I= PRIMARY STRUCTURE OF HOMOLOGIES FROM SEA SNAKE (microcephalophis gracilis gracilis) ROSE RIGNED PARAKEET (Psittacula Krameri).
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Title of Thesis
PRIMARY STRUCTURE OF HOMOLOGIES FROM SEA SNAKE (microcephalophis gracilis gracilis) ROSE RIGNED PARAKEET (Psittacula Krameri).

Alia Islam
Institute/University/Department Details
University of Karachi./H.E.J. Research Institute of Chemistry
Number of Pages
Keywords (Extracted from title, table of contents and abstract of thesis)
rose-ringed parakeet, psittacula krameri, microcephalophis gracilis gracilis (serpentes, reptilia), reptilian hemoglobin, hemoglobin, blood, psittaciformes

The present study on sea snake Microcephalophis gracilis gracilis (Serpentes, Reptilia) and Rose-ringed parakeet Psittacula krameri (Psittaciformes, Aves) hemoglobins were undertaken to characterize the primary structure of globin chains. Both animals contain a single hemoglobin component HbA.

Native globins were separated on CM-cellulose 52 in the presence of dissociating agent 8M urea, as well as on reversed phase high performance liquid chromatography. The carboxymethylated sub-units were further degraded by enzymatic and chemical cleavages into small peptides. The complete primary structure of the polypeprides were established by sequence determination of the intact globins chains, the CNBr fragments, peptides obtained by Glu-specific protease and tryptic digestion before and after citraconylation, manually by DABTIC method as well as automatic Edman degradation using the liquid and gas phase sequencers.

The first complete sequence determination of a-and B-chains of Sea snake hemoglobin allows comparison of different characterized species of reptiles. Comparison of a-and B-chains with the corresponding chain of human hemoglobin shows 58 exchanges in the a and 51 in the B-chain involving 17 a1B1, and 4 heme contact residues. Many positions (20 in a and 13 in B-chain) of the Sea snake hemoglobin contain residues unique to this form. Comparison of the Sea snake hemoglobin with other reptilian species shows 52-65 exchanges in the and 37-62 exchanges in the B chain. The corresponding Sea snake versus avian differences being 56-65 in the a and 31-39 in the B-chain.

Primary structures of 29 avian species are known, permitting comparison of the protein structure function relationships. The primary structure of Rose ringed parakeet hemoglobin defines variations within an additional avian order Psittaciformes, (II exchanges in a and 11 in B-chain). Homologous comparison of Rose-ringed parakeet with human hemoglobin revealed 48 difference in a and 2 in the B-chain are unique to Rose ringed parakeet. Comparison of Rose-ringed parakeet with other avian species shows 11-33 in the a and 9-15 in the B-chain, whereas with reptilian species the corresponding differences were 37-67 in the a and 26-58 in the B-chain. B-chain is more conserved than a-chain in both species. The various exchanges are discussed with reference to evolution of hemoglobin molecule. Phylogenetic trees have been constructed using the sequence data of all characterized a and B-chains of avian and reptilian species with respect to Sea snake and Rose-ringed parakeet globin chains. The evolutionary relationship between avain and reptilian species is discussed.

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S. No. Chapter Title of the Chapters Page Size (KB)
1 1 Introduction 1
297.76 KB
  1.1 General Description 1
  1.2 Hemoglobin Structure 1
  1.3 Function of Hemoglobin 4
  1.4 Reptilian Hemoglobin 7
  1.5 Avian Hemoglobin 15
  1.6 Evolution and Phylogeny 21
  1.7 Objective of the Present Study 25
2 2 Experimental 25
118.01 KB
  2.1 Collection of Blood samples 26
  2.2 Extraction of Hemoglobin 26
  2.3 Determination of Hemoglobin concentration 26
  2.4 Preparation of Globin 26
  2.5 Electrophoresis 27
  2.6 Separation by ion-Exchange chromatography 28
  2.7 Purification of the a-chain of sea snake 29
  2.8 Separation of Globin chains by RP-HPLC 29
  2.9 Modification of protein chains 30
  2.10 Chemical cleavage 30
  2.11 Enzymatic cleavages 30
  2.12 Fractionation of cyanogens bromide peptides 31
  2.13 Separation of peptides by RP-HPLC 31
  2.14 Amino Acid analysis 31
  2.15 Sequence analysis 32
  2.16 Monitoring of radioactivity 35
3 3 Results 36
263.53 KB
  3.1 Sea Snake ( Microcephalophis gracilis gracilis ) Hemoglobin 36
  3.2 Rose-ringed parakeet ( Psittacula krameri ) Hemoglobin 51
4 4 Discussion 67
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  4.1 Sea Snake ( Microcephalophis gracilis gracilis ) Hemoglobin 67
  4.2 Rose-ringed parakeet ( Psittacula krameri ) Hemoglobin 101
5 5 References 135
284.3 KB