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Isothermal calorimetry A natural complementation to scattering methods ??

Isothermal calorimetry A natural complementation to scattering methods ?? . ITC and membrane partitioning. Ligand in cell – titrate with membrane (NB the other way around won’t work since there is no saturation – it is partitioning between two phases ). Lipid membrane; 47.4mM.

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Isothermal calorimetry A natural complementation to scattering methods ??

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  1. IsothermalcalorimetryA naturalcomplementation to scatteringmethods ??

  2. ITC and membranepartitioning Ligand in cell – titratewithmembrane (NB the otherwayaroundwon’tworksincethere is nosaturation – it is partitioningbetweentwophases) Lipid membrane; 47.4mM Octanol 0.61mM 1-octanol OcOH depletion Rowe et al (1998) Biochem. 37, 2430

  3. ”Null balance ITC” Cell: Ethanol Syringe: Ethanol + POPC When the freeethanol in the lipid suspension is equal to the ethanolconcentration in the cell – weobservezeroenthalpychange.

  4. The resolution of ITC relates to the DIFFERENTIAL nature of the measurement CMC Slope (differentiate with respect to concentration)

  5. An asset of calorimetry: High resolution in compositionMicelle formation De-micellization of SDS CMC readily determined to within 10-50mM Otzen et al In press

  6. High resolutionand protein surfactant interactions Andersen et al Langmuir in press

  7. Semi-empiricalanalysis of complexisotherms (SDS – a-lactalbumin)

  8. Isothermal Calorimetry and enzyme activity • Detecting the heat of the catalyzed reaction: heat flow J/s = DH x rate • Rate –not concentration – is the primary observable – sensitivity ~pmol sec-1 • Real time – complex time courses readily characterized • No probe, fluorophore etc required • Works in ”complex systems” (opaque, multi-phase (colloid), insol. Substrate, side reactions) Substarte (syringe) → enzyme (cell) Constant substrate concentration Syringe: 40mM Urea, 3ml injections (88mM in cell) Cell: 50pM H.pylori Urease Substarte (syringe) → enzyme (cell) Substrate depletion Syringe: 25mM Urea, 18ml injections (513mM in cell) Cell: 4nM H.pylori Urease No product inhibition Todd & Gomes 2001

  9. Enzyme activity: Crowding Hexokinase in buffer and 250 mg/ml serum albumin Diffusion, binding, enzyme flexibility etc is affected by crowded surroundings Crowding decreases kcat and KM Inside a red cell Olsen 2006

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