Protein Behavior Directed by Heparin Charge and Chain Length
نویسندگان
چکیده
.................................................................................................................vii LIST OF TABLES ....................................................................................................... xiv LIST OF FIGURES ....................................................................................................... xv CHAPTER 1. BACKGROUND ON HEPARIN AND HEPARIN -PROTEIN INTERACTIONS ................................................................................................ 1 1.1 Glycosaminoglycans (GAGs) ......................................................................... 1 1.1.1 Heparan Sulfate and Heparin ........................................................... 2 1.2 Polyelectrolyte properties of GAGs ................................................................ 4 1.3 GAG-Protein Interactions ............................................................................... 5 1.3.1 Conventional models in protein-Hp binding ..................................... 6 1.3.2 Alternative models in protein-Hp binding ........................................ 8 1.4 Roles of electrostatics in Hp-Protein binding ................................................ 10 1.5 Low Molecular Weight Heparins: Therapeutic Significances ........................ 12 1.6 Aim and Outline of the Thesis ...................................................................... 13 2. COUNTERION CONDENSATION ON HEPARIN OLIGOMERS ................... 15 2.1 Abstract ........................................................................................................ 15 2.2 Introduction .................................................................................................. 16 x 2.3 Experimental ................................................................................................ 20 2.3.1 Materials ........................................................................................ 20 2.3.2 Methods ......................................................................................... 20 2.3.2.1 Capillary Electrophoresis (CE) ........................................ 20 2.3.2.2 Mass Spectrometry (MS) ................................................. 21 2.3.2.3 Size Exclusion Chromatography (SEC) ........................... 22 2.4 Results ......................................................................................................... 22 2.5 Discussion .................................................................................................... 27 2.6 Conclusions .................................................................................................. 35 ACKNOWLEDGEMENTS ............................................................................... 36 3. A HEPARIN DECAMER BRIDGES A GROWTH FACTOR AND AN OLIGOLYSINE BY DIFFERENT CHARGE-DRIVEN INTERACTIONS ....... 37 3.1 Abstract ........................................................................................................ 37 3.2 Introduction .................................................................................................. 38 3.3 Experimental ................................................................................................ 43 3.3.1 Materials ........................................................................................ 43 3.3.2 Methods. ........................................................................................ 43 3.3.2.1 Mass Spectrometry .......................................................... 43 3.3.2.2 Computational ................................................................. 44 3.3.2.3 Cell Culture and Proliferation Assays .............................. 44 3.3.2.4 Statistical Analysis .......................................................... 45 3.4 Results and discussion .................................................................................. 46 3.4.1 K4 binding to dp 10 ....................................................................... 46 3.4.2 FGF binding to dp10 ...................................................................... 48 3.4.3 Ternary complex formation ............................................................ 53 3.4.4 Biological activity of FGF vs FGF·dp10·K4 ................................... 57 xi 3.5 Conclusions .................................................................................................. 58 4. STRUCTURAL DETERMINANTS OF ELECTROSTATIC INTERACTIONS BETWEEN PROTEINS AND POLYANIONS: AN ESI-MS STUDY OF FIBROBLAST GROWTH FACTOR BINDING TO HEPARIN OLIGOMERS .................................................................................. 60 4.1 Abstract ........................................................................................................ 60 4.2 Introduction .................................................................................................. 61 4.3 Experimental ................................................................................................ 64 4.3.1 Materials ........................................................................................ 64 4.3.2 Methods ......................................................................................... 64 4.3.2.1 Electrospray Ionization Mass Spectrometry (ESIMS) ................................................................................. 64 4.3.2.2 DelPhi Calculations ......................................................... 65 4.3.2.3 Monte-Carlo Simulations................................................. 65 4.4 Results and Discussion ................................................................................. 66 4.4.1 Heparin facilitates formation of FGF-1 multimeric complexes ....... 66 4.4.2 Higher sulfated chains promote formation of multimeric complexes of FGF-1 ................................................................... 70 4.4.3 When bulk stoichiometry leads to a significant amount of bound heparin, the charge density of the free heparin is low relative to the initial heparin ........................................................ 73 4.5 Conclusions .................................................................................................. 77 ACKNOWLEDGMENTS .................................................................................. 77 5. ELECTROSPRAY IONIZATION MASS SPECTROMETRY STUDY TO REVEAL THE EFFECT OF HEPARIN CHAIN LENGTH ON ANTITHROMBIN AND FACTOR XA COMPLEX FORMATION.................. 78 5.
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