-
-
0.98
0.91
-
1.80
2.03
-
-
-
-
-
-
-
-
1.03
0.82
-
4.62
1.20
-
-
1.14
0.90
3.94
0.79
2.85
2.10
1.86
-
-
-
-
0.91
-
-
-
0.96
1.91
-
-
-
-
1.02
1.06
1.77
-
-
1.92
2.72
-
-
-
-
-
0.98
-
1.10
0.80
1.89
2.89
-
2.21
-
-
2.15
3.52(4)
-
-
-
-
-
-
-
-
-
1.13
1.07
1.05
1.09
-
1.01
1.12
1.07
-
1.04
-
-
-
-
0.93
-
-
-
1.01
2
7
28
12
15
9
21
7
22
-
2
* Determined after performic acid oxidation. Values in brackets are taken from sequence data.
Among the 2,3-diphosphoglycerate binding sites
/?NA1 and /3NA2 were found to be substituted with
Ac—Ser and Phe respectively. The ß\l chain was
found with free N-terminal Gly, such exchanges results in distortion of the secondary structure of the
hemoglobin. All members of the family Felidae
studied as far have identical substitutions at these
phosphate binding sites [1—3, 15 — 16].
Comparision of the amino acid sequences with
those of other available sequences of the family
Felidae revealed high degree of homology both in a
and ß chains. An interesting result of our study on
the hemoglobins of the North Persian Leopard is that
all three globin chains (a, ßl, ßlT) have identical
amino acid sequences when aligned with that of Jaguar globin chains. Among the order Carnivora the
only other known example of identical sequences
found in the family Ursidae where the hemoglobins
from Asiatic Black Bear, Polar Bear and Malayan
Sun Bear show identical primary structure [17].
We should like to thank Mr. R. Mentele, Ms. B.
Schrank, Ms. R. Gautsch and Ms. E. Wottawa for
their work on sequenator and analyser. A. Ahmed is
thankful to D A A D for a fellowship, and M. Jahan to
Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. for the award of a fellowship.
1344
A. A h m e d et al. • The Primary Structure of the Major and Minor Hemoglobin Components
Table III. A m i n o acid composition of peptides from ß\ chain of Leopard.
Tpl
Pos. 1 - 8
Asx _
Thr Ser 1.66
Glx 1.86
Pro Gly Ala 0.92
Cys* Val Met* lie Leu 0.97
Tyr Phe 0.96
His Trp Lys 1.00
Arg Sum 8
Tp2 Tp3
9 - 1 7 18-30
Tp4
31-40
Tp6
Tp5
Tp7
Tp8
4 1 - 5 9 6 0 - 6 1 6 2 - 6 5 66
Tp9a Tp9b Tp 10 a
67-76 77-82 83-87
Tp 10b
88-95
Tp 12/13 Tp 14
Tp 15
Tp 11
96-104 105-132 133-144145 — 14«
1.21
1.89
_
2.90
_
_
_
1.89
2.96
_
0.87
1.94
2.15
_
_
-
-
1.00
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
2.10
-
-
-
-
-
-
-
-
0.79
1.07
-
-
-
-
-
-
-
-
1.04
0.89
-
-
-
-
0.80
-
-
-
2.12
-
-
0.97
0.86
3.68
0.86
1.81
-
2.50(3)
1.05
-
-
-
-
-
1.01
2.88
1.59(2)
-
0.99
-
-
-
-
-
-
-
2.01
1.00
0.98
0.85
1.00
-
1.29
3.17
-
0.87
-
-
-
-
0.96
1.83
-
-
-
-
-
0.93
-
-
-
0.79
-
-
-
0.93
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
2.04
0.89
1.03
-
1.86
1.12
3.81
1.20
-
-
-
-
0.83
3.05
3.01
-
-
1.05
0.98
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
3.16
-
-
-
0.98
-
0.88
-
-
-
-
-
-
1.03
-
-
-
-
0.96
1.06
0.91
-
0.79
-
-
-
-
-
-
-
-
-
-
-
0.98
-
9
13
1.13
10
-
-
0.99
1.02
-
2.13
0.85
0.80
0.85
1.13
4.21
1.37(1)
1.89
3.12
0.96
3.69
-
-
-
-
1.14
4.06
-
-
-
2.30(3)
-
-
-
0.99 0.87
1.02
1.02
0.95
1.00
0.89
0.97
-
0.95
-
-
-
-
-
-
-
-
-
-
1.04
-
1.14
-
2
4
1
6
5
8
9
19
10
28
12
2
* Determined after performic acid oxidation. Values in brackets are taken from sequence data.
Table IV. Amino acid composition of peptides from ß\\ chain of Leopard.
Tpl
Pos. 1 - 8
Asx
Thr
Ser
Glx
Pro
Gly
Ala
Cys*
Val
Met*
lie
Leu
Tyr
Phe
His
Trp
Lys
Arg
Tp2 T p 3
9 - 1 7 18-30
Tp4
31-40
Tp7 Tp8
Tp5
Tp6
4 1 - 5 9 6 0 - 6 1 6 2 - 6 5 66
Tp9a T p 9 b Tp 10 a
67-76 77-82 83-87
Tp 10b
88-95
Tpl5
Tp 11
Tp 12/13 Tp 14
96-104 105-132 133-144 145-14(
_
0.98
1.88
_
3.00
_
_
_
2.12 2.81
_
1.18
2.13
2.10
-
-
-
-
-
1.00
-
-
-
-
-
-
-
-
-
-
-
-
0.87
1.78
0.86
-
-
-
-
-
1.84
-
-
-
1.04
-
2.17
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
0.89
0.88
-
-
0.98
0.87
0.95
1.00
-
-
3.67
0.87
-
-
0.88
0.82
1.87
-
1.12
-
-
-
-
-
0.92
1.71
-
-
3.04
1.30(1)
-
-
-
-
-
-
0.98
2.68
-
-
-
-
-
-
-
-
1.01
1.89
1.04
-
-
0.96
-
-
0.97
1.05
-
-
-
-
-
-
-
0.85
-
-
1.04
-
-
0.93
-
-
-
0.97
3.97
1.04
2.27
2.95
0.83
3.87
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
2.03
1.03
1.06
-
-
2.10
0.90
0.86
0.77
1.20
-
-
-
-
-
-
-
-
2.86
-
-
-
-
-
-
1.00
2.72
-
-
1.62(2)
-
-
-
-
-
-
-
-
-
-
1.96
1.08
4.01
1.20
-
-
-
-
-
-
1.01
1.01
-
0.86
-
3.04
3.09
-
-
-
-
-
-
1.01
-
-
-
-
1.01
-
0.89
-
-
-
-
-
-
-
-
-
0.95
-
-
-
-
1.01
0.89
Sum 8
9
13
10
1.10
19
-
2.41(3)
0.76
1.11
-
-
-
0.98
0.99
-
0.95
4.03
0.98
1.01
1.01
1.07
-
-
-
-
2
4
1
10
-
0.96
1.14
-
-
1.13
-
0.93
1.00
-
-
-
-
0.98
-
-
-
6
5
8
9
28
12
1.00
1.00
* Determined after performic acid oxidation. Values in brackets are taken from sequence data.
2
1345 A. A h m e d et al. • The Primary Structure of the M a j o r and Minor Hemoglobin Components
NA
(X
a
Val-
A
AB
B
Pro
Thr
10
Ala
Val
Ala 20
26
-Leu-Ser-Ser-Ala-Asp-Lys-Asn-Asn-Val-Lys-Ala-Cys-Trp-Gly-Lys-Ile-Gly-Ser-Hls-Ala-Gly-Glu-Tyr-Gly-Ala-
ßl AcSer-Phe-Leu-Ser-Ala-Glu-G1u-Lys-Gly-Leu-Va 1 -Asn-Gly-Leu-Trp-Ser-Lys-Va 1 BA
Val-His
Thr-Pro
Ser-Ala
Thr-Ala
NA
-Asn-Val-Asp-Glu-Val-Gly-Gly20
A
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-His-
Glu-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 Gly
Lys
Asn
70
Val-Asp-Gly-Ser-Ala-Gln-Val-Gln-Thr-Hls-Gly-Gln-Lys-Val-Ala-Asp-Ala-Leu-Thr-Lys-Ala-Val-Ala-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-IIe-AspVal
Gly
Pro
60
Gly-Ala 71
Ala-Hls-Leu
E
EF
F
FG
G
Met
80
His 90
Leu
Asp-Leu-Pro-Asn-Ala-Leu-Ser-Asp-Leu-Ser-Asp-Leu-Hls-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-H1s-Cys-Asp-Lys-Leu-H1s-Val-Asp-Pro-Glu-Asn-Phe-Arg-Leu-LeuAsn
Thr
Thr
90
100
F
FG
GH
G
H
110 Ala
Leu
Ala
120
Ser-H1s-Cys-Leu-Leu-Val-Thr-Leu-Ala-Cys-H1s-H1s-Pro-Glu-Glu-Phe-Thr-Pro-A1a-Val-H1s-Ala-Ser-Leu-Asp-Lys-PheGly-Asn-Val-Leu-Val-Cys-Val-Leu-Ala-Hls-Hls-Phe-Gly-Hls-Glu-Phe-Asn-Pro-Gln-Val-Gln-Ala-Ala-Phe-Gln-Lys-Val110
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-AIa-GIy-Val-Ala-Ser-Ala-Leu-Ala-Hls-Arg-Tyr-Hls
Asn 140
Lys
HC
Fig. 3. Amino acid sequences of North Persian Leopard
(Pp) globin chains in alignment with the corresponding
chains of human (Hu) globin. In case of human only the
exchanges are given. The Hb-II differs from the Hb-I at the
following positions.
ßl/ßU: ß~NA \ A c - S e r / G l y , /3HC1 Arg/Lys.
1346
A. A h m e d et al. • The Primary Structure of the M a j o r and Minor Hemoglobin Components
1] M. Jahan, A. A h m e d , G. Braunitzer, Z. H. Zaidi,
and R. Goltenboth, Z. Naturforsch. 42b, 1465 (1987).
2] A. A h m e d , M. Jahan, Z. H. Zaidi, G. Braunitzer,
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