docCarnivora: The Primary Structure of the Major and Minor
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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 0932 - 0776/88/1000 - 1 3 4 1 / $ 01.00/0 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 Dieses Werk wurde im Jahr 2013 vom Verlag Zeitschrift für Naturforschung in Zusammenarbeit mit der Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. digitalisiert und unter folgender Lizenz veröffentlicht: Creative Commons Namensnennung-Keine Bearbeitung 3.0 Deutschland Lizenz. This work has been digitalized and published in 2013 by Verlag Zeitschrift für Naturforschung in cooperation with the Max Planck Society for the Advancement of Science under a Creative Commons Attribution-NoDerivs 3.0 Germany License. Zum 01.01.2015 ist eine Anpassung der Lizenzbedingungen (Entfall der Creative Commons Lizenzbedingung „Keine Bearbeitung“) beabsichtigt, um eine Nachnutzung auch im Rahmen zukünftiger wissenschaftlicher Nutzungsformen zu ermöglichen. On 01.01.2015 it is planned to change the License Conditions (the removal of the Creative Commons License condition “no derivative works”). This is to allow reuse in the area of future scientific usage. 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, and R. Goeltenboth, Biol. Chem. Hoppe-Seyler 368, 1385 (1987). 3] A. Abbasi and G. Braunitzer, J. Prot. Chem. 4, 57 (1985). 4] B. J. Davis, A n n . N. Y. Acad. Sei. 121, 404 (1964). 5] B. P. Alter, S. C. Goff, G. D. Efremov, M. E. Gravely, and T. H. J. Huisman, Br. J. Hematol. 44, 527 (1980). 6] C. H . W. Hirs, W. H . Stein, and S. Moore, J. Biol. Chem. 221, 151 (1956). 7] C. H. W. Hirs, Methods in Enzymology 11, 218 (1967). 8] H. Kratzin, C. Yang, J. U. Krusche, and N. Hilschm a n , Hoppe-Seyler's Z . Physiol. Chem. 361, 1591 (1980). 9] P. E d m a n and G . Begg, Euro. J. Biochem. 1, 80 (1967). [10] G. Braunitzer, B. Schrank, and A. Ruhfus, HoppeSeyler's Z. Physiol. Chem. 351, 1589 (1970). [11] J. Pfletschinger and G. Braunitzer, Hoppe-Seyler's Z. Physiol. Chem. 361, 925 (1980). [12] G. Braunitzer. B. Schrank. A. Stangl, und U. Scheithauer, Hoppe-Seyler's Z. Physiol. Chem. 359, 137 (1978). [13] G. Begg. P. Rucknagel, J. Godovac-Zimermann. G. Braunitzer, and W. Schuster, Biol. Chem. HoppeSeyler's 367, 81 (1986). [14] C. L. Z i m m e r m a n n and J. J. Pisano, Methods in Enzymology 47, 45 (1977). [15] F. Taketa, A. G. Mauk, and J. L. Lessard, J. Biol. Chem. 246, 4471 (1971). [16] A. Abbasi and G. Braunitzer, Biol. Chem. HoppeSeyler's 366, 699 (1985). [17] O. H o f m a n , G. Braunitzer, and R. Goeltenboth, Biol. Chem. Hoppe-Seyler's 368, 507 (1987).
