I= INHIBITION OF CONFORMATIONAL CHANGES DUE TO CARBAMYLATION IN LENS CRYSTALLINS BY ASPIRIN
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Title of Thesis
INHIBITION OF CONFORMATIONAL CHANGES DUE TO CARBAMYLATION IN LENS CRYSTALLINS BY ASPIRIN
Author(s)
Syed Azeem ul Hassan
Institute/University/Department Details
H.E.J. Research Institute of Chemistry/ University of Karachi
Session
1993
Subject
Chemistry
Number of Pages
126
Keywords (Extracted from title, table of contents and abstract of thesis)
carbamylation, lens crystallins, aspirin, anticataract agent, peptides, enzymatic digestions, water soluble proteins

Abstract
The present study describes a new and more accurate technique, based on Fast atom bombardment mass spectrometry, to identify the types and quantitate the extent of modifications which have been produced due to the reactions of αA crystallin with isocyanate and aspirin. These findings would be helpful in understanding the phenomenon of carbamylation of lens crystallins in detail and also to check effectiveness of aspirin as an anticataract agent. The purified αA crystallin was incubated with KNCO and aspirin separately for 24 hours. Modified proteins then subjected to enzymatic digestions. Peptides generated by enzymatic digestions were separated using reverse phase HPLC` and analyzed by FAB-MS. For reaction conditions used in this investigation, carbamylated and acetylated lysyl residues were the principal products. For determination of the extent of carbamylation and acetylated, aliquotes of α-crystallin were incubated with isocyanate and aspirin for different time(i.e. 6 hours, 12 hours, 24 hours and 48 hours). After each incubation the modified α -crystallin was separated into αA and αB using RP HPLC. Each modified αA then subjected to enzymatic digestions, peptides generated by enzymatic digestions were recovered on Vydac C18 column and analyzed by FAB-MS. The extent of modifications was quantitated from the intensities of the peaks in the FAB spectra of modified and unmodified peptides. Both isocyanate and aspirin have modified all lysyl residues of αA crystallin hut with variable extent. Plots of the extent of carbamylation and acetylation versus time were used to calculate rate constants for reactions at each lysyl residue. Rate constants for carbamylation and acetylation of all lysyl residues except lysines 166 and 11 were found to be similar. The extent of modification" at Iysyl residues of αA crystallin were also estimated from concurren and sequential incubations. Both of these incubations caused a mark reduction in the extent of carbamylation and acetylation as compared to the extent of carbamylation and acetylation of Iysyl residues while α-crsytallin was incubated either with isocyanate or aspirin alone. Similarity in the rate constants and reduction in the extent of modifications due to concurrent and sequential incubations support the notion that "isocynate and aspirin compete for the same residue". But comparison of the yield of acetylated α-crystallin with the yield of carbamylated α-crystallin that might occur due to renal failure indicates that aspirin is not an effective inhibitor of cataract due to carbamylation of lysyl residues.
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818.82 KB
S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
122.49 KB
2 1 Introduction 1
122.48 KB
  1.1 Lens 1
  1.2 Lens Protein And Cataract 9
  1.3 Risk Factors 11
  1.4 Aspirin As Anticataract Agent 13
  1.5 Objective Of The Present Study 15
3 2 Experimental 16
71.86 KB
  2.1 Lens 16
  2.2 Extraction Of Water Soluble Proteins 16
  2.3 Modifications Of Α- Crystallin 19
4 3 Results 25
474.48 KB
  3.1 Gel Chromatography 25
  3.2 Reverse Phase Hplc 25
  3.3 Electrophoresis 25
  3.4 Enzymatic Cleavage 25
  3.5 Fab -Ms 25
  3.6 Confirmation Of Peptides 25
  3.7 Modifications Of Α- Crystallin 26
5 4 Discussion 92
158.1 KB
  4.1 Isolation Of Αa Crystallin 92
  4.2 Enzymatic Digestions And Peptide Mapping 92
  4.3 Modifications Of Α- Crystallin 94
  4.4 Conclusions 103
6 5 References 104
135.5 KB