David,
That is indeed a large difference, although not without precedent (10-25%
changes are common).
I guess, like you, I suspect an artifact is lurking here. My first
thoughts are:
1) Are you getting a major contribution from light scattering in the
spectrum in formic acid? A quick way to assess this is to look at the
apparent absorbance around 350 nm. Assuming, as I do, that you have no
prosthetic groups absorbing there, then the value should be very low. If
it isn't, you've probably got light scatter.
The magnitude of light scatter increases as a power function as the
wavelength decreases. Thus, the contribution at 270-280 nm is considerably
greater than at 350 nm. This will increase the apparent extinction
coefficient artifactually.
2) I would guess that 60% formic acid has a rather significant absorbance
around 260 nm and maybe higher.
How big is the absorbance of the "solvent"? If it's large, then you may be
subtracting a mountain in order to see the molehill.
3) What's the detergent, its concentration, and its CMC? Are there light
scatter problems due to the detergent?
Rod Levine
NIH
At 10:37 AM 5/17/2000 -0400, you wrote:
>see below...
>
>David W. Andrews, Ph. D.
>Director, Analytical Research & Development
>BioChem Pharma Inc.
>30 Bearfoot Road
>Northborough, MA 01532
>
>508-351-1004 (voice)
>508-351-9675 (fax)
>
>andrewsd@biochempharma.com
>
> > -----Original Message-----
> > From: Rod Levine [SMTP:rlevine@nih.gov]
> > Sent: Wednesday, May 17, 2000 10:26 AM
> > To: abrf@aecom.yu.edu; andrewsd@biochempharma.com
> > Subject: Re: (protein) extinction coefficient anomalies....
> >
> > David,
> >
> > Before I join in the discussion, it would be helpful to have a few more
> > details:
> >
> > 1) What are the absorbances under the two conditions? (I ask because a
> > "large difference" to one of us might not be considered large to another.)
> > [Andrews, David] the "native" molecule has an epsilon of ~1.0, which is
> > very close to the theoretical value, 1.04; when denatured in 60% formic
> > acid, the new value is 1.6.
> >
> > 2) What are the buffers, especially the pH? [Andrews, David] the pH of
> > the formic is around 2...I should add that this is a membrane protein, and
> > the "native" buffer contains detergent...
> >
> > Rod Levine
> > NIH
> >
> >
> >
> >
> >
> > David W. Andrews wrote:
> >
> > Has anyone ever encountered a protein in which the extinction
> > coefficient
> > (or the UV absorbance, if you prefer) was markedly different in the
> > denatured versus the native state? I am talking about the absorbance
> > at
> > 280nm, and I have a protein that, when dissolved in acidic buffers,
> > gives a
> > large difference in UV absorbance from its "native" state in
> > non-denaturing
> > buffers.
> >
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