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

S2

S1

S0

hABS hABS hFLUOR

internal conversion

Quenching

Energy gets transferred to the quencher,usually through collisions with a

nearby residue or molecule. This reducesphoton emissions and decreases

fluorescence intensity.

3 amino acids emit UV fluorescence:

Tryptophan

Tyrosine

Phenylalanine

Fluorescence Measurements

1. Increased sensitivity, 10-5 M abs vs. 10-8 M flour.

2. Rapid process for measuring kinetics, 10-9 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, 2dhkl 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 morenarrow range and better cutoffs on either side

4. Solution filters - used mostly in photochemistry

Lactose Repressor Protein

I P Z Y AO

The E. coli lac Operon and lac Repressor

lacR -regulatory

controlelements structural genes

lac operon

Calculations and Write-up

1. Determination of Kd - we have a function in IGOR

2. When your results differ from published results, postulate reasons this may be true.