I= MOLECULAR BASIC OF RESPIRATION IN BIRDS: PRIMARY STRUCTURE OF HEMOGLOBIN FROM TUFTED DUCK (AYTHYA FULIGULA, NASRIFORMES)
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Title of Thesis
MOLECULAR BASIC OF RESPIRATION IN BIRDS: PRIMARY STRUCTURE OF HEMOGLOBIN FROM TUFTED DUCK (AYTHYA FULIGULA, NASRIFORMES)

Author(s)
Ghosia Haroon
Institute/University/Department Details
University of Karachi./H.E.J Research Institute of Chemistry
Session
1999
Subject
Chemistry
Number of Pages
133
Keywords (Extracted from title, table of contents and abstract of thesis)
respiration in birds, tufted duck (aythya fuligula, nasriformes), anseriformes, anatidae, respiratory pigments, aa-chain, ad-chains, b-chains

Abstract
The primary structure of aA-,aD-and B-chains from the hemoglobin of Tufted duck (Aythya fuligula) has been elucidated. Tufted duck belongs to the avian order Anseriformes, family Anatidae. This order includes aquatic birds like ducks, geese and swans commonly known as waterfowl. Members of this order are well known for their exceptional tolerance to hypoxia.

Separation of the polypeptide subunits was achieved by ion exchange chromatography. The globin chains were degraded by enzymatic and chemical cleavages into peptides. The primary structure of hemoglobin was established by sequence determination of native chains and its tryptic and hydrolytic peptides by automatic Edman degradation in a Gas-phase sequencer.

The hemoglobin of Tufted duck is heterogeneouos, having two hemoglobin components, like all other Anseriformes representatives. Positions aA13 Ile, aA99 Arg, aA135 Thr, aD120 Asp, B10 and B11 Ile are specific to Tufted duck hemoglobin. Alignment of the primary structure with Anserformes representatives indicates a high degree of homology within the order. Homologous comparison with other avian species reveals much conservation in the B-chain as compared to aA –and aD-chain. The automated homology model building was performed using the protein structure modeling package, WHAT IF. The three dimensional models of the two components i.e. HbA and HbD were calculated using crystal structure coordinates of Bar-headed goose (PDB id=A4F) and Chicken (PDB id=I hbr) hemoglobin. The various exchanges at positions critical for structure and function of the hemoglobin are discussed.

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S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
137.67 KB
2 1 Introduction 1
334.01 KB
  1.1 General Description 1
  1.2 Respiratory pigments 1
  1.3 Hemoglobin 2
  1.4 Avian Hemoglobin 9
  1.5 Multiple avian hemoglobins 9
  1.6 Oxygen affinity of avian hemoglobin 12
  1.7 Expression of aD-chain in avian hemoglobins 20
  1.8 Primary structure of avian hemoglobins 21
  1.9 Tufted duck 23
  1.10 Evolution and phylogeny 30
  1.11 Objective of the study 32
3 2 Experimental 33
136.36 KB
  2.1 Collection of blood samples 33
  2.2 Isolation of Hemoglobin 33
  2.3 Preparation of globin 33
  2.4 Electrophoresis 34
  2.5 Sepration by ion-exchange chromatography 36
  2.6 Modification by oxidation 37
  2.7 Cleavage of polypeptides 37
  2.8 Fractionation of tryptic peptide by gel filtration 38
  2.9 Separation of tryptic peptides on rephplc 38
  2.10 Separation of ASP-PRO peptides 39
  2.11 Amino acid analysis 39
  2.12 Mass spectrometry 39
  2.13 Sequence analysis 40
  2.14 Molecular modeling studies 42
  2.15 Phylogenetic analysis 44
4 3 Results 46
474.22 KB
  3.1 Electrophoresis 46
  3.2 Chromatography on CM-cellulose 46
  3.3 Fractionation of tryptic peptides by gel filtration HPLC 46
  3.4 Separation of peptides by RP-HPLC 46
  3.5 Amino acid analysis 47
  3.6 Chemical cleavage of aA-chain 47
  3.7 ESI-MS 47
  3.8 N-Terminal determination by dabitc’s method 47
  3.9 Automatic sequence analysis 67
  3.10 Evaluation of the homology models 73
5 4 Discussion 82
378.21 KB
  4.1 Heterogeneity of hemoglobins 82
  4.2 Sequence determination of globin chains 82
  4.3 Primary structure of the aA-, Ad- and B-chains 84
  4.4 The over all structures of HBA and HBD homology models 87
  4.5 The intersubunit binding sites in HBA and HBO models of tufted duck hemoglobin 88
  4.6 Phylogenetic analysis 103
  4.7 Tufted duck hemoglobin 108
6 5 Conclusion 112
377.85 KB
  5.1 References 114
  5.2 Appendix 115