1. Absorption Spectroscopy Topic 4 (abridged) Biophysics

2. Energy and Absorption Absorption is proportional to Get resonance at hf = ħ  = E b – E a www.andor.com/chemistry/?app=189

3. Absorption Broadening pubs.rsc.org/ej/DT/2000/B002123G/ Every electronic state (eg S 0 and S 1 ) has several vibrational and rotational substates. Each of the sharp peaks is further broadened by Doppler shifts and solvent effects and quantum probabilities.

4. Extinction Coefficient Sample with pathlength l Light Source I 0 monochromator Detector I Or monochromator Light Source Beam Splitter Reference I 0 Detector I1I1 Sample with pathlength l I 0 Detector I2I2 A( ) = log (I 0 /I) A( ) = C  l

5. Biopolymer absorption Protein Secondary Structure Protein Aromatics Blood http://life.nthu.edu.tw/~lslpc/biophys/hyperchorism.gif

6. Chemical Kinetics Zero Order Rate independent of concentrations -dC/dt = k C(t) = C0 – kt C t Reaction of nitrite with deoxyhemoglobin

7. Chemical Kinetics First Order -dCA/dt = kCA, C A = C A 0 e -kt t 1/2 = ln(2)/k;  = 1/k = lifetime ln(C A ) t NO binding to Hb

8. Chemical Kinetics Second Order -dC A /dt = -dC B /dt = kC A C B Make one species in excess so get pseudofirst order kinetics, k obs = kC B so C A = C A 0 exp(-k obs t)

9. Hemoglobin  Cooperative Binding of Oxygen  Linked to quaternary structure  Explained by MWC Model

10. On the Nature of Allosteric Transitions: A Plausible Model Jacques Monod, Jeffries Wyman, Jean-Pierre Changux J. Mol. Biol. 1965

11. Time Resolved Optical Density TROD http://fig.cox.miami.edu/~cmallery/255/255enz/ecb3x28.jpg Stopped-flow Pump-probe Perturb System, measure change in absorption over time.

12. 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

13. 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 2 nd ary Structure) http://upload.wikimedia.org/wikipedia/com mons/thumb/d/d1/Circular_dichroism.png/6 22px-Circular_dichroism.png CD ORD

14. Polarized Absorption Arrangements can be made to measure elements directly, using PEM or using ellipsometric techniques.

15. Ellispometric Technique Key is crossed (or nearly crossed) polarizers as in this schematic for TRORD. Used for protein folding (Goldbeck paper).