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Title of Thesis

Nadeem Wajih
Institute/University/Department Details
H.E.J Research Institute of Chemistry/ University of Karachi
Number of Pages
Keywords (Extracted from title, table of contents and abstract of thesis)
novel protein, peptides, rabbit brain, ubiquitin, amino acid, myelin, protein, brain peptides, polypeptides

The present study on rabbit brain was undertaken to isolate, purify and characterize new proteins/peptides from rabbit brain employing gel filtration, reverse phase HPLC, cation exchange FPLC, electrophoresis and electroblotting for the isolation, purification and characterization of new proteins/peptides.

The present study is the first report on the primary structure of variant ubiquitin from rabbit brain two successive reverse phase HPLC of rabbit brain extract resulted in the isolation of two ubiquitin components A and B. Both the components have indistinguishable amino acid composition and identical in sequence. Separation of the ubiquitin preparation into two components on reverse phase chromatography therefore appears to reflect non-covalent binding with the reverse phase support or a post translational modification of ubiquitin that did not hinder in the sequence study.

The primary structure of the ubiquitin has been established by sequence analysis of the intact protein and its Lys-C endoprotease peptides. The structure has 76 amino acid residues. It shows 98.6% homology with human, bovine and mouse with only one exchange at position 6, while with Yeast and D. discoideum, it is 94.7% with four exchanges.

Furthermore, partial sequence of a new isoform of rabbit myelin basic protein is also elucidated. The purification of this new isoform of myelin basic protein has been carried out by the combination of gel filtration, reverse phase HPLC and cation exchange FPLC. New isoform show strong homology towards its other counter parts isolated from rabbit and other species. It shows 68% identity with human, 80% with bovine, 87.7% with mouse, 32.5% with chick and 73.9% identity with rat myelin basic proteins.

Present investigation also resulted in the purification of a 5kDa protein from rabbit brain. Its partial sequence structure shows strong homology (59%) to human ubiquitin. Another protein having 30kDa molecular mass was also purified from rabbit brain. Sequence study shows blocked N-terminus. The amino acid composition reveals high concentration of hydrophobic amino acids (34.6%).

Present investigation also describes the Electroblotting of rabbit brain proteins. 3M ammonium sulfate ppt. fraction(base soluble ) was electrophoresed, 14, 20 and 25kDa proteins were cut and electroblotted onto polybrene treated glass fiber filter discs and subjected to microsequence analysis. Sequence data show good repetitive yields in the range of 92-95%. N-terminal sequences of the blotted proteins were searched for sequence homology and it was found that these proteins do not resembles to any of the proteins reported ftill date from brain proteins.

Download Full Thesis
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S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
91.2 KB
2 1 Introduction 1
73.45 KB
  1.1 General Introduction 1
  1.2 Historical Perspective 1
  1.3 Categories Of Brain Peptides And Polypeptides 2
  1.4 Current Methods For Deciphering Unknown Peptides 2
  1.5 Brain Peptides: Evolutionary Aspects 4
  1.6 Studies On Rabbit Brain Proteins And Peptides 5
3 2 Experimental 10
58.25 KB
  2.1 Preparation Of White New Zealand Rabbits 10
  2.2 Separation And Purification Of Proteins From Extract 10
  2.3 Sds-Polyacrylamide Gel Electrophoresis ( Sds -Page ) 11
  2.4 Modification Of Proteins 12
  2.5 Enzymatic Cleavage 13
  2.6 Peptides Purification By Rp-Hplc 13
  2.7 Amino Acid Analysis 13
  2.8 Amino Acid Sequencing Of Protein And Peptides 13
  2.9 Electroblotting Of Proteins 14
4 3 Results 17
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  3.1 Separation Of The Brain Base Extract 17
  3.2 Separation And Characterization Of Ubiquitin 14
  3.3 5kda Protein From Rabbit Brain 31
  3.4 Electroblotting Of Rabbit Brain Protein 31
  3.5 Separation Of The Brain Acid Soluble Extract 37
  3.6 Purification Of 30kda Protein From Rabbit Brain 49
5 4 Discussion 50
117.74 KB
  4.1 Ubiquitin From Rabbit Brain 50
  4.2 Myelin Basic Protein 57
  4.3 5kda Protein From Rabbit Brain 60
  4.4 30kda N-Terminal Blocked Protein From Rabbit Brain 62
  4.5 Electroblotting Of Protein From Rabbit Brain 64
6 5 References 66
65.61 KB