[RASMB] One pure sample, three wavelengths, and differing apparent elution volumes !

[Jacob Lebowitz]

Another possibility is that the RNase A preparation has contamination with 
nucleotides or small RNA/ single strand DNA fragments and there is 
sufficient binding of the latter to generate a mixture of eluting species. 
Can you detect any small molecule elution from the column?

At 02:22 PM 2/4/2006 +1300, Richard Kingston wrote:

>Recently, when running molecular weight standards over a size
>exclusion column I made a puzzling observation.
>
>I routinely monitor  column outflow at three wavelengths, since our
>chromatography system allows it (I'm using an Amersham Biosciences
>Äkta). This is to ensure that an undistorted elution profile is
>obtained at at least one wavelength (For concentrated, and highly
>absorbing samples, the detector response may become non-linear). So
>typically I would monitor the signal at 280 nm (the peak due to Trp/ 
>Tyr/Cys) and at then on the shoulder of this peak (293 and 300nm) for
>reduced sensitivity. Or when increased sensitivity is required, I
>often monitor at 220 or 230 nm, on the far shoulder of the peptide
>bond absorbance peak.
>
>In the past,  when using the instrument in this way, traces have
>always been identical (up to some scaling factor) when the absorbance
>is < 1.0.
>
>But last week, when running standards over a Superdex 75 column, one
>of them (Ribonuclease A) gave different apparent elution volumes at
>different wavelengths (280, 293 and 300nm). The behavior was
>reproducible, and the shifts in the peak position were small yet
>significant (far beyond the inherent uncertainty in the peak
>position). Apparent elution volume at 280 nm > 293 nm > 300 nm. This
>also corresponds to the order in which the wavelengths are cycled by
>the monochomator.
>
>Yet the remaining standards (Chymotrypsinogen A, Ovalbumin, Albumin,
>Blue Dextran 2000, L-Tryptophan) , run before and afterwards, in an
>identical fashion, did not exhibit this behavior. In these cases the
>traces at different wavelengths were all equivalent, accounting for
>the differing absorbance by the sample. These standards are both
>smaller and larger than Ribonuclease A.
>
>So the behavior is both puzzling and concerning (what wavelength
>should I 'believe'  in the case of Ribonuclease A?) and I'm left
>scratching for an explanation. I'm hoping somebody on the list might
>suggest something.
>
>The UV Monitor on the Akta has a 1 cm path length continuous flow
>cell. Light is provided by a xenon flash lamp,  monochromated with a
>grating, and there is a beam splitter which sends 50% pf the light
>through the cell, and the remainder through a reference fiber. Up to
>three wavelengths can be recorded 'simultaneously'  (in practice the
>wavelength is rapidly switched, with the reported switch time < 500 ms).
>
>Thanks for any suggestions
>
>Rich.
>
>Richard Kingston, PhD
>School of Biological Sciences
>The University of Auckland
>Auckland
>New Zealand.
>
>rl.kingston at auckland.ac.nz
>
>
>_______________________________________________
>RASMB mailing list
>RASMB at rasmb.bbri.org
>http://rasmb.bbri.org/mailman/listinfo/rasmb

Jacob Lebowitz, PhD
Molecular Interactions Resource
Division of Bioengineering and Physical Science, ORS
National Institutes of Health
Mail: Bldg. 13 Rm. 3N17
Office Bldg. 13 Rm. 3E49
13 South Drive
Bethesda, MD 20892 - 5766
Tel: (301) 435-1955
Fax: (301) 480-1242
email: lebowitz at helix.nih.gov