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Bchem 471 > Fluorescense 2 > Flashcards

Fluorescense 2 Flashcards

1
Q

Why is flouresnce important

A

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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2
Q

Why is flouresnce important

A

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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3
Q

During the long time between excitation and fluorescence what are the ways that the molecule can lose energy

A

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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4
Q

What is an example of a negative charged noncovalently bound fluorophore

What is it characteristics

A

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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5
Q

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

A

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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6
Q

What is an example of a postitwvly charged fluorophore

What are its characteristics

A

EtBr

Positively charged, Has a hydrophobic region

binds to hydrophobic regions between bases in DNA

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7
Q

What makes EtBr have a large quantum yeild

A

Because it intercalates within dna to cause a lot of fluorescence

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8
Q

What are examples of covalently bound fluorophore?

A

1,5-IADANS

FITC

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9
Q

What are the characteristics of 1,5-IAEDANS

A

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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10
Q

What are the conditions for IAEDANS binding to the cysteine of proteins

A

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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11
Q

If you wanted to only label one cys residue with IAEDANS what would you do

A

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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12
Q

What makes IAEDANS a specific fluorophore

A

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

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13
Q

What are the characteristic of FITC

A

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

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14
Q

Why would you choose to label lysines

A

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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15
Q

For which molecules/probes is the quantum yield higher for

A

More rigid molecules

Less polar environments

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16
Q

What’s a great source for finding info on probes

A

Molceuclar probes website

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17
Q

Many excited fluorpohres ______

A

Don’t emit a photon

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18
Q

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

A

The excited state of that fluorophore clashes into another molecule

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19
Q

What is an example of self quenching

A

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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20
Q

What is fluorescence quenching

A

A process where the fluorescence intensity of a fluorophore is reduced

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21
Q

What is an example of fluorescence quenching

A

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

22
Q

What is a stern volmer plot

A

A plot of If/IQ vs [Q]

Can tell us the amount of quenching occurring in a solution

23
Q

What are the two types of quenching

A

Static

Dynamic

24
Q

What is static quenching

A

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

25
Q

What is dynamic quenching

What are the quenchers

A

The M gets excited and the M* collides with the Q to get deactivated

Cl-, br-

26
Q

Stern volmer equation

A

Slide 13

27
Q

What does Ksv depend on

A

The lifetime of the floruophore (tau)

The size and diffusion coefficients of both the quencher and the fluorophore

28
Q

How do you make a stern volmer plot

What does the slope of this plot tell us

A

Plot the If/IQ vs [Q]

Ksv (constant)

29
Q

What happens to the diffusion coefficient and Ksv if you have a buried fluorophore

How did this show the usefulness of the Ksv

A

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

30
Q

If something does not have a linear trend on a stern volmer plot what does this mean

A

The quencher of that sample is not a good quencher

Must be linear

At higher concentrations of that quencher the trend is not linear

31
Q

What is Kq also called

A

The bimolecular quenching constant

32
Q

The longer the lifetime of the excited state ____

A

The greater chance of collision between the Q and the M*

33
Q

The longer the lifetime of the excited state ____

A

The greater chance of collision between the Q and the M*

34
Q

If the quencher or molecule are bigger what does this mean

A

There are less chances of collisions because they are too big to move and collide

So Ksv and Kq are lower

35
Q

Why would we choose to use N-ac-Trp-NH2 as a fluorophore instead of tryptophan

A

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)

36
Q

Why would we choose to use N-ac-Trp-NH2 (NAWA) as a fluorophore instead of tryptophan

A

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)

37
Q

What is guanidinium chloride

A

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

38
Q

Since the Kq for native proteins with I or oxygen quenchers is lower than the kq for just trp what does this mean

A

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

39
Q

Would the Kq of a protein with iodide be bigger or smaller than that with oxygen
Why

A

Kq for iodide would be smaller than that for oxygen

Kq for oxygen would be bigger

This is because oxygen is smaller than Iodide

40
Q

If smaller quencher what happens to Kq

A

Higher kq

41
Q

Point three of slide 16

A

Idk

42
Q

What does the Kq (10^10) of a free chromophore tell us

A

Tells us that the reaction is diffusion controlled

Meaning As quickly as the molecules can come together, then reaction happens

43
Q

Review question slide 18

A

Ok

44
Q

If given the stern volmer plot what do you do to find Ksv

A

Find the slope (y2-y1/x2-x1)

That is Ksv

45
Q

If the predicted (slope from graph) Ksv is lower than the one from the equation what does this mean

Slide 19

A

Less than 10 percent of the encounters between the quencher and fluorophore lead to quenching

There is no indication of static quenching

46
Q

Fluorescent probes have ___ flurorescent life times

A

Long

47
Q

Why does ethidium bromide have a large tau F (fluorescent lifetime)

A

Because it’s rigid and less polar

48
Q

Why does ethidium bromide have a large tau F (fluorescent lifetime)

A

Because it’s rigid and less polar

49
Q

Is 10ns lifetime of fluoresce a long or short time

A

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)

50
Q

What type of curvature do buried fluorophores give on a stern vomer plot

A

Downward since they aren’t accessible to the quencher

51
Q

Equations for fraction of fluorophore accessible to quencher

A

Slide 22

Bchem 471 (13 decks)

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