Fluorescense 2 Flashcards by Zunaira Ali

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)

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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)

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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

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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

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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

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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

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What makes EtBr have a large quantum yeild

Because it intercalates within dna to cause a lot of fluorescence

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What are examples of covalently bound fluorophore?

1,5-IADANS

FITC

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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

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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.

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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

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What makes IAEDANS a specific fluorophore

The reaction of it with his, lys, or met is much slower than with cys

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What are the characteristic of FITC

Binds to lysines through a isocyanate (N=C=S)

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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

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For which molecules/probes is the quantum yield higher for

More rigid molecules

Less polar environments

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What’s a great source for finding info on probes

Molceuclar probes website

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Many excited fluorpohres ______

Don’t emit a photon

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What is the collision part of something losing energy after being excited

The excited state of that fluorophore clashes into another molecule

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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

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What is fluorescence quenching

A process where the fluorescence intensity of a fluorophore is reduced

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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

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