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

[John Philo]

Richard,

What you are describing is exactly what one would expect if your 'pure'
RNase A peak is in fact really composed of multiple unresolved species with
different absorbance spectra, so the leading part of the peak has a
different spectrum from the trailing part. 

Is it possible your RNase has gotten clipped by a protease that is removing
some fragments containing aromatic amino acids? (This might happen for
example if it is contaminated by bacteria.)

Other than that I can't really fully explain the apparent elution time
shift, but the following points may be relevant:

1) Unlike your other protein standards RNase A contains no tryptophan. Thus
its absorbance maximum will occur well below 280 nm, and that absorbance
peak will be narrower (essentially that of tyrosine). RNase will also have
proportionally much less absorbance at 293 and 300 nm than the other
proteins.

2) I don't know the details on an Akta, but typically these chromatography
monitors have a much broader band pass than a bench top spectrophotometer (5
nm is typical). 

John

-----Original Message-----
From: rasmb-bounces at rasmb.bbri.org [mailto:rasmb-bounces at rasmb.bbri.org] On
Behalf Of Richard Kingston
Sent: Friday, February 03, 2006 5:22 PM
To: rasmb at rasmb.bbri.org
Subject: [RASMB] One pure sample, three wavelengths,and differing apparent
elution volumes !



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


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