[RASMB] Why is my interference data different from teh absorbance

[Paul Leonard]

Dear Borries Demeler,

Thank-you for replying so quickly.

In response to your suggestions I have a few comments (although these
responses are not directed directly to you but to the RASMB mailing list
as a whole - please enjoy the labor day weekend rather than responding to
me).

I collected abs measurements at both 280nm and 250nm.  For the 250nm
curves there was no problem with the absorbance going above 1.5 abs units
so I used the same radial range as I used with the interference optics.

I believe this rules out aggregation as being responsible for the
difference in curvature between abs and interference.

I really do not know how BeF3- would behave in the experiment.  Actually I
should clarify this a little.  BeF3- is actually the addition of 5.3 mM
BeCl2 and 33 mM NaF.  Previous studies by NMR have found that at these
concentrations the major beryllium fluoride species present is BeF3- (and
not BeF2, BeF42- etc).  However BeF2 is relatively insoluble as is MgF2
which would also be present in small amounts in my sample.

However I guess I over complicated the issue (or at least me greatest
concern) by mentioning the BeF3- experiments.  Even in the experiments
where BeF3- was not present I still do not get the same answer when
fitting the absorbance and interference optics so there must be something
fundamentally wrong with what I am doing as the two techniques are viewing
the exact same protein distribution.


kind regards

Paul



> Paul,
>
> A couple of thoughts: You may have some aggregation in your equilibrium
> experiment (perhaps because of the longer run time). Perhaps in the
> absorbance
> experiment you are clipping the higher OD's because they are out of
> the dynamic range of the optics, but in the interference experiment
> the dynamic range is larger, and you are including the portion of the
> scan that correlates most with the aggregates.
>
> Another possibility is the presence of time invariant noise, but that
> should have been subtracted from the data by your baseline scan.
>
> Finally, I wonder if the BeF3 refractive index is properly handled by
> having a meniscus-matched BeF3 subtracted. I don't know the BeF3 density
> off hand, but dependening on density it could for a gradient and
> significantly contribute to the refractive index signal.
>
> Others may have addtl. ideas...
>
> -borries
>
>> Dear all,
>>
>> I have been running both sedimentation velocity and sedimentation
>> equilibrium AUC experiments on a relatively simple protein system.
>>
>> The sedimentation velocity experiments at multiple protein
>> concentrations
>> show that the protein is just a monomer unless BeF3- is present (to
>> mimic
>> phosphorylation) and then all of the protein becomes dimeric.
>>
>> However if I run sedimentation equilibrium experiments at multiple
>> speeds
>> I find that if I use the absorbance optics it is in agreement with the
>> sedimentation velocity result - protein is just a monomer in the absence
>> of BeF3- but fits as a dimer if BeF3- is present.
>>
>> However if I look at the interference scans for the same cells (for
>> sedimentation equilibrium AUC) the protein looks considerably bigger -
>> i.e. it would appear that it is in equilibrium between monomer and dimer
>> in the absence of BeF3-
>>
>> I have also record analytical size exclusion chromatography profiles for
>> concentrations of the protein all the way from 10uM upto 500 uM and if
>> BeF3- is absent I cannot see any evidence of dimer formation.  If I add
>> BeF3- the protein is all dimer.
>>
>> Therefore I am pretty certain that I should only be seeing monomer in
>> the
>> absence of BeF3- and dimer if BeF3- is present so I'd like to know why
>> my
>> interference data makes the protein look larger than it should.  Is
>> there
>> some correction that I need to apply.  I have recorded water blanks at
>> each speed and it makes virtually no difference if I subtract these
>> scans
>> (Not enough of a correction to account for the additional curvature in
>> the
>> scans).
>>
>> Could it be that the radial calibration is wrong?  Is something wrong
>> with
>> the interference optics?  Any explanation would be greatly appreciated.
>>
>> I fear that if we cannot get reliable results for this simple case using
>> this equipment for more complex systems will become impossible (at least
>> for sedimentation equilibrium experiments).
>>
>> Thank-you for any help you can provide.
>>
>> Paul
>


Department of Biochemistry
UMDNJ-Robert Wood Johnson Medical School
Center for Advanced Biotechnology and Medicine
679 Hoes Lane
Piscataway, New Jersey 08854-5627

Phone: (732) 235-4206
Email: leonard at cabm.rutgers.edu