Fluorescense 2 Flashcards
Why is flouresnce important
Because the time length of fluorescence is long in comparison to absorbance
Absorbances is femtoseconds
Fluorescence is nanoseconds (more things have more time to happen)
Why is flouresnce important
Because the time length of fluorescence is long in comparison to absorbance
Absorbances is femtoseconds
Fluorescence is nanoseconds (more things have more time to happen)
During the long time between excitation and fluorescence what are the ways that the molecule can lose energy
Can emit
Can to intersystem crossing (to a triplet state)
Can transfer energy to an acceptor
Can do thermal inactivation
Can collide with other molecules
What is an example of a negative charged noncovalently bound fluorophore
What is it characteristics
ANS
It has a hydrophobic region (due to conjugated rings) to bind to hydrophobic regions of other proteins
Has a negative charge (SO3-) so that it can be soluble in water
When you put ANS in water vs n-Octanol (a thing mimicking the hydrophobic environment of proteins is might bind to)
What do you observe in the spectra of ANS
The quantum yeild in the n-octonol is higher than in the water
This means that you see more flourescnse when ANS is bound to proteins
What is an example of a postitwvly charged fluorophore
What are its characteristics
EtBr
Positively charged, Has a hydrophobic region
binds to hydrophobic regions between bases in DNA
What makes EtBr have a large quantum yeild
Because it intercalates within dna to cause a lot of fluorescence
What are examples of covalently bound fluorophore?
1,5-IADANS
FITC
What are the characteristics of 1,5-IAEDANS
Binds to cysteine residues on the surface of proteins through its haloacteamide group
The halo means an iodine (halogen)
It attaches the fluorophore onto it and then to the protein
What are the conditions for IAEDANS binding to the cysteine of proteins
We need the solution that the protein is in to be at ph 8.
When this happens, the cys is S- and the haloacetamide of the IAEDANS reacts with it to bind.
If you wanted to only label one cys residue with IAEDANS what would you do
You could do site directed mutagenesis of other cysteines to make it so IAEDANS can bind to them
Or you could add in a modified cysteine that the IAEDANS will bind to only
What makes IAEDANS a specific fluorophore
The reaction of it with his, lys, or met is much slower than with cys
What are the characteristic of FITC
Binds to lysines through a isocyanate (N=C=S)
Why would you choose to label lysines
Lysine is on the surface of proteins at the aqueous environment (interaction with water unfavourably)
Can label the surfaces of proteins
For which molecules/probes is the quantum yield higher for
More rigid molecules
Less polar environments
What’s a great source for finding info on probes
Molceuclar probes website
Many excited fluorpohres ______
Don’t emit a photon
What is the collision part of something losing energy after being excited
The excited state of that fluorophore clashes into another molecule
What is an example of self quenching
A fluorescent dye (like fluorescein)
When dilute its green
And when concentrated it’s dark red
It’s changes it’s own fluoresce depending on its concentration
What is fluorescence quenching
A process where the fluorescence intensity of a fluorophore is reduced
What is an example of fluorescence quenching
A solution of tonic water has quinine in it
Quinine fluoresces
But when nacl is in the solution with quinine, the fluorescence drops
This means either the na or cl is quenching
What is a stern volmer plot
A plot of If/IQ vs [Q]
Can tell us the amount of quenching occurring in a solution
What are the two types of quenching
Static
Dynamic
What is static quenching
The protein and quencher complex (M•Q) gets formed before excitation
The bound quencher makes The M* (excited M) gets deactivated by routes other than fluorescence
That absorption spectrum might be different because it’s not the M that’s absorbing it’s the MQ complex
What is dynamic quenching
What are the quenchers
The M gets excited and the M* collides with the Q to get deactivated
Cl-, br-
Stern volmer equation
Slide 13
What does Ksv depend on
The lifetime of the floruophore (tau)
The size and diffusion coefficients of both the quencher and the fluorophore
How do you make a stern volmer plot
What does the slope of this plot tell us
Plot the If/IQ vs [Q]
Ksv (constant)
What happens to the diffusion coefficient and Ksv if you have a buried fluorophore
How did this show the usefulness of the Ksv
smaller diffusion coefficient and smaller Ksv
Is you have a lower Ksv, you can tell that the fluorophore (ex. Trp) is buried and not on the surface of the protein
If something does not have a linear trend on a stern volmer plot what does this mean
The quencher of that sample is not a good quencher
Must be linear
At higher concentrations of that quencher the trend is not linear
What is Kq also called
The bimolecular quenching constant
The longer the lifetime of the excited state ____
The greater chance of collision between the Q and the M*
The longer the lifetime of the excited state ____
The greater chance of collision between the Q and the M*
If the quencher or molecule are bigger what does this mean
There are less chances of collisions because they are too big to move and collide
So Ksv and Kq are lower
Why would we choose to use N-ac-Trp-NH2 as a fluorophore instead of tryptophan
There is an acetyl on the n term and a NH2 on the carboxy term
This masks the charge of tryptophan and makes it look more like it would in a protein (in a peptide bond on either side)
Why would we choose to use N-ac-Trp-NH2 (NAWA) as a fluorophore instead of tryptophan
There is an acetyl on the n term and a NH2 on the carboxy term
This masks the charge of tryptophan and makes it look more like it would in a protein (in a peptide bond on either side)
What is guanidinium chloride
It denatures proteins without having to change the temp or anything
The guanidinium group in it looks like the guanidinium in Argenine
Positively charged and small meaning it can go in to proteins to disrupt h bonding and dentature them
Since the Kq for native proteins with I or oxygen quenchers is lower than the kq for just trp what does this mean
This means that with the native proteins, the quencher couldn’t reach the fluororphore (trp) and thus didn’t quench as much
This means the trp was buried in the protein
The trp(aq) is higher because nothing it blocking it from being quenched
Would the Kq of a protein with iodide be bigger or smaller than that with oxygen
Why
Kq for iodide would be smaller than that for oxygen
Kq for oxygen would be bigger
This is because oxygen is smaller than Iodide
If smaller quencher what happens to Kq
Higher kq
Point three of slide 16
Idk
What does the Kq (10^10) of a free chromophore tell us
Tells us that the reaction is diffusion controlled
Meaning As quickly as the molecules can come together, then reaction happens
Review question slide 18
Ok
If given the stern volmer plot what do you do to find Ksv
Find the slope (y2-y1/x2-x1)
That is Ksv
If the predicted (slope from graph) Ksv is lower than the one from the equation what does this mean
Slide 19
Less than 10 percent of the encounters between the quencher and fluorophore lead to quenching
There is no indication of static quenching
Fluorescent probes have ___ flurorescent life times
Long
Why does ethidium bromide have a large tau F (fluorescent lifetime)
Because it’s rigid and less polar
Why does ethidium bromide have a large tau F (fluorescent lifetime)
Because it’s rigid and less polar
Is 10ns lifetime of fluoresce a long or short time
A long time for collisions to take places
Long time for solvent to reorient around the fluorophore (and allow for blue shift or red shift to happen because there’s time for the alignment of the dipoles)
What type of curvature do buried fluorophores give on a stern vomer plot
Downward since they aren’t accessible to the quencher
Equations for fraction of fluorophore accessible to quencher
Slide 22
