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Chemical Modification

Chemical Modification. Variety Oxidation Nitrosylation Dissociation Effects Folding Controls Enviromental Reactive. Protein structure - function. AA sequence O-N pairing of backbone Ionic/hydrophobic interaction of side chains Chemical environment Ionic strength pH – ie: H + ions

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Chemical Modification

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  1. Chemical Modification • Variety • Oxidation • Nitrosylation • Dissociation • Effects • Folding • Controls • Enviromental • Reactive

  2. Protein structure - function • AA sequence • O-N pairing of backbone • Ionic/hydrophobic interaction of side chains • Chemical environment • Ionic strength • pH – ie: H+ ions • Reactive • Side chain modification

  3. Chemistry • A + B  AB • AB increases with either A or B • Equilibrium constant Ka=[AB]/([A][B]) • With total A constant • A/AB switch • A/AB indicator • Complexchemistry alterssensitivity

  4. Multiple modifications • For fixed “A” the amount of product is • One “B”: AB • Two “B”: AB+AB2 • More • Hill equation: • n: cooperativity • Kd: apparent dissociation constant

  5. Chemical sensors 2-3 log dynamic range Decreasing Kd increasing affinity, increases sensitivity Increasing cooperativity increases gain

  6. pH • Charged amino acid side chains • H+ movement can dramatically alter molecular folding • pK=-log( ) [B-][H+] [HB]

  7. [HCO3-][H+] [CO2] [HCO3-] [CO2] [HCO3-] [CO2] Bicarbonate buffers • CO2 solubility ~0.03mM/Torr • CO2 Hydration • Carbonic anhydrase • Henderson-Hasselbalch equation K= pK= -log( ) pK = pH – log( ) pH = pK + log( ); pK=6.1 [HCO3-][H+] [CO2]

  8. [HCO3-][H+] [CO2] Ka = Bicarbonate buffers pH 2 pH 7.2 5% CO2 pH 7.4 pH 7.6 pH 12

  9. Open vs Closed Buffer Systems • Bicarbonate • Physiological • pCO2 = 40 mmHg • [CO2] = 1.2 mM • [CO2]+[HCO3]=31 mM • HEPES • Equivalent Buffer • [HEPES]+[HEPES-]=31 mM • [HEPES-]=14.7 • [HEPES]=16.3

  10. Open vs Closed Buffers • Bicarb • Add 10 mM HCl • Immediate • [HCO3]=20 mM • [CO2]=11 mM • pH = 6.4 (400 nM) • HEPES • Add 10 mM HCl • Immediate • [HEPES-]=4.7 mM • [HEPES]=26 mM • pH=7.2 (63 nM) • Much better pH control near pKa

  11. Open vs Closed Buffer System • Bicarb • CO2 solubility 1.2 mM • [HCO3]=20 mM-350 nM • [CO2] = 1.2mM+350 nM • pH=7.3 (50 nM) • Much better than 6.4 w/o exchange • HEPES • No mass exchange • pH =7.2 Could do bicarb in one step:

  12. pH Control • Cell membranes impermeable to H+ • Compartmentalization of pH • Cytoplasm 7.15 • Nucleus 7.2 • Mitochondria 8.0 • Golgi ~6.3 • Lysosome 5.5 • Transporters • H+/K+ • HCO3-/Cl-

  13. Dissociation of amino acid side chains In cytoplasm, pH=7.15, so 80% of histidine is in base form. 0.1% of lysine is in its base form. In Golgi, pH=6.3, and 40% of histidine is in base form.

  14. Reactive modification • NO S-nitrosylation • Cysteines in hydrophobic acid/base pockets • Hemoglobin • S-NO forms in oxidative environment • Allows NO release in low oxygen • Targets vasodilating NO to oxygen starved tissue -S-H -S-N=O

  15. Reactive oxidation • Oxidized oxygen: O2·, H2O2, OH· • Protein modification • Cys, His, Phe, Tyr, Met • Sulfur • Ring structures • Chain break • Cross-linking • Chain reaction • DNA/Lipid modification

  16. Reactive modification Thymine Thymine glycol • Amino acid modification changes local polarity • Crosslinking • Strand break Thymine glycol distorts DNA structure Kung & Bolton 1997

  17. Electrochemistry • Redox reactions describe electron transfer • Zn + CuSO4Cu + ZnSO4 • Zn + Cu2+Cu + Zn2+ • Zn Zn2+ + 2e- and Cu2+ + 2e- Cu • 2 GSH + H2O2 GSSG + 2H2O • 2 GSH GSSG + 2e- + 2H+ andH2O2 + 2e- + 2H+ 2H2O Inorganic Biological

  18. Electrochemistry-free energy • Electrical • DG = -nFDE • Faraday constant 9.65 104 C/mol • Concentration • DG = RT ln( Q) • Gas constant 8.31 J/K/mol • Whole reaction • DG = DG0 + RT ln(QProd) – RT ln(Qreac) • -nFDE = -nFDE0 + RT ln(Qprod/Qreac) • DE = DE0 - RT/nF ln(Qprod/Qreac) • Nernst Equation for redox reaction • Equilibrium at DG= DE = 0

  19. Electrochemistry-half cells • Standard Reduction Potential E0 • Metals (Daniell cell) • Zn Zn2+ + 2e- Zn2+ + 2e- Zn E0=-0.76V • Cu2+ + 2e- Cu E0=+0.34V • DE0=0.34-(-0.76) = 1.1V • Biological (glutathione) • 2 GSH GSSG + 2e- + 2H+ GSSG + 2e- + 2H+  2 GSH E0=+0.18 • H2O2 + 2e- + 2H+ 2H2O E0=+1.78 • DE0=1.78-(0.18) = 1.6V

  20. GSSG GSSG GSSG GSSG GSH2 H2O2 GSH2 H2O2 GSH2 H2O2 GSH2 H2O2 Cellular Redox State • Biological • 2 GSH + H2O2 GSSG + 2H2O • DE0= 1.6V • DG = -nF (1.6V) + RT ln( ) • DE = 1.6V – RT/nF ln( ) • Steady state trend • 0 = 1.6 –(8.31*310)/(2*9.6e4) ln( ) • = 1052 • ie: Not a lot of free peroxide in a cell • Still needs a catalyst • Real cells have many potential half-cells

  21. GSH2 GSH2 GSSG H2 GSSG H2 GSH:GSSG redox buffer • GSH is abundant reducing agent • GSSG + 2e- + 2H+  2 GSH E0=+0.18 • DE = 0.18 – RT/nF ln( ) • DE = 0.18 – 0.03 log( ) • GSH:GSSG ratio as marker of redox state • More GSH, more negative DE, more reducing • GSSG reduction appears as negative in whole reaction • Whole reaction more favorable with positive DE • More H+, more positive DE, more oxidizing • Neutral [H+]2 ~ 10-14 • Many biological oxidations include H+

  22. Cellular redox cascade • Oxygen radicals are not equivalent • ROS generation • Mitochondria • Photons (UV & ionizing radiation) • Inflammatory cells (NADPH oxidase) • Radical scavengers • O2•-H2O2 superoxide dismutase • H2O2H2O Catalase • H2O2 + GSH GSSG glutathione peroxidase • OH• hydroxyl (uncharged OH-)

  23. Redox state • Intracellular reductive • Low free oxygen, relatively negative • Extracellular oxidative • High O2, relatively positive Extracellular antioxidants Extracellular signals that promote oxidative stress Cytoplasmic oxidants Cytoplasmic antioxidants

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