absorption spectroscopy topic 4 (abridged) biophysics
TRANSCRIPT
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Absorption Spectroscopy
Topic 4 (abridged)
Biophysics
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Energy and Absorption
• Absorption is proportional to
• Get resonance at hf = ħ = Eb – Ea
2
ab
ab22
2
2
0ab2
bb
2tEE2
2tEEsintE~
CP
0ba E~
www.andor.com/chemistry/?app=189
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Absorption Broadening
pubs.rsc.org/ej/DT/2000/B002123G/
•Every electronic state (eg S0 and S1) has several vibrational and rotational substates.
•Each of the sharp peaks is further broadened by Doppler shifts and solvent effects and quantum probabilities.
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Extinction Coefficient
Sample with pathlength lLight Source
I0
monochromator
Detector
I
Or
monochromator
Light Source
Beam Splitter
Reference I0
Detector
I1
Sample with pathlength l
I0
Detector
I2
A() = log (I0/I)
A() = Cl
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Biopolymer absorption
Protein Secondary Structure Protein Aromatics
Bloodhttp://life.nthu.edu.tw/~lslpc/biophys/hyperchorism.gif
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Chemical KineticsZero Order
• Rate independent of concentrations• -dC/dt = k • C(t) = C0 – kt
C
t
0.00E+00
2.00E-01
4.00E-01
6.00E-01
8.00E-01
1.00E+00
1.20E+00
1.40E+00
450 500 550 600 650 700
Wavelength (nm)
Ab
sorp
tio
n
A
B
Reaction of nitrite with deoxyhemoglobin
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Chemical KineticsFirst Order
• -dCA/dt = kCA , CA = CA0 e-kt
• t1/2 = ln(2)/k; = 1/k = lifetime
ln(CA)
t
NO binding to Hb
ZA k
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Chemical KineticsSecond Order
• -dCA/dt = -dCB/dt = kCACB
• Make one species in excess so get pseudofirst order kinetics, kobs = kCB so
CA = CA0 exp(-kobst)
0B
0A0
B0A
B
A
CClnktCC
C
Cln
oductsPrBA k
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Hemoglobin
Cooperative Binding of Oxygen
Linked to quaternary structure
Explained by MWC Model
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On the Nature of Allosteric Transitions:
A Plausible ModelJacques Monod, Jeffries Wyman,
Jean-Pierre Changux
J. Mol. Biol. 1965
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Time Resolved Optical DensityTROD
http://fig.cox.miami.edu/~cmallery/255/255enz/ecb3x28.jpg
Stopped-flow Pump-probe
Perturb System, measure change in absorption over time.
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SVD and data analysis(eg. for Hofrichter paper)
• Truncated X (or A) data matrix is fit to exponentials or other model• Get noise filtering
Basis Specta Time Course of Basis Specta
www.math.tau.ac.il/~nin/Courses/BioInfo04/svd.ppt
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Circular Dichroism and Optical Rotary Dispersion
• CD is the differential absorption of circularly polarized light• ORD rotates linearly polarized light (and other)• Both result from chiral structures as can be used as
structural probes (eg Protein 2ndary Structure)
http://upload.wikimedia.org/wikipedia/commons/thumb/d/d1/Circular_dichroism.png/622px-Circular_dichroism.png
CD ORD
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Polarized Absorption
1'
1'
'1
'1
LBLBCD
LBCBLD
LBCBLD
CDLDLD
e AM
A A A
LB n n l LB n n l CB n n l
LD A A LD A A CD A A
h v
h v l r
h v l r
ln ( ) / ,
( ) / , ' ( ) / ( ) / ,
ln ( ) / , ' ln ( ) / , ln ( ) / ,
10 2
2 2 2
10 2 10 2 10 2
,
Arrangements can be made to measure elements directly, using PEM or using ellipsometric techniques.
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Ellispometric Technique
• Key is crossed (or nearly crossed) polarizers as in this schematic for TRORD.
• Used for protein folding (Goldbeck paper).