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Chapter 1

CHEM 437 Chapter Notes - Chapter 1: Förster Resonance Energy Transfer, Surface Plasmon, Scintillation Counter


Department
Chemistry
Course Code
CHEM 437
Professor
Hoff
Chapter
1

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Introduction:
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pI: Isoelectric point = where molecule has not
neutral charge!
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Section 2: Assays and the Meanings of their outputs:
Kassociation (Kassoc) = inverse of the dissociation constant"Ka= [RL]/[R]*[L]!
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Kd = dissociation constant"Kd= 1/Kassoc!
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Ki= inhibitory constant !
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"-The smaller the Ki, the greater the binding anity and the smaller amount of medication needed in order to""""
""" inhibit the activity of that enzyme.$!
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IC50=The IC50 is the concentration of an inhibitor where the response (or binding) is reduced by half. [I] when
Vmax is 50% of the original Vmax!
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EC50= The EC50 is the concentration of a drug that gives half-maximal response.!
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LD50 = Lethal dosage for 50% of animals die !
""-> extrapolate to humans!
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Titration/ Dose Response:!
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!!!!!!!!!!!Stronger (decreased Kd)
!!!!!!!!!!!Weaker (increased Kd)!
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% Bound!
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"""""[I]!
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""% Bound!
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"""""""EC50!
-stronger curve earlier endpoint!
-in any case the change in [TI] up titration is characteristic of the Keq!
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2.1: Direct assays of reversible drug-protein binding
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2.1.1 Isothermal Titration Calorimetry (ITC)!
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"T+I <-> TI" ΔH!
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-Power required to keep sample cell at same temperature a reference cell!
-If ligand binding is exothermic then power is decreased upon heat evolution!
"-Thus ΔH of binding is measured directly!
-Dierence in power is measured between cells!
-Signal out=dierential power!
-T is known!
- ΔG and ΔS are known from: ΔG=ΔH-TΔS!
-Kd from shape of curve!
"- a strong binding interaction will give a squared titration curve !
"-indicates a soon as you titrate molecule binds!
"-weaker shape is more distorted b/c only some binding sites get bound!
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Advantages:!
-No label!
-Full info from one experiment!
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Disadvantages:
-Lots of sample needed (0.2-2.0 mL)!
-one at a time, experiment takes hours --> one of the lowest throughput methods!
-Finicky and hard to get reproducible data!
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2.1.2 NMR!
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-Protein NMR is usually:!
""""""""Signal!
"""""15N (0.36%)"YES!
"""""14 N (>99%)"NO!
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-Grow in presence of lots of 15N!
"-> gives protein with enriched 15N!
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T* + I <-> T*I""" T*=observable!
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Advantages:!
-No label!
-Native protein that is unmodified!
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Disadvantages:
-low sensitivity!
-lots of protein needed!
-magnets cost $$$$$!
-minutes to hours per sample!
-one at a time samples!
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2.1.3 Surface plasmon resonance (SPR)!
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-SPR is the basis of many standard tools for measuring adsorption of material onto planar metal (typically gold or
silver) surfaces!
-Silver/gold common metals b/c inert --> gelatinous biomaterial on surface gold!
-molecules on gold surface aect the energy of the surface plasmon!
-The mechanism of detection is based on that the adsorbing molecules cause changes in the local index of
refraction!
-The changes in the absorption wavelength is followed!
-Biacore experiments = flow experiments!
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Plasmon: a plasmon
is a quantum of
plasma oscillation. !
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Kd= kd/ka!
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Advantages:!
-small volumes!
-small sample demand!
-label free!
-native like bilayer for chemical attachment !
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