Fwd: Re: [RASMB] peak in sedimentation velocity scans at high protein conc.

[Peter Schuck]

Hi Everybody,

I agree with everything said, but since there seems to be a developing 
general interest in this topic, I'd like to add one observation we made 
repeatedly with the interference optics.

First - and this is certainly not new - one can easily visualize the 
lensing effect of the boundary.  I am attaching a powerpoint slide showing 
the camera image from a run with several mg/ml thyroglobulin at 50000 
rpm.  There's an obvious lack of light intensity in the boundary when using 
the 12 mm centerpiece, which is absent from the image of the 3 mm 
centerpiece in the same run, as John mentioned.  The lack of light 
intensity is what causes the artificial peaks in the absorbance scans.

But the interesting point is that one can frequently still acquire fringe 
shift data through the boundary - in fact even through the one shown in the 
picture.  They do not exhibit an artificial peak, but they have different 
problems.  At very high gradients, the scans seem to 'skip' fringes, i.e. 
the algorithm to assign fringe shifts from neighboring points fails and 
instead of an increase in fringe shift from one radial point to the next, 
one may see even a slight decrease from one point to the next (I suspect 
the XLI software simply can't deal with more than 0.5 fringes shift for 
neighboring radial points).  As Jack mentioned, we've seen this a lot also 
in CsCl gradients, and at this occasion I've built into SEDFIT some limited 
capability to repair this problem afterwards in software (with an algorithm 
assuming that the boundary and its derivatives are monotonic).  However, 
there also seems to be a range of boundary steepness that is already 
producing peaks in absorbance, but not yet exhibiting the fringe 'skipping' 
in interference optics.  Of course, the fringe profiles from these steep 
gradients can still be questioned with regard to their faithful 
representation of the concentration gradient, which I doubt.

However, the absence of the artificial peak at least allows us to load the 
data and conveniently determine an empirical s-value from the 
midpoint.  Assuming that the distortion of the boundary shape is not 
modifying the integral over the boundary in a time-dependent way, one might 
even be able to get a fairly good precision on the weight-average s.

Best,
Peter




>X-SBRS: 5.5
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>Date: Fri, 28 Oct 2005 08:59:27 -0400
>From: Peter Schuck <pschuck at helix.nih.gov>
>User-Agent: Mozilla Thunderbird 1.0.6 (Windows/20050716)
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>To: Norbert.Muecke at dkfz-heidelberg.de
>Subject: Re: [RASMB] peak in sedimentation velocity scans at high protein
>  conc.
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>Hi Norbert,
>this is Wiener skewing from refraction of light within the boundary. If 
>you use interference optics, you should not observe this. Also, you can 
>probably minimize it by running slower rotor speed and not generating as 
>steep gradients. Peter
>
>
>Norbert Muecke wrote:
>
>>Hi,
>>I have done some velocity runs at high protein conc. (up to 6g/l).
>>A new peak within the boundary could be observed.
>>I have attached the scans of three different concentrations.
>>Absorbance was measured at 284nm.
>>Any idea of how to explain that phenomena would be appreciated.
>>
>>Best regards
>>Norbert
>>
>>
>>Division Biophysics of Macromolecules, B040
>>German Cancer Research Center (DKFZ), TP3
>>Im Neuenheimer Feld 580
>>D-69120 Heidelberg, Germany
>>
>>
>>
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