Bios 532 Fluorescence

Vocabulary Fluorophore - molecule that emits fluorescence. Excitation - absorption of a photon. Emission - release of a photon.

Luminescence - the emission of light from electronically excited states of a molecule: Fluorescence Phosphorescence

Efficiency of fluorescence is measured in terms of the quantum yield.  = # of photons emitted # of photons absorbed S2S2 S1S1 S0S0 h ABS h FLUOR internal conversion

Quenching Energy gets transferred to the quencher, usually through collisions with a nearby residue or molecule. This reduces photon emissions and decreases fluorescence intensity.

3 amino acids emit UV fluorescence: Tryptophan Tyrosine Phenylalanine

Fluorescence Measurements 1.Increased sensitivity, M abs vs M flour. 2.Rapid process for measuring kinetics, sec. 3.Measure at 90º to incident beam. 4.Dirt, lint, air bubbles, all scatter light significantly. 5.Photobleaching of sample can be a problem. 6.Light passes through a prism or diffraction grating to set excitation wavelength. The setting is chosen according to Bragg’s Law, 2d hkl sin  =n. The problem is n - you get, 2, 3, etc., so filters and are sometimes used.

Filters 1.Longpass - block shorter, pass longer 2.Bandpass - allows only one region of spectrum through, blocks both longer and shorter 3.Interference - similar to bandpass, but with a more narrow range and better cutoffs on either side 4.Solution filters - used mostly in photochemistry

Lactose Repressor Protein

IPZYA O The E. coli lac Operon and lac Repressor lacR - regulatory control elements structural genes lac operon

Calculations and Write-up 1.Determination of K d - we have a function in IGOR 2.When your results differ from published results, postulate reasons this may be true.