fluorescence
Frank Fürst
frank at fuerst.de
Tue Mar 23 13:44:47 EST 1999
Eugene Gussakovsky wrote:
>
> Eugene Gussakovsky <gussak at ias.agri.gov.il> wrote:
> [base-64 encoded Word 8 snipped; here is the contents. Please note
> forged From: and Reply-To: header. I did not write this. - CK]
>
> Dear Mr. Bhupest,
>
> On your questions about thermal unfolding measurements with tryptophan
> fluorescence.
>
[...]
>
> finished yet. So you'll have two linear equations y1=a1+b1*T/v (native) and
> y1=a2+b2*T/v. Using these equations you can construct logK-vs-1/T plot in
> temperature range between these two linear regions, where K=(1/q-y1)/(y2-1/q)
> for each temperature in this range.
Yes, that seems to be the complete treatment of the thing.
> If you have a two-state transition, this
> plot will be linear
But the opposite is not true:
If the plot is linear you cannot be sure that you have a real
equilibrium two-state-transition.
You should at least test reversibility by cooling down the heated
solution and again monitoring flourescence. Only if you observe
reversibility (that means that the curve measured upon refolding
coincides with that of unfolding) you know that you've measured an
equilibrium transition (and not some combination of equilibrium
unfolding and aggregation), and only then can you calculate the
thermodynamic parameters. As proteins are also subject to chemical
degradation at high temperatures, maybe a linear decrease of the
signal is tolerable. I'm not sure about that.
> and gives you thermodynamic parameters of unfolding:
> slope=-enthalpy/R and intercept=entropy/R. If you'll find the plot is
> non-linear, you have more complicate transition.
>
--
****************************************************
Frank Fuerst, Institut für Biochemie der Uni Potsdam
Im Biotechnologiepark, 14943 Luckenwalde
Tel.: +49-3371-681334; Fax.: +49-3371-681339
ffrank at rz.uni-potsdam.de
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