so welcome to the lecture number twenty five so let us continue our discussion on fluorescence
quenching that we were doing for last few lectures and we have already get some idea
about the types of quenching we got the idea of static quenching dynamic quenching combine
static and dynamic quenching then we have seen the quenching sphere of action so in
that case the apparent static quenching term is coming in where we are getting a smaller
value of k s which is because of the immediate presence of quencher next to the fluorophore
and then we have also seen the quenching resolved emission spectra and now today i
wanted to show you some application of this fluorescence quenching method
so let us start with one by one so there is several things we can do with this fluorescence
quenching method like the characterisation of protein lets say
in this case fluorescence quenching method is being used for example let me take a protein
right i can just draw my protein like this way as usual and there was a debate between
two diffractions one diffraction is said that ok i have a two tryptophan residue as i told
you this tryptophan residues are kind of amino acid which are fluorescent in nature right
so one diffraction is said that i have two tryptophan residue one is present at the surface
and another is also present at the surface of this protein and the second diffraction
is come in and he told no my tryptophan results tells me that both the sorry one of them
are present at the surface and another is buried right and
now what to do right but using using a fluorescence quenching you can resolve this and suppose
suppose you are doing this quenching experiment for the tryptophan both are present at the
surface the quenching will be much more now if the tryptophan one of them is present in
the buried location and another is exposed to the surface then you will going to see
a lesser amount of quenching because that tryptophan which is present in the buried
location will not contribute to to the fluorescence quenching right i mean that quencher will
not be able to reach that buried tryptophan and it will not able to quench the tryptophan
emission so the fluorescence intensity will not decrease that way that it would be for
the case when both the tryptophan will be present at the surface right ok
number two so you can also do this protein conformational change you can also study by
fluorescence quenching method for example suppose you have a protein lets say this is
you protein and you have this tryptophan residing over here now if you add a quencher molecule
from outside that quencher molecule will not be able to reach this tryptophan residue hundred
percent so the fluorescence intensity of this tryptophan which is the intensity fluorescence
of this protein will not going to be affected by addition of the quencher in the solution
now consider that you have done something on this protein so that this protein chain
right the secondary structure of the protein is getting distorted so thats what is referred
as the unfolding of the protein or the change in the conformation of this project right
so you do something you put some perturbation so that the protein is unfolded
now as the protein is unfolded right the structure of the protein will change suppose you have
this structure and this structure as it is unfolding the structure is getting changed
like this way ok so that tryptophan which was originally
here on the buried location this is my t r p it is now over here still again in the buried
location now it is open somewhat and now it is fully open this is my tryptophan residue
so depending on the condition depending on the secondary structure of this protein the
accessibility of this quencher will be different so in this case the quencher cannot interact
with this tryptophan here also it cannot interact with this tryptophan but here somewhat it
can interact with this tryptophan and here the interaction with the tryptophan is quite
easy now it tells me that if i do this quenching
experiment here the quenching will be low here also low here moderate moderate quenching
but here i will get high quenching of fluorescence now consider that as your giving
this perturbation this perturbation could be anything this perturbation could be the
raise in the temperature this perturbation could be the adding of some foreign agents
like guanidine in hydro chloride urea these are the well known well known chemical denature
and which actually unfold some protein so this perturbation could be anything and this
perturbation also could be the lower in the p h so if you change the p h of the medium
from neutral p h seven point four to to or one then also this is also access the perturbation
so the protein is getting denatures or unfolded in this case right if the now
now if i plot the quenching right i can plot the k a d right because this is typical and
dynamic quenching so if the k d value is more right you remember here so when i plot i zero
by i then versus q i get a straight line with intercept equal to one and my slope is k d
right so k d more means the quenching is more so in this case i will get for this case where
the quenching is low i will get a low value of k d then as i will go in this direction
then the k d value will larger and larger so large value of k d so now if i plot the
observed k d value as a function of this perturbation right if it Is the temperature then what i
can simply do i can simply plot the measured k d value as a function of temperature so
here temperature is my perturbation it could be other thing also right so then what i will
going to see the value of k d k d value was really small right over here initial temperature
or the temperature is lower so lets say here is my room temperature and as i increase the
temperature so it may suddenly increase like this and then remains same like such kind
of feature right the feature is it is almost similar here then suddenly increase and then
remains same right such kind of feature by looking at such kind of feature you can comment
that this unfolding of these protein when you raise the temperature is a two step process
right here is your native state in this part is
your native state and this part is your denatured or unfolded state so you see by simply doing
this quenching experiment right of such kind of molecule we can comment on half of the
structure of the protein is changing by increasing the temperature suppose you you have done
this exactly same experiment but not temperature as your perturbation but the change p h as
your perturbation so in this case let us assume that here you have used p h as your perturbation
so and you have measured this k d value at different different p h
and your plotting it this versus p h and what you have seen is that such kind of feature
at neutral p h lets say here is my seven at neutral p h the value of k d what you have
observed from your experiment is really low value right and as and little bit of higher
p h seven point four seven point six seven point eight this value is like this as you
increase the p h probably you have seen such kind of data
and the trend is like this it clearly tells you that when your perturbation
is this p h here is your perturbation then the change of k d is two step here is a transition
number one and here is a transition number two if i assign these you can do so na assignment
if obviously the physiological p h is my native state i assign this as native state and if
this is my denatured or unfolded state i can clearly tell that i have some state which
is not denatured not denatured not native right intermediate state so i would have the
signature of intermediate state in this case right so in this case the denaturation is
not a two step process this is a three step process so i can write the native state of
the protein is first changing to some several intermediate state and then this is converting
to the unfolded state so this is a three state process ok
we can also study the protein drug interaction by this method number four which i will discuss
in elaborately is you can also use this fluorescence quenching for the determination of aggregation
number of micelle so
for the aggregation number of micelle so let me first tell you that what is micelle so
micelle is a supra molecular aggregate ok so this supra molecular aggregates made of
surfactant molecules what is surfactant molecules we have a long alive tic chain with a charge
head group either positive or it could looks something like this negative so this is charge
head group like the famous name of this surfactant is s d s right sodium dodecyl sulphate so
you see this sulphate this is negatively charge head group and dodecyl this is alive tic chain
right similarly c t a b acetyl trimetheyl ammonium bromide so here is my positive head
group and here is the long chain right so this is a positively charged surfactant head
group this is the negatively charged and so on
so if you keep on adding this surfactant in water lets say in water then after a certain
concentration this surfactants will form supra molecular aggregate right then after a certain
concentration this surfactants right will forming supra molecular aggregate that looks
something like this for this surfactant all the head groups of the surfactant will come
outside and these hydro carbon chain of the surfactant will come together to form a kind
of spherical structure is a kind of spherical structure as i have
drawn over here like these are all the positively charge so this positively charge is exposed
to the water and this hydro carbon part i am making a core like of thing so basically
this part is like oil so this inside part is like a oil and the outside is water and
the outside is water so this is like a i have a droplet of oils but these are very tiny
very tiny right in size is nanometre size droplet oil droplet in water and because of
these actually the surfactants like our detergents actually helps to wash our clothes right ok
thats is the different story let us proceed this surfactant molecules form such kind of
supra molecular aggregate only after a certain concentration right if you may if you add
this surfactant lets say this is your water and your keep on adding this surfactant just
for a very small concentration it will not form this micelle only after a certain concentration
after a certain concentration those kind of micelles will form like this these balls like
micelles will form and that concentration is known as critical micelle concentration
and generally denoted written as c m c so for this different different surfactant has
a different values of c m c for this as i said this s d s the c m c of s d s is eight
mill molar so after it cross the eight mill molar concentration in water then only such
kind of micelles will form on the other hand for the c t a p it is about
one mill molar so after one mill molar this c t a b will be form such kind of micelles
right ok now as i have drawn this figure like it consists of several surfactants molecules
is number one two three dot dot dot dot dot dot dot dot n number of surfactant molecule
actually makes this micelle the question is how i will going to determine these c m c
value and the aggregation number so for this micelle formation from the surfactant i encounter
two thing one is that critical micelle concentration and after it the critical micelle concentration
n number of surfactant will molecule this micelle and that is called the aggregation
number of this micelle and this called aggregation number and denoted as n a g g now my question
is that how will going to experimentally determine this c m c and this n aggregation
so there are several method to determine a for the determination of the same c m c for
example surface tension the surface tension of the air water interface right will keep
on increase as you as add the surfactant but after the c m c that will remains same the
if you measure the molar conductance will will be almost similar right before c m c
but once the surfactant will form the micelle the molar conductance will decrease because
of the bulky nature of this micelle however as we are doing the basic course on
the fluorescence spectroscopy we will try to tell you a method based on the fluorescence
how to determine this c m c and the n aggregation for the measurement of this c m c right you
see the situation i have solution lets say this is water and let us take one fluorophore
inside this water right some concentration lets say these are my fluorophore these are
my fluorophore and this structure of this fluorophore is this is my fluorophore and
this looks something this something like this
looks something like this so you see this is not a polar molecule but it is a low solubility
in a water so the solubility of this molecule is higher in non polar medium than in the
polar medium now you are keep on adding this surfactant molecule lets say these are your
surfactant molecule so you are keep on adding so surfactant molecules are they are as you
increase the concentration the concentration of this surfactant molecule is keep on increasing
and there is not much change in the local environment of this fluorophore and in this
case if i now plot the florescence spectra of this molecule how will get you just excite
with a suitable light lambda excitation and then measure the fluorescence intensity as
a function of wavelength then you will get the fluorescence spectra
so i will just plot intensity versus lambda emission so you will get the fluorescence
spectra of this molecule lets say the fluorescence spectra looks something like this you are
not changing the number of molecules in this medium so its remains same because you are
not adding more number of this fluorophore you are not adding more number of fluorophore
with time so the number is remains same what you adding is a surfactant right so the fluorescence
intensity will not change that way right ok now as your adding the surfactant you keep
on measure the fluorescence spectra of this molecule so when there is no surfactant lets
say it looks like this no surfactant and as you are adding surfactant you are measuring
initially there is no change it will remain like this remain like this remain like this
but once there is a formation of those micelles when it will be once the concentration
of the surfactants will cross or will reach the c m c value so then there will be formation
of supra molecular aggregates of this surfactant molecule and automatically as these fluorophores
likes to stay in oil so then this fluorophore will go inside it will go inside this micelle
because inside the micelle as i said oil light outside is water so it will preferred to go
inside the micelle right so it will go inside once it will go inside it will not going to
see that may much of water in inside it as that inside the micelle there is no water
it is like a oil hydrocarbon so when you will going to excite this fluorophore in the excited
state we will not going to be stabilised in the same way that it was in case of water
right when it was present in the water then the stabilisation of the excitation state
was quite large because of the high polarity of the water but inside this micelle micelle
environment the polar the polarities low so it will not going to stabilise so what will
going to see you will see a sudden change of emission maxima why because the emission
maxima tells me that how much it is solo rated if it is solo rated more it is same as leapat
mathaga right when you increase this is the case of excited state dipole moment is more
than the ground state dipole moment so here you see there is a change and when all the
fluorophores will be inside then there will be not much change in the shape of the emission
spectra so if i know plot in a different way the concentration
of surfactant versus lambda emission maximum then for the low value of the surfactant right
these lambda was high value right lower energy as as as you seen here so you will get such
kind of thing and then it will once there is formation of a micelles this value will
decrease and then it it will be again constant so from here what you can get the midpoint
is nothing but the value of this c m c we can use this fluorescence quenching now to
get this aggregation number or this micelle now let us see how we can use this fluorescence
quenching to find out the aggregation number right like we have found the c m c value by
fluorescence method but in this case we will use the fluorescence quenching method to find
the aggregation number of the micelle we will find the n aggregate ok let me define all
the terms step by step and then it will very easy for you to understand
so here first term i will going to define is lets say the concentration of surfactant
let me use this symbol s to denote this aggregation number
lets say n a g g as i said earlier concentration of quencher lets denote s q is usual that
critical micelle concentration that is my c m c now i can write from here
the concentration of micelle or i can write here concentration of micelles this will be
equal to lets represent this as m this is equal to concentration of surfactant minus
that c m c value because only after c m c this surfactant will form the micelle and
divided by aggregation number that means n a g g right and i can now also try to define
this quantity that number of quencher molecule for micelle right represent by this average
q this is nothing but the concentration of q concentration of the quencher divided by
the concentration of the micelle and that will going to be the average its not necessarily
that you all this micelle will have that exact number of quencher but that is my average
right so i will just simply write here concentration of q divided by this m right and i know how
to write this probability so let me write that the probability that a particular micelle
having thats the same way earlier i have done right having n quencher molecule that using
my poison distribution i can write p n this is my average q right to the power n factorial
n e to the power minus average q right now i will take two assumptions which are
obviously valid assumptions right let us take this assumptions with this other colour here
the first one i will take as the the fluorophores and quenchers are such that the can be present
in the micelle it is not that the fluorophore is completely hydro it is a charge and micelle
surface is positively charged fluorophore is also positively charge so fluorophore will
never come to the micelle i will not take such kind of situation i will take a neutral
molecule neutral fluorophore neutral quencher so that it has a hydro part hydro fabric interaction
is favour favourable in that case those all these fluorophores and quenchers will go and
sit the micelle so that depends on me i I can choose the type of fluorophore and
quencher which will go and sit on the micelle will not remain in the solution right so the
first assumption is valid assumption i will write here both the fluorophore and the quencher
stay in the micelle and the second one is that even i have a single quencher present
in the micelle that is good enough to quench the fluorescence completely of the fluorophore
which is present in the micelle clear ok so even a single quencher will deactivate
all the fluorophores in the micelle in this case i will not create such kind of situation
at all i will use the fluorophore concentration is low so that each micelle probably we will
have on and average less than one it means that some micelle will have fluorphores some
are not right but i will use a quencher more that means the micelle which are having fluorophore
that must contain a quencher i will create the condition like that way right and there
is a very little chance that a micelle is having more than one fluorophore but only
one quencher that condition i will not let let it come right so in this case both will
be satisfied if so then let us see what will happen so
then i can write the probability that a micelle having no quencher that is
simply p zero which is q that average q to the power zero by factorial zero e to the
power minus this average q which is nothing but e to the power minus this average q right
so here this means right i can write i by i zero is equal to e to the power minus q
right this means this so this is equal to e to the power minus q by m which is equal
to e to the power minus q n aggregation because i know this means that right s minus that
c m c value whatever it is so if i take ln so ln i zero by i is equal to q n aggregation
divided by s minus c m c right now i can make a plot easily so the plot i will do like this
way i will plot ln this measured quantity right i zero and i they are measured quantity
i zero is the fluorescence intensity without quencher and i is with quencher so i can take
the ln of that and i plot the ln i zero by i versus q so then this this should be a straight
line passing through the origin so then this should be a straight line passing through
the origin and the slope would be n a g g divided by concentration of the surfactant
minus the c m c value i know this concentration of the surfactant
right for a particular experiment and the slope is known c m c you already have determined
from other experiment so you can simply get the n aggregation value so see that using
fluorescence quenching easily we can determined the n aggregation value ok so we will finish
here and thank you for listening