Absorption Spectroscopy Topic 4 (abridged) Biophysics.

Absorption Spectroscopy

Topic 4 (abridged)

Biophysics

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

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.

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

Biopolymer absorption

Protein Secondary Structure Protein Aromatics

Bloodhttp://life.nthu.edu.tw/~lslpc/biophys/hyperchorism.gif

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

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

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

Hemoglobin

Cooperative Binding of Oxygen

Linked to quaternary structure

Explained by MWC Model

On the Nature of Allosteric Transitions:

A Plausible ModelJacques Monod, Jeffries Wyman,

Jean-Pierre Changux

J. Mol. Biol. 1965

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.

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

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

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.

Ellispometric Technique

• Key is crossed (or nearly crossed) polarizers as in this schematic for TRORD.

• Used for protein folding (Goldbeck paper).