🚀 go-pugleaf

Dominic-Luc Webb wrote:
> I have worked for some time with fluroescence polarization (FP)
> in studies of drug-protein binding. My drug, which is
> hydrophobic, typically has a fluorescent adduct and the
> solution has 0.01% Tween 20 to keep down non-specific binding
> to test tubes, multiwell plates, etc. Assay usually works well.
> 
> I usually do a check with a series of glycerol concentrations
> to ensure that FP is working OK. I expect at high glycerol
> concentration, the higher viscosity lowers molecular rotation
> which is detected as an increase in the amount of polarized
> light in the static (parallel) channel. This usually works
> fine, fluorescein for instance. I recently have found some
> labelled drugs that show no increase in polarization, and
> even a small decrease (about 20 mP) even when adding glycerol
> to 50%. Again, Tween 20 is 0.01% throughout. Fluorescence
> intensity remains relatively constant with only a slight
> parallel increase in both channels. The underlying physics of
> FP always seemed pretty straightforward, but now I am
> confronted with such data that has been reproduced several
> times and it is not clear how this came about.
> 
> Strangely, when a protein that is known to be a drug target is
> present, there is a strong increase in polarization (100-300 mP).

That is what I'd expect.  Binding to a large molecule (i.e., 
the protein) reduces the rotation rate of the labeled drug, 
thus increasing the polarization.

> The glycerol data has created an uncertainty about negative
> data for test proteins that I am screening for drug binding.
> 
> Is there anyone in here that might have some hints how this
> lack of change in polarization in glycerol might have come
> about, maybe some suggestions how to troubleshoot this?

It could be that the change of solvent to 50% glycerol 
reduces the fluorescence lifetime, thus increasing the 
measured polarization.

> 
> Dominic
> 

Thanks Marvin for your response, and sorry about any confusion I
might have created in the use of the word "strangely". The
"strangeness" had only to do with the disjunct between the
unexpected glycerol data and the expected protein binding data
using the same labelled drug. Until now, I regarded both of
these as positive control tests.

Regarding 50% glycerol, I could be convinced that the fluorescence
lifetime was changed, but I would need to bring this in line with
recent experiments. I went back and made some further measurements
and quantifications together with data I collected earlier. I
calculate a 1.4 fold increase in fluorescence intensity (FI) when
measured with normal FI filter set. Using the FP filters to measure
same samples, there was also an increase in fluorescence intensity in
both of the two channels (1.35 in the static channel, 1.41 in the
perpendicular channel). Fluorescence yield must have increased
concommitantly with a differential shift towards the perpendicular
channel.

Considering that last sentence, this assay has thus far not been
very sensitive to different intensities (e.g., fluorescein over a
few orders of magnitude). However, the differential shift towards
the perpendicular channel (1.41/1.35), although small, approaches
significance and seems in line with your suggestion that fluorescence
lifetime was decreased. Does this make sense?

Dominic


> > Strangely, when a protein that is known to be a drug target is
> > present, there is a strong increase in polarization (100-300 mP).
>
> That is what I'd expect.  Binding to a large molecule (i.e.,
> the protein) reduces the rotation rate of the labeled drug,
> thus increasing the polarization.
>
> > The glycerol data has created an uncertainty about negative
> > data for test proteins that I am screening for drug binding.
> >
> > Is there anyone in here that might have some hints how this
> > lack of change in polarization in glycerol might have come
> > about, maybe some suggestions how to troubleshoot this?
>
> It could be that the change of solvent to 50% glycerol
> reduces the fluorescence lifetime, thus increasing the
> measured polarization.
>
> >
> > Dominic
> >
>

Dominic-Luc Webb wrote:
> Thanks Marvin for your response, and sorry about any confusion I
> might have created in the use of the word "strangely". The
> "strangeness" had only to do with the disjunct between the
> unexpected glycerol data and the expected protein binding data
> using the same labelled drug. Until now, I regarded both of
> these as positive control tests.
> 
> Regarding 50% glycerol, I could be convinced that the fluorescence
> lifetime was changed, but I would need to bring this in line with
> recent experiments. I went back and made some further measurements
> and quantifications together with data I collected earlier. I
> calculate a 1.4 fold increase in fluorescence intensity (FI) when
> measured with normal FI filter set. Using the FP filters to measure
> same samples, there was also an increase in fluorescence intensity in
> both of the two channels (1.35 in the static channel, 1.41 in the
> perpendicular channel). Fluorescence yield must have increased
> concommitantly with a differential shift towards the perpendicular
> channel.
> 
> Considering that last sentence, this assay has thus far not been
> very sensitive to different intensities (e.g., fluorescein over a
> few orders of magnitude). However, the differential shift towards
> the perpendicular channel (1.41/1.35), although small, approaches
> significance and seems in line with your suggestion that fluorescence
> lifetime was decreased. Does this make sense?
> 
> Dominic
When you change the environment around a molecule, energy 
levels change.  Try scanning the absorption and emission 
spectra, which should be different in 50 glycerol vs. water. 
  It isn't easy to generalize about specific changes in 
optical properties.