[RASMB] interference optics

Sat Apr 5 13:05:09 PDT 2008

Hi Fumio-
John is right about the Weiner skewing and I agree that using thinner 
cells is a good idea.
I believe the jogs in your data may have a slightly different origin. At 
each radial position the software can only determine the fractional 
fringe shift (relative to the first radial position, defined as 0 fringe 
shift). In order to keep track of the total fringe shift, the software 
accumulates the total fringe shift, by adding or subtracting the 
increment between the current calculation and the preceding fractional 
fringe determination. There is a limit to the maximum fringe shift 
between adjacent points that can be accounted for. If the data are 
steeper than that, i.e. the fringes shift by 0.9 fringe between two 
radial positions (or in cases where the fringes are of poor quality) the 
software gets confused and you get the sorts of jumps seen in your data. 
In general, the software-caused jumps occur at concentration gradients 
that are below the Weiner skewing limit.

Beckman sells 3 mm centerpieces and Spin Analytical sells 3 mm and 1.2 
mm centerpieces. The spacers for these centerpieces place the solution 
approximately at the midpoint of the cell. In principle, the 
interference optics should be focused at the 2/3 plane of the solution 
to minimize the effects of Weiner skewing. Fortunately, the shorter 
optical path reduces the effects if Weiner skewing (see Yphantis, DA 
[1964] Biochemistry 3:297-317, for a full discussion). There is not a 
way to focus the "stock" XLA interference optics with sufficient 
accuracy. Jeff Lary has modified the XLA lens mounts with micrometer 
screws, which do have sufficient accuracy (though using Gropper's spurs, 
the preferred method of determining the proper focus, is not available 
on the XLA).

John's second point also is true. Most current software have limited 
capabilities to handle nonideality in sedimentation velocity data. This 
means that Weiner skewing has a relatively small effect on boundary 
shape compared to nonideality. SedFit/SedPhat uses two proportionality 
constants to account for the effects of thermodynamic and hydrodynamic 
nonideality. Note that these two terms have opposite effects on boundary 
shape- hydrodynamic nonideality sharpens boundaries, repulsive 
thermodynamic nonideality broadens them. For symmetrical, globular 
proteins with low to moderate charge and in solvents having an ionic 
strength ~0.1 M, the model seems to work adequately for a single 
component up to ~15 - 20 mg/ml (hydrodynamic nonideality dominates). The 
models cannot handle systems that have significant concentrations of 
multiple components (separate cross terms are needed that reference the 
individual concentrations, e.g. the Johnston-Ogston effect) or systems 
where both attractive and repulsive thermodynamic terms are significant. 
Walter Stafford is working adding the ability to handle these cross 
terms to Sedanal.

With the Aviv fluorescence detection system, it is possible to acquire 
data in very complex, very concentrated solutions (e.g. serum, sputum, 
urine, cell lysates) that exhibit all sorts of nonideality. For systems 
like serum, etc. while the nonideality is complicated, it also is 
relatively constant from sample to sample, and you often can answer some 
very interesting qualitative questions deductively.

Best wishes,
Tom

John Philo wrote:
>
> Fumio,
>
> There is a limit on the maximum gradient that can be measured 
> (fringes/mm) due to Weiner (? spelling) skewing, and you are exceeding 
> that limit.  As Ariel said you should switch to 3 mm centerpieces, and 
> then at some point to get to higher concentrations you would have to 
> drop the rotor speed too to broaden the boundaries. If you look 
> through the old RASMB postings you will find a number of 
> excellent posts from others about Weiner skewing and proper optical 
> alignment to minimize distortion of strong gradients.
>
> But probably the more important question is what are you going to be 
> able to do with such data even if you collect it? Remember, in 
> velocity there is hydrodynamic non-ideality as well as thermodynamic 
> non-ideality, so the non-ideality effects will kill you at much lower 
> concentrations than in equilibrium. To my knowledge the only model 
> available to analyze a velocity experiment at 10 mg/mL is a single 
> non-ideal species. At high concentrations you cannot even correctly 
> measure the fractions of different components (e.g. aggregates) in a 
> multi-component mixture due to the Johnston-Ogston effect.
>
> John
>
> -----Original Message-----
> From: rasmb-bounces at rasmb.bbri.org 
> [mailto:rasmb-bounces at rasmb.bbri.org] On Behalf Of Fumio Arisaka
> Sent: Saturday, April 05, 2008 4:20 AM
> To: rasmb at server1.bbri.org
> Subject: [RASMB] [Fwd: interference optics]
>
> Dear RASMBers,
>
> This concerns measurement of high concentrations of IgG with 
> interference optics. I have not much experience with IF, but have 
> started to use it for the necessity to measure high concentration 
> samples. Concentrations less than 5 mg/mL had no problem.
> Attached jpg file is to show the problem at 10mg/mL. Somehow, the 
> boudaries tend to go horizontally before reaching the plateau.
>
> I thought this was due to the misalignment of optics or something, 
> because the fringe pattern is not so good as I expect, but the 
> BeckmanCoulter person who came to fix it could not make it better.
>
> I would like to know what is the common highest concentration that you 
> could measure by IF. As I understand one could measure SE at higher 
> concentrations than 100 mg/mL, but when the boundary gets too steep in 
> SV measurement, the fringes get too much squeezed to count precisely.
>
> Any comments and advice are highly appreciated.
>
> Best wishes, Fumio
>
>
>
>
> ------------------------------------------------------------------------
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