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Title of Thesis

Jaweed Shafqat
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)
venom, naja naja naja, neurotoxins, cytotoxins, haemotoxins, proteases, phospholipases, oxidases, rnases, dnases, elapidae, phospholipase a2, kunitz, trypsin, chymotrypsin, snake, peptides

Snake venoms are complex mixture of various toxic and nontoxic proteins like; neurotoxins, cytotoxins, haemotoxins, inhibitor of proteases, enzymes like proteases, phospholipases, oxidases, RNases, DNases, etc. However, no single venom contain all of them and each family has its own characteristic venom. Snake naja naja naja is a member of family Elapidae and possesses a potent neurotoxic venom

Present study is carried out in order to characterize several toxic, as well as nontoxic protein components present in the venom of Naja naja naja. The objectives has been to isolate, purify and to elucidate the primary structure of component proteins not yet characterized from its venom. The investigations resulted in complete characterization of several components like phospholipase A2, a Kunitz type trypsin inhibitor, a Kunitz type chymotrypsin inhibitor-like peptide, several isoforms of miscellaneous neurotoxins, and a cytotoxin-like basic protein

Brief summary of the components characterization is given below: (1) Phospholipase A2 Phospholipase A2 was purified by a combination of reverse phase HPLC and ion exchange HPLC. The complete primary structure of phospholipase A2 determined by sequencing the intact protein and its Lys-specific protease digested peptides showed 119 positions in the protein and several involve charged residues. However, the Naja naja naja subform has no unique residue at the differing positions, thus the functional properties of Naja naja naja phospholipase A2 remains unaltered, is clearly related with other subspecies

(II) Trypsin inhibitor Naja naja naja venom trypsin inhibitor bystep of reverse phase HPLC. The protein strongly inhibits trypsin (K1=3.5X10-12M). The primary phase HPLC. The protein strongly inhibits trypsin (Ki=3.5X10-12M). The primary structure was established by peptide analyses of the 14C-carboxymethylated inhibitor. The 57 residue polypeptide chain belongs to the familyof Kunitz type inhibitors and exhibit significant residue identity with corresponding peptides from other resources. Functionally, a basic residue at position P3 (in the region of main contact with trypsin) conrrelates with strong inhibition

(III) Chymotrypsin Kunitz inhibitor-type peptide The chymotrypsin Kuntiz inhibitor type of polypeptide was isolated from the venom of Naja naja naja by reverse phase HPLC followed by cat-ion exchange FPLC. It wasd present in a considerably lower amount than that of the corresponding trypsin inhibitor. The primary structure, determined by the sequence analysis of whole molecule and its tryptic peptides showed 57 residues with an apparent molecular mass of 6.200 KDa. The main contact site with the protease (P1) has a phe residue, showing the specificity of the inhibitor of residues considered functionally important in Kunitz-type inhibitor, Gly-36 is repiaced by Ser in a segment of weak contact with the protease

(IV) Cytotoxin-like Basic Peptide cytotoxin-like basic peptide was isolated by a single step of reverse phase HPLC from the venom of Naja naja naja. The primary structure was determined and consist of 62 amino acid residue in a single polypeptide chain. The structure is highly similar to that of the cytotoxin-like basic peptides isolated from other Naja species but differ in two of the SS-loop structures from that of cytotoxins

(V) Miscellaneous Neurotoxin Several miscellaneous type neurotoxins forms are detected in the venom of Naja naja naja by the use of reverse phase HPLC and FPLC. The complete primary structure of major form (MS 8) is determined whiles fou other similar toxins (MS5-1 and II, MS6 and MS&) are only partially determined (upto 46 residues). The toxin MS8 showed 65 residues, with 10 half cystine and resembles the miscellaneous type of neurotoxins from other Naja species. Structures of other toxins upto 46 amino acid residues showed similar half cystine pattern and 53% sequence homology confirming them the member of miscellaneous type neurotoxin group having two additional cysteins at positions 6 and II. This typical cysteine pattern and difference in the sequence with long neurotoxins suggests an structure with altered confirmation. Existence of various different form of neurotoxins in the venom is related to the toxin gene multiplicity

Download Full Thesis
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S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
119.8 KB
2 1 Introduction 1
119.64 KB
  1.1 The Snakes 1
  1.2 Snake Venom And Their Compositions 2
  1.3 Neurotoxins 3
  1.4 Miscellaneous Neurotoxins 6
  1.5 Phospholipase A2 6
  1.6 Cytotoxins 9
  1.7 Cytotoxin-Like Basic Proteins 11
  1.8 Proteinase Inhibitor 11
  1.9 Enzymes Of Snake Venoms 13
3 2 Materials And Methods 15
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  2.1 Extraction And Methods 15
  2.2 Purification Of Venom 15
  2.3 Polyocrylamide Gel Electrophoresis 16
  2.4 Modification Of Proteins 16
  2.5 Enzymatic Cleavages 17
  2.6 Peptides Purifications 17
  2.7 Amino Acid Analyses 18
  2.8 Amino Acid Sequence Analysis Of Proteins And Peptides 19
  2.9 Monitoring Of Radioactivity 19
  2.10 Inhibitor Of Tryptic Activity 20
4 3 Results 21
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  3.1 Separation Of Crude Venom By Reverse Phase Hplc Phospholipase A2 From Naja Naja Naja Venom 21
  3.2 Purification Of Phospholipase A2 21
  3.3 Trypsin Inhibitor Of Naja Naja Naja Venom 27
  3.4 Chymotrypsin Inhibitor-Like Peptide Of Naja Naja Naja Venom 39
  3.5 Miscellaneous Neurotoxins Of Naja Naja Naja Venom 47
  3.6 Naja Naja Naja Venom Cytotoxin -Like Basic Protein 56
5 4 Discussion 69
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  4.1 Phospholipase A2 69
  4.2 Trypsin Inhibitor 74
  4.3 Kunitz Type Chymotrypsin Inhibitor-Like Peptide 80
  4.4 Miscellaneous Neurotoxins 83
  4.5 Cytotoxin-Like Basic Protein From Naja Naja Naja Venom 91
6 5 References 95
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  5.1 Appendix I (one and three letter symbols of amino acids 108