Atiya Abbasi | University Of Pune'S AFFILIATED INDIRA COLLEGE OF COMMERCE AND SCIENCE, PUNE (original) (raw)

Papers by Atiya Abbasi

Biochemical and Biophysical Research Communications, 2002

The primary structure of the major hemoglobin component, HbA (␣ A -and ␤-chain), from Tufted duck... more The primary structure of the major hemoglobin component, HbA (␣ A -and ␤-chain), from Tufted duck (Aythya fuligula) is presented. The separation of the globin subunits was achieved by ion exchange chromatography on CM-cellulose in 8 M urea. The amino acid sequence was determined by automatic Edman degradation of native chains as well as tryptic and hydrolytic peptides in a gas-phase sequencer. The automated homology model was generated by the protein structure modeling package WHAT IF using the crystal structure coordinates of Bar-headed goose hemoglobin. The 3D structure prediction enables ␣99Arg and ␤101Glu to emerge as a new intersubunit contact site not found in the hemoglobin structure of any other species. ␣99Arg forms a complex salt bridge network involving ␣99Arg-␤101Glu-␤104Arg-␤108Asp. Also the substitution at ␣34 3 Ile, ␣38 3 Gln and ␤55 3 Leu serves to stabilize the oxy-structure, leading to higher oxygen affinity. © 2002 Elsevier Science (USA)

Biophysical Journal, 2000

Synchrotron x-ray scattering measurements were performed on dilute solutions of the purified hemo... more Synchrotron x-ray scattering measurements were performed on dilute solutions of the purified hemocyanin subunit (Bsin1) from scorpion (Buthus sindicus) and the N-terminal functional unit (Rta) from a marine snail (Rapana thomasiana). The model-independent approach based on spherical harmonics was applied to calculate the molecular envelopes directly from the scattering profiles. Their molecular shapes in solution could be restored at 2-nm resolution. We show that these units represent stable, globular building blocks of the two hemocyanin families and emphasize their conformational differences on a subunit level. Because no crystallographic or electron microscopy data are available for isolated functional units, this study provides for the first time structural information for isolated, monomeric functional subunits from both hemocyanin families. This has been made possible through the use of low protein concentrations (< or = 1 mg/ml). The observed structural differences may offer advantages in building very different overall molecular architectures of hemocyanin by the two phyla.

Journal of Protein Chemistry, 1985

The complete amino acid sequence of the major component of hemoglobin from amur-leopard (Panthera... more The complete amino acid sequence of the major component of hemoglobin from amur-leopard (Panthera pardus orientalis) is presented. The major component accounts for more than 90% of the total hemoglobin. Separation of the globin subunits was achieved by ion-exchange chromatography on CM-cellulose in urea. The sequence was studied by automatic Edman degradation of tryptic and hydrolytic peptides. Alignment was carried out with human hemoglobin sequence. The β NH2 terminus is blocked with Ac-serine. The data are compared with other mammalian hemoglobins and results are discussed with respect to sequence and physiology.

Archives of Biochemistry and Biophysics, 2000

and §Abteilung fü r Transfusionsmedizin mit Blutbank, Otfried-Mü ller-Straße 4/1, The enzyme L-am... more and §Abteilung fü r Transfusionsmedizin mit Blutbank, Otfried-Mü ller-Straße 4/1, The enzyme L-amino acid oxidase (LAO) from the leaf-nosed viper (Eristocophis macmahoni) snake venom was purified to homogeneity in a single step using high performance liquid chromatography on a Nucleosil 7C 18 reverse phase column. The molecular mass of the purified enzyme was 58734.0 Da, as determined by matrix-assisted laser desorption/ionization mass spectrometry. The N-terminal amino acid se-

Biochemical and Biophysical Research Communications, 2004

Avian hemoglobins have attracted much attention in view of the unique oxygen transport characteri... more Avian hemoglobins have attracted much attention in view of the unique oxygen transport characteristics. The present study describes the primary structure of minor hemoglobin component HbD from Tufted duck (Aythya fuligula), a migratory bird seen in Pakistan during the winter season. Separation of the polypeptide subunits was achieved by ion exchange chromatography in the presence of 8 M urea. Molecular masses of the intact protein as well as peptides obtained from chemical and enzymatic cleavages were determined by electrospray ionization mass spectrometry. The sequence was studied by automatic Edman degradation of the native chains and their tryptic/hydrolytic fragments in a gas-phase sequencer. Comparison of the hemoglobin sequence with the corresponding sequences of Anseriform representatives and other avian species shows residues like a D 23 Asp, a D 120 Asp as being specific to Tufted duck. The three-dimensional structure analyzed with the protein structure modeling package, WHAT IF, using the crystal structure coordinates of chicken hemoglobin (PDB code = 1hbr) shows a D 34 Val, a D 38 Gln, and a D 94 Asp as possible mediators offering alternate pathway for oxygen uptake and release thereby leading to distinct hypoxia tolerance in the Tufted ducks. Results are discussed with reference to function and evolution in the Anseriform representatives.

Journal of Protein Chemistry, 1991

The complete amino acid sequence of the αA-chain of major hemoglobin component from Cuckoo (Eudyn... more The complete amino acid sequence of the αA-chain of major hemoglobin component from Cuckoo (Eudynamys scolopaceae) is presented. Separation of the polypeptide subunits was achieved by ion exchange chromatography in the presence of 8 M urea. The sequence was studied by automatic Edman degradation of the native chain and its tryptic fragments in a gas-phase sequencer. Comparison with other avian hemoglobins shows residues α21, α30, α96, α110, and α114 as being specific to Cuckoo. The functional significance of these is discussed.

Toxicon, 1999

Crude venom from Eristocophis macmahoni was demonstrated to exert a potent inhibition of human bl... more Crude venom from Eristocophis macmahoni was demonstrated to exert a potent inhibition of human blood platelet aggregation mediated by adenosine diphosphate (ADP), platelet activating factor (PAF) and arachidonic acid (AA). The venom caused lysis of the platelets, however, the red blood cells were not lysed by the venom. Substantial oedema was produced upon injection of the venom into the rat hind paw. Contrarily, the intraperitoneal injection of the venom to the rats caused an inhibition of the carrageenin-induced rat paw oedema. However, an 100% lethality within 24 h was observed with a dose of 40 mg/kg body weight. The venom was fractionated by reverse phase high pressure liquid chromatography (HPLC) and the fractions were analyzed for their eect on ADP-induced platelet aggregation. The fraction eluted at 15.5 min (20% acetonitrile concentration) exhibited an inhibitory eect of several-fold greater potency than that of the crude venom. Fractions eluted at 18.5 min (25.4% acetonitrile concentration) and onward showed a proaggregatory but insigni®cant eect. It is suggested that although the venom contains pro-Toxicon 37 (1999) 1095±1107

Journal of Protein Chemistry, 1991

Primary structure of β-chain of pigeon is presented. It was determined by amino acid sequence ana... more Primary structure of β-chain of pigeon is presented. It was determined by amino acid sequence analysis of intact β-chain and its peptides obtained by the enzymatic and chemical cleavage. Comparison of amino acid sequence of the chain with other available data shows β 14 Ile, β61 Lys, and β113 Ile as residues specific to pigeon. One important replacement at α1β1 contact is β55 Met→Ser.

Journal of Protein Chemistry, 1989

Primary structure of hemoglobin of α-chain ofColumba livia is presented. The separation of α-chai... more Primary structure of hemoglobin of α-chain ofColumba livia is presented. The separation of α-chain was obtained from globin by ion-exchange chromatography (CMC-52) and reversed-phase HPLC (RP-2 column). Amino acid sequence of intact as well as tryptic digested chain was determined on gas-phase sequencer. Structure is aligned homologously with 21 other species. Among different exchanges, positions α24 (Tyr→Leu), α26 (Ala→Gly), α32 (Met→Leu), α64 (Asp→Glu), α113 (Leu→Phe), and α129 (Leu→Val) are unique to pigeon hemoglobin. The various exchanges in α-chain are discussed with reference to evolution and phylogeny. The results show that the order Columbiformes is evolutionarily closer to the order Anseriformes. Since the pigeon is homogeneous, having HbA (αA-chain) and lacks αD-chain, its phylogenetic placement could be established among birds having single hemoglobin components.

Archives of Biochemistry and Biophysics, 2006

Scorpion venoms are among the most widely known source of peptidyl neurotoxins used for calliperi... more Scorpion venoms are among the most widely known source of peptidyl neurotoxins used for callipering different ion channels, e.g., for Na + , K + , Ca + or Cl À . An a-toxin (Bs-Tx28) has been purified from the venom of scorpion Buthus sindicus, a common yellow scorpion of Sindh, Pakistan. The primary structure of Bs-Tx28 was established using a combination of MALDI-TOF-MS, LC-ESI-MS, and automated Edman degradation analysis. Bs-Tx28 consists of 65 amino acid residues (7274.3 ± 2Da), including eight cysteine residues, and shows very high sequence identity (82-94%) with other long-chain a-neurotoxins, active against receptor site-3 of mammalian (e.g., Lqq-IV and Lqh-IV from scorpions Leiurus sp.) and insect (e.g., BJa-IT and Od-1 from Buthotus judaicus and Odonthobuthus doriae, respectively) voltage-gated Na + channels. Multiple sequence alignment and phylogenetic analysis of Bs-Tx28 with other known a-and a-like toxins suggests the presence of a new and separate subfamily of scorpion a-toxins. Bs-Tx28 which is weakly active in both, mammals and insects (LD 50 0.088 and 14.3 lg/g, respectively), shows strong induction of the rat afferent nerve discharge in a dose-dependent fashion (EC 50 = 0.01 lg/mL) which was completely abolished in the presence of tetrodotoxin suggesting the binding of Bs-Tx28 to the TTX-sensitive Na + -channel. Three-dimensional structural features of Bs-Tx28, established by homology modeling, were compared with other known classical a-mammal (AaH-II), a-insect (Lqh-aIT), and a-like (BmK-M4) toxins and revealed subtle variations in the Nt-, Core-, and RT-CT-domains (functional domains) which constitute a ''necklace-like'' structure differing significantly in all a-toxin subfamilies. On the other hand, a high level of conservation has been observed in the conserved hydrophobic surface with the only substitution of W43 (Y43/42) and an additional hydrophobic character at position F40 (L40/A/V/G39), as compared to the other mentioned a-toxins. Despite major differences within the primary structure and activities of Bs-Tx28, it shares a common structural and functional motif (e.g., transRT-farCT) within the RT-CT domain which is characteristic of scorpion a-mammal toxins.

Archives of Biochemistry and Biophysics, 2001

Four depressant insect-selective neurotoxin analogs (termed Bs-dprIT1 to 4) from the venom of the... more Four depressant insect-selective neurotoxin analogs (termed Bs-dprIT1 to 4) from the venom of the scorpion Buthus sindicus were purified to homogeneity in a single step using reverse-phase HPLC. The molecular masses of the purified toxins were 6820.9, 6892.4, 6714.7, and 6657.1 Da, respectively, as determined by mass spectrometry. These long-chain neurotoxins were potent against insects with half lethal dose values of 67, 81, 103, and 78 ng/100 mg larva and 138, 160, 163, and 142 ng/100 mg cockroach, respectively, but were not lethal to mice even at the highest applied dose of 10 g/20 g mouse. When injected into blowfly larvae (Sarcophaga falculata), Bs-dprIT1 to 4 induced classical manifestations of depressant toxins, i.e., a slow depressant flaccid paralysis. The primary structures of Bs-dprIT 1 to 4 revealed high sequence homology (60 -75%) with other depressant insect toxins isolated from scorpion venoms. Despite the high sequence conservation, Bs-dprIT1 to 4 showed some remarkable features such as (i) the presence of methionine (Met 6 in Bs-dprIT1 and Met 24 in Bs-dprIT2 to 4) and histidine (His 53 and His 57 in Bs-dprIT1) residues, i.e., amino acid residues that are uncommon to this type of toxin; (ii) the substitution of two highly conserved tryptophan residues (Trp43 3 Ala and Trp53 3 His) in the sequence of Bs-dprIT1; and (iii) the occurrence of more positively charged amino acid residues at the C-terminal end than in other depressant insect toxins. Multiple sequence alignment, sequence analysis, sequence-based structure prediction, and 3D homology modeling studies revealed a protein fold and secondary structural elements similar to those of other scorpion toxins affecting sodium channel activation. The electrostatic potential calculated on the surface of the predicted 3D model of Bs-dprIT1 revealed a significant positive patch in the region of the toxin that is supposed to bind to the sodium channel.

Biochemical and Biophysical Research Communications, 2003

Cytotoxic lymphocytes (CTLs), the key players of cell mediated immunity, induce apoptosis by enga... more Cytotoxic lymphocytes (CTLs), the key players of cell mediated immunity, induce apoptosis by engaging death receptors or through exocytosis of cytolytic granules containing granzyme (proteases) and pore-forming protein (perforin). The crystal structure of granzyme B from human (B h ) and rat (B r ), as well as that of pro-granzyme K ðK h Þ has been reported recently. In the present communication, we describe the homology modeling of granzyme family (in particular Gzm A h , M h , B m , and C m from human and mouse) based on the crystal structural coordinates of trypsin, granzyme K ðK h Þ, and granzyme B ðB h Þ. These models have been used for establishing phylogenetic relationship as well as identifying characteristic features for designing specific inhibitors. The paper also highlights key residues at the S1, S2, and S2 0 binding subsites in all granzyme, which may be involved in the structure-function relationship of this enzyme family. The predicted 3D homology models show a conserved two similar domain structure, i.e., an Nterminal domain and a C-terminal domain comprising predominantly of b-sheet structure with a little a-helical content. Microheterogeneities have been observed in the vicinity of the active site in all granzymes as compared to granzyme B h . For example, in granzyme M h , valine is present at the S1 subsite instead of arginine. Similarly differences at S2 (Leu ! Phe), S3 (Ser ! Gly), and S4 (Arg ! Asn) subsites are quite apparent and appear to hold the potential for selective designing of inhibitors for possible therapeutic applications. Furthermore, analysis of the electrostatic surface potential on the shape of granzyme-inhibitor binding groove reveals clear differences at the reactive site. Additionally the different posttranslational modification sites such as phosphorylation (e.g., in granzyme M Thr101, Ser109), myristoylation (Gly22, 117, and 131), and glycosylation (Ser160) have been identified, as very little is known about the functional significance of these modifications in the granzyme family. Thus, glycosylation at Ser160 in granzyme M may influence the net charge of the enzyme, resulting in altered substrate binding as compared to granzyme B. Also this modification may influence the rate of complexation and binding affinity with proteoglycans. These studies are expected to contribute towards the basic understanding of functional associations of the granzymes with other molecules and their possible role in apoptosis.

Biochemical and Biophysical Research Communications, 2003

Cytotoxic lymphocytes (CTLs), the key players of cell mediated immunity, induce apoptosis by enga... more Cytotoxic lymphocytes (CTLs), the key players of cell mediated immunity, induce apoptosis by engaging death receptors or through exocytosis of cytolytic granules containing granzyme (proteases) and pore-forming protein (perforin). The crystal structure of granzyme B from human (B h ) and rat (B r ), as well as that of pro-granzyme K ðK h Þ has been reported recently. In the present communication, we describe the homology modeling of granzyme family (in particular Gzm A h , M h , B m , and C m from human and mouse) based on the crystal structural coordinates of trypsin, granzyme K ðK h Þ, and granzyme B ðB h Þ. These models have been used for establishing phylogenetic relationship as well as identifying characteristic features for designing specific inhibitors. The paper also highlights key residues at the S1, S2, and S2 0 binding subsites in all granzyme, which may be involved in the structure-function relationship of this enzyme family. The predicted 3D homology models show a conserved two similar domain structure, i.e., an Nterminal domain and a C-terminal domain comprising predominantly of b-sheet structure with a little a-helical content. Microheterogeneities have been observed in the vicinity of the active site in all granzymes as compared to granzyme B h . For example, in granzyme M h , valine is present at the S1 subsite instead of arginine. Similarly differences at S2 (Leu ! Phe), S3 (Ser ! Gly), and S4 (Arg ! Asn) subsites are quite apparent and appear to hold the potential for selective designing of inhibitors for possible therapeutic applications. Furthermore, analysis of the electrostatic surface potential on the shape of granzyme-inhibitor binding groove reveals clear differences at the reactive site. Additionally the different posttranslational modification sites such as phosphorylation (e.g., in granzyme M Thr101, Ser109), myristoylation (Gly22, 117, and 131), and glycosylation (Ser160) have been identified, as very little is known about the functional significance of these modifications in the granzyme family. Thus, glycosylation at Ser160 in granzyme M may influence the net charge of the enzyme, resulting in altered substrate binding as compared to granzyme B. Also this modification may influence the rate of complexation and binding affinity with proteoglycans. These studies are expected to contribute towards the basic understanding of functional associations of the granzymes with other molecules and their possible role in apoptosis.

Journal of Basic Microbiology, 2001

Bacillus sp. RM16 was isolated from a hot spring in Karachi and screened for the production of αa... more Bacillus sp. RM16 was isolated from a hot spring in Karachi and screened for the production of αamylase. The enzyme was obtained after 72 h cultivation of strain in Luria broth containing 1% starch (w/v). Enzyme Amy RM16 was purified to electrophoretic homogeneity by a series of sequential steps including precipitation with ammonium sulfate at 70% saturation, Q-Sepharose, Phenyl Sepharose and reversed phase chromatography. The purified enzyme is made up of a single polypeptide chain of 66 kDa as established by a combination of SDS-PAGE and zymographic analysis. In our experimental conditions, a total yield of 1.35% with specific activity of 6380U/mg was obtained providing 17 fold final purification of the enzyme. Biochemical characterization of the Amy RM16 such as optimum temperature and pH, substrate specificity and enzymatic susceptibilities towards different metal ions and inhibitors were also performed. Results of these studies revealed that, the enzyme is active at wide temperature range with optimum activity at 80°C and retained 85% of the activity for 3 h at 50°C and around 50% of remaining activity for 1 h at 80°C. The enzyme showed optimum activity at pH 5.0. On the other hand, Ca +2 and EDTA (1 to 5 mM) did not significantly affect the enzyme activity. The main substrate for the enzyme was found to be starch but it could also hydrolyze raw starch, dextrin, γ γ γ γcyclodextrin and pullulan.

Comparative Biochemistry and Physiology A-molecular & Integrative Physiology, 1999

Out of eight different polypeptide chains, present in the native hemocyanin molecule of scorpion ... more Out of eight different polypeptide chains, present in the native hemocyanin molecule of scorpion Buthus sindicus, subunit Bsin1 was preparatively purified and characterized by amino acid composition, laser desorption mass spectrometry and spectroscopic techniques. Thermal stability of the intact respiratory protein and its subunit was studied by following the change in ellipticity at 222 nm as a function of temperature. The copper-dioxygen system at the binuclear active site has a stabilizing effect and the oxy-proteins are significantly more thermostable than the apo-forms. The thermal stability of the scorpion Hc from B. sindicus is considerably smaller than that of the tarantula (Eurypelma californicum) Hc [Sterner R et al., FEBS Lett 1995;364:9 -12], although both, scorpion and spider, belong to the subphylum chelicerata. The fluorescence properties of the scorpion Hc and Bsin1 suggest that the indol groups are in rather nonpolar environment, deeply 'buried' in the interior of the aggregates and structural subunits. At the same time, these chromophores are located on subunit interfaces in the C. sapidus and O. 6ulgaris Hcs, participating in protein-protein interactions [Ricchelli F et al., Arch Biochem Biophys 1984;235:461 -469; Stoeva S et al., Spectrochimica Acta A 1995;51:1965.

Journal of Basic Microbiology, 2001

Bacillus sp. RM16 was isolated from a hot spring in Karachi and screened for the production of αa... more Bacillus sp. RM16 was isolated from a hot spring in Karachi and screened for the production of αamylase. The enzyme was obtained after 72 h cultivation of strain in Luria broth containing 1% starch (w/v). Enzyme Amy RM16 was purified to electrophoretic homogeneity by a series of sequential steps including precipitation with ammonium sulfate at 70% saturation, Q-Sepharose, Phenyl Sepharose and reversed phase chromatography. The purified enzyme is made up of a single polypeptide chain of 66 kDa as established by a combination of SDS-PAGE and zymographic analysis. In our experimental conditions, a total yield of 1.35% with specific activity of 6380U/mg was obtained providing 17 fold final purification of the enzyme. Biochemical characterization of the Amy RM16 such as optimum temperature and pH, substrate specificity and enzymatic susceptibilities towards different metal ions and inhibitors were also performed. Results of these studies revealed that, the enzyme is active at wide temperature range with optimum activity at 80°C and retained 85% of the activity for 3 h at 50°C and around 50% of remaining activity for 1 h at 80°C. The enzyme showed optimum activity at pH 5.0. On the other hand, Ca +2 and EDTA (1 to 5 mM) did not significantly affect the enzyme activity. The main substrate for the enzyme was found to be starch but it could also hydrolyze raw starch, dextrin, γ γ γ γcyclodextrin and pullulan.

Spectrochimica Acta Part A-molecular and Biomolecular Spectroscopy, 1998

Circular dichroism spectroscopy is used to investigate the thermostability of six arthropod hemoc... more Circular dichroism spectroscopy is used to investigate the thermostability of six arthropod hemocyanins (Hcs), representatives of the subphyla Crustacea (infraorder Brachyura) and Chelicerate (infraorders Xiphosura and Arachnida), and three molluscan Hcs from gastropod organisms. Melting ...

Comparative Biochemistry and Physiology B-biochemistry & Molecular Biology, 2000

The hemocyanin (Hc) from Buthus sindicus, studied in the native state, demonstrated to be an aggr... more The hemocyanin (Hc) from Buthus sindicus, studied in the native state, demonstrated to be an aggregate of eight different types of subunits arranged in four cubic hexamers. Both, the 'top' and the 'side' views of the native molecule have been identified from the negatively stained specimens using transmission electron microscopy. Out of these, eight different polypeptide chains, the partial primary structure (68%) of a subunit Bsin1 (M r = 72422.7 Da) was established using a combination of automated Edman degradation and mass spectrometry. A multiple sequence alignment with other closely related cheliceratan Hc subunits revealed average identities of ca. 60%. Most of the structurally important residues, i.e. copper and calcium-binding ligands, as well as the residues involved in the presumed oxygen entrance pathway, proved to be strictly conserved in Bsin1. Sequence variations have been observed around the functionally important chloride-binding site, not only for the B. sindicus subunit Bsin1, but also for the subunit Aaus-6 of the scorpion A. australis and the subunit Ecal-a from the spider Eurypelma californicum Hcs. Deviation in the primary structure related to the chloride-binding site suggest that the effect of chloride ions may vary in different hemocyanins. Furthermore, the secondary structural contents of the Hc subunit Bsin1 were determined by circular dichroism revealing ca. 33% a-helix, 18% b-sheet, 19% b-turn, and 30% random coil composition. These values are in good agreement with the crystal structure of the closely related Hc subunit Lpol-II from horseshoe crab L. polyphemus. Electron microscopic studies of the purified Hc subunit under native conditions revealed that Bsin1 has self aggregation properties. Results of these studies are discussed. (S. Abid Ali) 0305-0491/00/$ -see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 3 0 5 -0 4 9 1 ( 0 0 ) 0 0 1 8 9 -9

Toxicon, 1999

Two phospholipases A 2 (PLA 2 , H1 and H2) from sea snake Hydrophis cyanocinctus venom were puri®... more Two phospholipases A 2 (PLA 2 , H1 and H2) from sea snake Hydrophis cyanocinctus venom were puri®ed to homogeneity in a single step using reversed-phase high performance liquid chromatography on a Nucleosil 7C 18 column. The molecular weights of H1 and H2, as estimated by MALDI MS, were 13588.1 and 13247.2 Da, respectively. The N-terminal 60 amino acid residues were determined by direct automated Edman degradation analysis. Since both PLA 2 s show close sequence homologies to those of PLA 2 s from other Elapid snakes (60±84%) they have been tentatively classi®ed as belonging to group-IA and Asp-49 phospholipases A 2 . Despite the sequence variation (18%) between H1 and H2, their general structural organization is very similar as shown by their clearly related CD spectra. Furthermore, both enzymes are quite thermostable (60±658C) as determined by temperature variable CD spectra, indicating that the enzymes contain compact folded structure, mainly based on the core structure of disul®de bridges. However, the major PLA 2 (H1) shows higher toxicity to albino rats (LD 50 i.p. 0.04 mg/kg) and puri®cation resulted in 18-fold 0041-0101/99/$ -see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 1 -0 1 0 1 ( 9 9 ) 0 0 0 9 1 -4 Toxicon 37 (1999) 1505±1520 www.elsevier.com/locate/toxicon

Journal of Protein Chemistry, 1989

The complete amino acid sequence of the αA-chain of major hemoglobin component from gray partridg... more The complete amino acid sequence of the αA-chain of major hemoglobin component from gray partridgeFrancolinus pondacerianus is presented. The major component HbA accounts for 75% of the total hemolysate. Separation of the globin subunits was achieved by ion-exchange chromatography on CM-Cellulose in 8 M urea. The sequence was studied by automatic Edman degradation of the native chain and its tryptic peptides in a gas-phase sequencer. The phylogenetic relationship of Galliformes with other avian orders is discussed.

Biochemical and Biophysical Research Communications, 2002

The primary structure of the major hemoglobin component, HbA (␣ A -and ␤-chain), from Tufted duck... more The primary structure of the major hemoglobin component, HbA (␣ A -and ␤-chain), from Tufted duck (Aythya fuligula) is presented. The separation of the globin subunits was achieved by ion exchange chromatography on CM-cellulose in 8 M urea. The amino acid sequence was determined by automatic Edman degradation of native chains as well as tryptic and hydrolytic peptides in a gas-phase sequencer. The automated homology model was generated by the protein structure modeling package WHAT IF using the crystal structure coordinates of Bar-headed goose hemoglobin. The 3D structure prediction enables ␣99Arg and ␤101Glu to emerge as a new intersubunit contact site not found in the hemoglobin structure of any other species. ␣99Arg forms a complex salt bridge network involving ␣99Arg-␤101Glu-␤104Arg-␤108Asp. Also the substitution at ␣34 3 Ile, ␣38 3 Gln and ␤55 3 Leu serves to stabilize the oxy-structure, leading to higher oxygen affinity. © 2002 Elsevier Science (USA)

Biophysical Journal, 2000

Synchrotron x-ray scattering measurements were performed on dilute solutions of the purified hemo... more Synchrotron x-ray scattering measurements were performed on dilute solutions of the purified hemocyanin subunit (Bsin1) from scorpion (Buthus sindicus) and the N-terminal functional unit (Rta) from a marine snail (Rapana thomasiana). The model-independent approach based on spherical harmonics was applied to calculate the molecular envelopes directly from the scattering profiles. Their molecular shapes in solution could be restored at 2-nm resolution. We show that these units represent stable, globular building blocks of the two hemocyanin families and emphasize their conformational differences on a subunit level. Because no crystallographic or electron microscopy data are available for isolated functional units, this study provides for the first time structural information for isolated, monomeric functional subunits from both hemocyanin families. This has been made possible through the use of low protein concentrations (< or = 1 mg/ml). The observed structural differences may offer advantages in building very different overall molecular architectures of hemocyanin by the two phyla.

Journal of Protein Chemistry, 1985

The complete amino acid sequence of the major component of hemoglobin from amur-leopard (Panthera... more The complete amino acid sequence of the major component of hemoglobin from amur-leopard (Panthera pardus orientalis) is presented. The major component accounts for more than 90% of the total hemoglobin. Separation of the globin subunits was achieved by ion-exchange chromatography on CM-cellulose in urea. The sequence was studied by automatic Edman degradation of tryptic and hydrolytic peptides. Alignment was carried out with human hemoglobin sequence. The β NH2 terminus is blocked with Ac-serine. The data are compared with other mammalian hemoglobins and results are discussed with respect to sequence and physiology.

Archives of Biochemistry and Biophysics, 2000

and §Abteilung fü r Transfusionsmedizin mit Blutbank, Otfried-Mü ller-Straße 4/1, The enzyme L-am... more and §Abteilung fü r Transfusionsmedizin mit Blutbank, Otfried-Mü ller-Straße 4/1, The enzyme L-amino acid oxidase (LAO) from the leaf-nosed viper (Eristocophis macmahoni) snake venom was purified to homogeneity in a single step using high performance liquid chromatography on a Nucleosil 7C 18 reverse phase column. The molecular mass of the purified enzyme was 58734.0 Da, as determined by matrix-assisted laser desorption/ionization mass spectrometry. The N-terminal amino acid se-

Biochemical and Biophysical Research Communications, 2004

Avian hemoglobins have attracted much attention in view of the unique oxygen transport characteri... more Avian hemoglobins have attracted much attention in view of the unique oxygen transport characteristics. The present study describes the primary structure of minor hemoglobin component HbD from Tufted duck (Aythya fuligula), a migratory bird seen in Pakistan during the winter season. Separation of the polypeptide subunits was achieved by ion exchange chromatography in the presence of 8 M urea. Molecular masses of the intact protein as well as peptides obtained from chemical and enzymatic cleavages were determined by electrospray ionization mass spectrometry. The sequence was studied by automatic Edman degradation of the native chains and their tryptic/hydrolytic fragments in a gas-phase sequencer. Comparison of the hemoglobin sequence with the corresponding sequences of Anseriform representatives and other avian species shows residues like a D 23 Asp, a D 120 Asp as being specific to Tufted duck. The three-dimensional structure analyzed with the protein structure modeling package, WHAT IF, using the crystal structure coordinates of chicken hemoglobin (PDB code = 1hbr) shows a D 34 Val, a D 38 Gln, and a D 94 Asp as possible mediators offering alternate pathway for oxygen uptake and release thereby leading to distinct hypoxia tolerance in the Tufted ducks. Results are discussed with reference to function and evolution in the Anseriform representatives.

Journal of Protein Chemistry, 1991

The complete amino acid sequence of the αA-chain of major hemoglobin component from Cuckoo (Eudyn... more The complete amino acid sequence of the αA-chain of major hemoglobin component from Cuckoo (Eudynamys scolopaceae) is presented. Separation of the polypeptide subunits was achieved by ion exchange chromatography in the presence of 8 M urea. The sequence was studied by automatic Edman degradation of the native chain and its tryptic fragments in a gas-phase sequencer. Comparison with other avian hemoglobins shows residues α21, α30, α96, α110, and α114 as being specific to Cuckoo. The functional significance of these is discussed.

Toxicon, 1999

Crude venom from Eristocophis macmahoni was demonstrated to exert a potent inhibition of human bl... more Crude venom from Eristocophis macmahoni was demonstrated to exert a potent inhibition of human blood platelet aggregation mediated by adenosine diphosphate (ADP), platelet activating factor (PAF) and arachidonic acid (AA). The venom caused lysis of the platelets, however, the red blood cells were not lysed by the venom. Substantial oedema was produced upon injection of the venom into the rat hind paw. Contrarily, the intraperitoneal injection of the venom to the rats caused an inhibition of the carrageenin-induced rat paw oedema. However, an 100% lethality within 24 h was observed with a dose of 40 mg/kg body weight. The venom was fractionated by reverse phase high pressure liquid chromatography (HPLC) and the fractions were analyzed for their eect on ADP-induced platelet aggregation. The fraction eluted at 15.5 min (20% acetonitrile concentration) exhibited an inhibitory eect of several-fold greater potency than that of the crude venom. Fractions eluted at 18.5 min (25.4% acetonitrile concentration) and onward showed a proaggregatory but insigni®cant eect. It is suggested that although the venom contains pro-Toxicon 37 (1999) 1095±1107

Journal of Protein Chemistry, 1991

Primary structure of β-chain of pigeon is presented. It was determined by amino acid sequence ana... more Primary structure of β-chain of pigeon is presented. It was determined by amino acid sequence analysis of intact β-chain and its peptides obtained by the enzymatic and chemical cleavage. Comparison of amino acid sequence of the chain with other available data shows β 14 Ile, β61 Lys, and β113 Ile as residues specific to pigeon. One important replacement at α1β1 contact is β55 Met→Ser.

Journal of Protein Chemistry, 1989

Primary structure of hemoglobin of α-chain ofColumba livia is presented. The separation of α-chai... more Primary structure of hemoglobin of α-chain ofColumba livia is presented. The separation of α-chain was obtained from globin by ion-exchange chromatography (CMC-52) and reversed-phase HPLC (RP-2 column). Amino acid sequence of intact as well as tryptic digested chain was determined on gas-phase sequencer. Structure is aligned homologously with 21 other species. Among different exchanges, positions α24 (Tyr→Leu), α26 (Ala→Gly), α32 (Met→Leu), α64 (Asp→Glu), α113 (Leu→Phe), and α129 (Leu→Val) are unique to pigeon hemoglobin. The various exchanges in α-chain are discussed with reference to evolution and phylogeny. The results show that the order Columbiformes is evolutionarily closer to the order Anseriformes. Since the pigeon is homogeneous, having HbA (αA-chain) and lacks αD-chain, its phylogenetic placement could be established among birds having single hemoglobin components.

Archives of Biochemistry and Biophysics, 2006

Scorpion venoms are among the most widely known source of peptidyl neurotoxins used for calliperi... more Scorpion venoms are among the most widely known source of peptidyl neurotoxins used for callipering different ion channels, e.g., for Na + , K + , Ca + or Cl À . An a-toxin (Bs-Tx28) has been purified from the venom of scorpion Buthus sindicus, a common yellow scorpion of Sindh, Pakistan. The primary structure of Bs-Tx28 was established using a combination of MALDI-TOF-MS, LC-ESI-MS, and automated Edman degradation analysis. Bs-Tx28 consists of 65 amino acid residues (7274.3 ± 2Da), including eight cysteine residues, and shows very high sequence identity (82-94%) with other long-chain a-neurotoxins, active against receptor site-3 of mammalian (e.g., Lqq-IV and Lqh-IV from scorpions Leiurus sp.) and insect (e.g., BJa-IT and Od-1 from Buthotus judaicus and Odonthobuthus doriae, respectively) voltage-gated Na + channels. Multiple sequence alignment and phylogenetic analysis of Bs-Tx28 with other known a-and a-like toxins suggests the presence of a new and separate subfamily of scorpion a-toxins. Bs-Tx28 which is weakly active in both, mammals and insects (LD 50 0.088 and 14.3 lg/g, respectively), shows strong induction of the rat afferent nerve discharge in a dose-dependent fashion (EC 50 = 0.01 lg/mL) which was completely abolished in the presence of tetrodotoxin suggesting the binding of Bs-Tx28 to the TTX-sensitive Na + -channel. Three-dimensional structural features of Bs-Tx28, established by homology modeling, were compared with other known classical a-mammal (AaH-II), a-insect (Lqh-aIT), and a-like (BmK-M4) toxins and revealed subtle variations in the Nt-, Core-, and RT-CT-domains (functional domains) which constitute a ''necklace-like'' structure differing significantly in all a-toxin subfamilies. On the other hand, a high level of conservation has been observed in the conserved hydrophobic surface with the only substitution of W43 (Y43/42) and an additional hydrophobic character at position F40 (L40/A/V/G39), as compared to the other mentioned a-toxins. Despite major differences within the primary structure and activities of Bs-Tx28, it shares a common structural and functional motif (e.g., transRT-farCT) within the RT-CT domain which is characteristic of scorpion a-mammal toxins.

Archives of Biochemistry and Biophysics, 2001

Four depressant insect-selective neurotoxin analogs (termed Bs-dprIT1 to 4) from the venom of the... more Four depressant insect-selective neurotoxin analogs (termed Bs-dprIT1 to 4) from the venom of the scorpion Buthus sindicus were purified to homogeneity in a single step using reverse-phase HPLC. The molecular masses of the purified toxins were 6820.9, 6892.4, 6714.7, and 6657.1 Da, respectively, as determined by mass spectrometry. These long-chain neurotoxins were potent against insects with half lethal dose values of 67, 81, 103, and 78 ng/100 mg larva and 138, 160, 163, and 142 ng/100 mg cockroach, respectively, but were not lethal to mice even at the highest applied dose of 10 g/20 g mouse. When injected into blowfly larvae (Sarcophaga falculata), Bs-dprIT1 to 4 induced classical manifestations of depressant toxins, i.e., a slow depressant flaccid paralysis. The primary structures of Bs-dprIT 1 to 4 revealed high sequence homology (60 -75%) with other depressant insect toxins isolated from scorpion venoms. Despite the high sequence conservation, Bs-dprIT1 to 4 showed some remarkable features such as (i) the presence of methionine (Met 6 in Bs-dprIT1 and Met 24 in Bs-dprIT2 to 4) and histidine (His 53 and His 57 in Bs-dprIT1) residues, i.e., amino acid residues that are uncommon to this type of toxin; (ii) the substitution of two highly conserved tryptophan residues (Trp43 3 Ala and Trp53 3 His) in the sequence of Bs-dprIT1; and (iii) the occurrence of more positively charged amino acid residues at the C-terminal end than in other depressant insect toxins. Multiple sequence alignment, sequence analysis, sequence-based structure prediction, and 3D homology modeling studies revealed a protein fold and secondary structural elements similar to those of other scorpion toxins affecting sodium channel activation. The electrostatic potential calculated on the surface of the predicted 3D model of Bs-dprIT1 revealed a significant positive patch in the region of the toxin that is supposed to bind to the sodium channel.

Biochemical and Biophysical Research Communications, 2003

Cytotoxic lymphocytes (CTLs), the key players of cell mediated immunity, induce apoptosis by enga... more Cytotoxic lymphocytes (CTLs), the key players of cell mediated immunity, induce apoptosis by engaging death receptors or through exocytosis of cytolytic granules containing granzyme (proteases) and pore-forming protein (perforin). The crystal structure of granzyme B from human (B h ) and rat (B r ), as well as that of pro-granzyme K ðK h Þ has been reported recently. In the present communication, we describe the homology modeling of granzyme family (in particular Gzm A h , M h , B m , and C m from human and mouse) based on the crystal structural coordinates of trypsin, granzyme K ðK h Þ, and granzyme B ðB h Þ. These models have been used for establishing phylogenetic relationship as well as identifying characteristic features for designing specific inhibitors. The paper also highlights key residues at the S1, S2, and S2 0 binding subsites in all granzyme, which may be involved in the structure-function relationship of this enzyme family. The predicted 3D homology models show a conserved two similar domain structure, i.e., an Nterminal domain and a C-terminal domain comprising predominantly of b-sheet structure with a little a-helical content. Microheterogeneities have been observed in the vicinity of the active site in all granzymes as compared to granzyme B h . For example, in granzyme M h , valine is present at the S1 subsite instead of arginine. Similarly differences at S2 (Leu ! Phe), S3 (Ser ! Gly), and S4 (Arg ! Asn) subsites are quite apparent and appear to hold the potential for selective designing of inhibitors for possible therapeutic applications. Furthermore, analysis of the electrostatic surface potential on the shape of granzyme-inhibitor binding groove reveals clear differences at the reactive site. Additionally the different posttranslational modification sites such as phosphorylation (e.g., in granzyme M Thr101, Ser109), myristoylation (Gly22, 117, and 131), and glycosylation (Ser160) have been identified, as very little is known about the functional significance of these modifications in the granzyme family. Thus, glycosylation at Ser160 in granzyme M may influence the net charge of the enzyme, resulting in altered substrate binding as compared to granzyme B. Also this modification may influence the rate of complexation and binding affinity with proteoglycans. These studies are expected to contribute towards the basic understanding of functional associations of the granzymes with other molecules and their possible role in apoptosis.

Biochemical and Biophysical Research Communications, 2003

Cytotoxic lymphocytes (CTLs), the key players of cell mediated immunity, induce apoptosis by enga... more Cytotoxic lymphocytes (CTLs), the key players of cell mediated immunity, induce apoptosis by engaging death receptors or through exocytosis of cytolytic granules containing granzyme (proteases) and pore-forming protein (perforin). The crystal structure of granzyme B from human (B h ) and rat (B r ), as well as that of pro-granzyme K ðK h Þ has been reported recently. In the present communication, we describe the homology modeling of granzyme family (in particular Gzm A h , M h , B m , and C m from human and mouse) based on the crystal structural coordinates of trypsin, granzyme K ðK h Þ, and granzyme B ðB h Þ. These models have been used for establishing phylogenetic relationship as well as identifying characteristic features for designing specific inhibitors. The paper also highlights key residues at the S1, S2, and S2 0 binding subsites in all granzyme, which may be involved in the structure-function relationship of this enzyme family. The predicted 3D homology models show a conserved two similar domain structure, i.e., an Nterminal domain and a C-terminal domain comprising predominantly of b-sheet structure with a little a-helical content. Microheterogeneities have been observed in the vicinity of the active site in all granzymes as compared to granzyme B h . For example, in granzyme M h , valine is present at the S1 subsite instead of arginine. Similarly differences at S2 (Leu ! Phe), S3 (Ser ! Gly), and S4 (Arg ! Asn) subsites are quite apparent and appear to hold the potential for selective designing of inhibitors for possible therapeutic applications. Furthermore, analysis of the electrostatic surface potential on the shape of granzyme-inhibitor binding groove reveals clear differences at the reactive site. Additionally the different posttranslational modification sites such as phosphorylation (e.g., in granzyme M Thr101, Ser109), myristoylation (Gly22, 117, and 131), and glycosylation (Ser160) have been identified, as very little is known about the functional significance of these modifications in the granzyme family. Thus, glycosylation at Ser160 in granzyme M may influence the net charge of the enzyme, resulting in altered substrate binding as compared to granzyme B. Also this modification may influence the rate of complexation and binding affinity with proteoglycans. These studies are expected to contribute towards the basic understanding of functional associations of the granzymes with other molecules and their possible role in apoptosis.

Journal of Basic Microbiology, 2001

Bacillus sp. RM16 was isolated from a hot spring in Karachi and screened for the production of αa... more Bacillus sp. RM16 was isolated from a hot spring in Karachi and screened for the production of αamylase. The enzyme was obtained after 72 h cultivation of strain in Luria broth containing 1% starch (w/v). Enzyme Amy RM16 was purified to electrophoretic homogeneity by a series of sequential steps including precipitation with ammonium sulfate at 70% saturation, Q-Sepharose, Phenyl Sepharose and reversed phase chromatography. The purified enzyme is made up of a single polypeptide chain of 66 kDa as established by a combination of SDS-PAGE and zymographic analysis. In our experimental conditions, a total yield of 1.35% with specific activity of 6380U/mg was obtained providing 17 fold final purification of the enzyme. Biochemical characterization of the Amy RM16 such as optimum temperature and pH, substrate specificity and enzymatic susceptibilities towards different metal ions and inhibitors were also performed. Results of these studies revealed that, the enzyme is active at wide temperature range with optimum activity at 80°C and retained 85% of the activity for 3 h at 50°C and around 50% of remaining activity for 1 h at 80°C. The enzyme showed optimum activity at pH 5.0. On the other hand, Ca +2 and EDTA (1 to 5 mM) did not significantly affect the enzyme activity. The main substrate for the enzyme was found to be starch but it could also hydrolyze raw starch, dextrin, γ γ γ γcyclodextrin and pullulan.

Comparative Biochemistry and Physiology A-molecular & Integrative Physiology, 1999

Out of eight different polypeptide chains, present in the native hemocyanin molecule of scorpion ... more Out of eight different polypeptide chains, present in the native hemocyanin molecule of scorpion Buthus sindicus, subunit Bsin1 was preparatively purified and characterized by amino acid composition, laser desorption mass spectrometry and spectroscopic techniques. Thermal stability of the intact respiratory protein and its subunit was studied by following the change in ellipticity at 222 nm as a function of temperature. The copper-dioxygen system at the binuclear active site has a stabilizing effect and the oxy-proteins are significantly more thermostable than the apo-forms. The thermal stability of the scorpion Hc from B. sindicus is considerably smaller than that of the tarantula (Eurypelma californicum) Hc [Sterner R et al., FEBS Lett 1995;364:9 -12], although both, scorpion and spider, belong to the subphylum chelicerata. The fluorescence properties of the scorpion Hc and Bsin1 suggest that the indol groups are in rather nonpolar environment, deeply 'buried' in the interior of the aggregates and structural subunits. At the same time, these chromophores are located on subunit interfaces in the C. sapidus and O. 6ulgaris Hcs, participating in protein-protein interactions [Ricchelli F et al., Arch Biochem Biophys 1984;235:461 -469; Stoeva S et al., Spectrochimica Acta A 1995;51:1965.

Journal of Basic Microbiology, 2001

Bacillus sp. RM16 was isolated from a hot spring in Karachi and screened for the production of αa... more Bacillus sp. RM16 was isolated from a hot spring in Karachi and screened for the production of αamylase. The enzyme was obtained after 72 h cultivation of strain in Luria broth containing 1% starch (w/v). Enzyme Amy RM16 was purified to electrophoretic homogeneity by a series of sequential steps including precipitation with ammonium sulfate at 70% saturation, Q-Sepharose, Phenyl Sepharose and reversed phase chromatography. The purified enzyme is made up of a single polypeptide chain of 66 kDa as established by a combination of SDS-PAGE and zymographic analysis. In our experimental conditions, a total yield of 1.35% with specific activity of 6380U/mg was obtained providing 17 fold final purification of the enzyme. Biochemical characterization of the Amy RM16 such as optimum temperature and pH, substrate specificity and enzymatic susceptibilities towards different metal ions and inhibitors were also performed. Results of these studies revealed that, the enzyme is active at wide temperature range with optimum activity at 80°C and retained 85% of the activity for 3 h at 50°C and around 50% of remaining activity for 1 h at 80°C. The enzyme showed optimum activity at pH 5.0. On the other hand, Ca +2 and EDTA (1 to 5 mM) did not significantly affect the enzyme activity. The main substrate for the enzyme was found to be starch but it could also hydrolyze raw starch, dextrin, γ γ γ γcyclodextrin and pullulan.

Spectrochimica Acta Part A-molecular and Biomolecular Spectroscopy, 1998

Circular dichroism spectroscopy is used to investigate the thermostability of six arthropod hemoc... more Circular dichroism spectroscopy is used to investigate the thermostability of six arthropod hemocyanins (Hcs), representatives of the subphyla Crustacea (infraorder Brachyura) and Chelicerate (infraorders Xiphosura and Arachnida), and three molluscan Hcs from gastropod organisms. Melting ...

Comparative Biochemistry and Physiology B-biochemistry & Molecular Biology, 2000

The hemocyanin (Hc) from Buthus sindicus, studied in the native state, demonstrated to be an aggr... more The hemocyanin (Hc) from Buthus sindicus, studied in the native state, demonstrated to be an aggregate of eight different types of subunits arranged in four cubic hexamers. Both, the 'top' and the 'side' views of the native molecule have been identified from the negatively stained specimens using transmission electron microscopy. Out of these, eight different polypeptide chains, the partial primary structure (68%) of a subunit Bsin1 (M r = 72422.7 Da) was established using a combination of automated Edman degradation and mass spectrometry. A multiple sequence alignment with other closely related cheliceratan Hc subunits revealed average identities of ca. 60%. Most of the structurally important residues, i.e. copper and calcium-binding ligands, as well as the residues involved in the presumed oxygen entrance pathway, proved to be strictly conserved in Bsin1. Sequence variations have been observed around the functionally important chloride-binding site, not only for the B. sindicus subunit Bsin1, but also for the subunit Aaus-6 of the scorpion A. australis and the subunit Ecal-a from the spider Eurypelma californicum Hcs. Deviation in the primary structure related to the chloride-binding site suggest that the effect of chloride ions may vary in different hemocyanins. Furthermore, the secondary structural contents of the Hc subunit Bsin1 were determined by circular dichroism revealing ca. 33% a-helix, 18% b-sheet, 19% b-turn, and 30% random coil composition. These values are in good agreement with the crystal structure of the closely related Hc subunit Lpol-II from horseshoe crab L. polyphemus. Electron microscopic studies of the purified Hc subunit under native conditions revealed that Bsin1 has self aggregation properties. Results of these studies are discussed. (S. Abid Ali) 0305-0491/00/$ -see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 3 0 5 -0 4 9 1 ( 0 0 ) 0 0 1 8 9 -9

Toxicon, 1999

Two phospholipases A 2 (PLA 2 , H1 and H2) from sea snake Hydrophis cyanocinctus venom were puri®... more Two phospholipases A 2 (PLA 2 , H1 and H2) from sea snake Hydrophis cyanocinctus venom were puri®ed to homogeneity in a single step using reversed-phase high performance liquid chromatography on a Nucleosil 7C 18 column. The molecular weights of H1 and H2, as estimated by MALDI MS, were 13588.1 and 13247.2 Da, respectively. The N-terminal 60 amino acid residues were determined by direct automated Edman degradation analysis. Since both PLA 2 s show close sequence homologies to those of PLA 2 s from other Elapid snakes (60±84%) they have been tentatively classi®ed as belonging to group-IA and Asp-49 phospholipases A 2 . Despite the sequence variation (18%) between H1 and H2, their general structural organization is very similar as shown by their clearly related CD spectra. Furthermore, both enzymes are quite thermostable (60±658C) as determined by temperature variable CD spectra, indicating that the enzymes contain compact folded structure, mainly based on the core structure of disul®de bridges. However, the major PLA 2 (H1) shows higher toxicity to albino rats (LD 50 i.p. 0.04 mg/kg) and puri®cation resulted in 18-fold 0041-0101/99/$ -see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 1 -0 1 0 1 ( 9 9 ) 0 0 0 9 1 -4 Toxicon 37 (1999) 1505±1520 www.elsevier.com/locate/toxicon

Journal of Protein Chemistry, 1989

The complete amino acid sequence of the αA-chain of major hemoglobin component from gray partridg... more The complete amino acid sequence of the αA-chain of major hemoglobin component from gray partridgeFrancolinus pondacerianus is presented. The major component HbA accounts for 75% of the total hemolysate. Separation of the globin subunits was achieved by ion-exchange chromatography on CM-Cellulose in 8 M urea. The sequence was studied by automatic Edman degradation of the native chain and its tryptic peptides in a gas-phase sequencer. The phylogenetic relationship of Galliformes with other avian orders is discussed.